CN116265489A - Bifunctional fusion protein for simultaneously targeting human CD73 and human TGF beta, preparation method and application thereof - Google Patents

Bifunctional fusion protein for simultaneously targeting human CD73 and human TGF beta, preparation method and application thereof Download PDF

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CN116265489A
CN116265489A CN202111552939.5A CN202111552939A CN116265489A CN 116265489 A CN116265489 A CN 116265489A CN 202111552939 A CN202111552939 A CN 202111552939A CN 116265489 A CN116265489 A CN 116265489A
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fusion protein
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张学赛
李晴柔
黄浩旻
朱祯平
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Abstract

The invention provides a bifunctional fusion protein for simultaneously targeting human CD73 and human TGF beta, a preparation method and application thereof. In particular, a bifunctional fusion protein is provided comprising a first binding domain that specifically binds to the target molecule CD73 protein; and a second binding domain that specifically binds to a tgfβ protein of the target molecule. The dual-function fusion protein for resisting CD73 and TGF beta has the advantages of high binding affinity with dual targets, strong specificity and the like, can obviously improve tumor microenvironment immunity, enhance anti-tumor effect and has important clinical application prospect.

Description

Bifunctional fusion protein for simultaneously targeting human CD73 and human TGF beta, preparation method and application thereof
Technical Field
The present invention relates to the field of antibodies. In particular to a bifunctional fusion protein simultaneously targeting human CD73 and human TGF beta, a preparation method and application thereof.
Background
It has been found that one of the main reasons for poor tumor immunotherapy response is the presence of immune cell inhibiting substances in the tumor microenvironment, which lead to the tumor cells escaping from the killing of immune cells. Adenosine, one of the important substances for tumor immunosuppression in tumor microenvironment, activates Protein Kinase A (PKA) and Csk kinase by binding to adenosine receptor (A2 AR), inhibits LCK, MAPK, PKC and other signal pathways related to immune activation, and plays an immunosuppressive role. CD73 is an extracellular-5' -nucleotidase encoded by the NT5E gene, has a molecular weight of 70kD, and is the main rate-limiting enzyme catalyzing the dephosphorization of AMP to adenosine. Expression of CD73 is regulated by molecules such as hypoxia inducible factor-1 (HIF-1), TGF beta, EGFR, AKT, beta-catenin, etc., particularly HIF-1, which functions as a transcription factor, is the most critical. Hypoxia (Hypoxia) is an important feature of the tumor microenvironment, and Hypoxia induces HIF-1 upregulation, which in turn leads to the broad expression of CD73 in the tumor microenvironment. Thus, CD73 is found to be overexpressed on the surface of various tumor cells and is closely related to invasive migration of tumors and poor prognosis, including breast cancer, lung cancer, ovarian cancer, colorectal cancer, renal cancer, gastric cancer, head and neck cancer, and the like.
Preclinical studies have shown that inhibition of CD73 may stimulate T cell activity and enhance antitumor immune monitoring of adenosine regulated T cell and other immune cell levels. Relieving the inhibition of Tumor Microenvironment (TME) on immune effector cells is an important aspect for overcoming the drug resistance and improving the curative effect of immunotherapy. Thus, blocking the production of adenosine by inhibiting the enzymatic activity of CD73 would be a very promising therapeutic strategy to directly disrupt adenosine-mediated immunosuppression.
Transforming growth factor-beta (tgfβ) is a pleiotropic cytokine that can inhibit tumor progression by pre-cancerous cells, as well as promote the spread of advanced tumors. In advanced primary and metastatic tumors, high expression of tgfβ is associated with poor clinical prognosis, tumor treatment resistance, among which the following: 1) TGF beta can significantly inhibit dendritic cell function and CD4 + Or CD8 + Proliferation of T cells, while tgfβ promotes differentiation of regulatory T cells (tregs) and Myeloid Derived Suppressor Cells (MDSCs), forming an immunosuppressive microenvironment; 2) TGF beta can obviously improve the expression of VEGF/VEGF-R and promote the generation of tumor blood vessels by activating ALK1 and the like; 3) TGF beta induces expression of transcription factors such as snail1/2, ZEB1/2, HMGA2 and the like by activating classical smad pathway or non-smad pathway, thereby promoting Epithelial Mesenchymal Transition (EMT), enabling cells to obtain migration and infiltration, and realizing diffusion of cancer cells to other tissues.
In anti-tumor studies, tgfβ1 and CD73 were found to be intimately involved in the progression of tumor development. At the level of tumor cells themselves, it is found in cervical cancer that secretion of tgfβ1 by cervical cancer cells is dependent on the CD 73-adenosine pathway, and tgfβ1 in turn maintains CD73 expression on the cell surface. From the tumor microenvironment level, clinical studies have found that breast cancer cells can induce γδ T cells (γδ T cells are considered to be an important component of breast cancer tumor infiltrating lymphocytes) to express CD73, and their induction process is dependent on tgfβ1.
Therefore, in view of the obvious role of human CD73 and TGF beta in regulating tumor immune response, the design of medicines for simultaneously antagonizing CD73 and TGF beta has wide application prospect in the aspect of cancer treatment.
Disclosure of Invention
The invention aims to provide a bifunctional fusion protein simultaneously targeting human CD73 and human TGF beta, a preparation method and application thereof.
In a first aspect of the present invention, there is provided a bifunctional fusion protein comprising:
a first binding domain D1; and
a second binding domain D2;
wherein D1 specifically binds to the target molecule CD73 protein;
d2 specifically binds to the target molecule tgfβ protein; wherein, the D1 is an anti-CD 73 antibody or antigen-binding fragment thereof; the anti-CD 73 antibody comprises a heavy chain variable region comprising the complementarity determining region CDRs:
HCDR1 shown in SEQ ID NO.1,
HCDR2 shown in SEQ ID NO.2, and
HCDR3 shown in SEQ ID No. 3; and
the light chain variable region has complementarity determining region CDRs selected from the group consisting of:
LCDR1 shown in SEQ ID NO.4,
LCDR2 as shown in SEQ ID NO.5, and
LCDR3 as shown in SEQ ID NO. 6.
In another preferred embodiment, the target molecule TGF-beta protein is selected from the group consisting of: TGF-beta 1 protein, TGF-beta 2 protein, TGF-beta 3 protein, or a combination thereof.
In another preferred embodiment, the antibody comprises: animal-derived antibodies (e.g., murine antibodies), chimeric antibodies, and humanized antibodies.
In another preferred embodiment, the antibody is an IgG class antibody.
In another preferred embodiment, the antigen binding fragment comprises a single chain variable region fragment (scFv), a double chain variable region fragment (dcFv).
In another preferred embodiment, the heavy chain variable region of said anti-CD 73 antibody comprises the amino acid sequence of SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.
In another preferred embodiment, the light chain variable region of said anti-CD 73 antibody comprises the amino acid sequence shown in SEQ ID NO. 17.
In another preferred embodiment, the D2 is a polypeptide fragment that specifically binds TGF-beta 1 protein, TGF-beta 2 protein, or TGF-beta 3 protein.
In another preferred embodiment, the D2 is a polypeptide fragment that specifically binds TGF-beta 1 protein, and the polypeptide fragment is derived from a TGF-beta receptor.
In another preferred embodiment, the D2 is a TGF-beta RII extracellular domain element.
In another preferred embodiment, the TGF-beta RII extracellular domain element comprises a TGF-beta RII extracellular domain partial fragment and mutants thereof.
In another preferred embodiment, the tgfβrii extracellular domain element is selected from one or more of the following groups:
(1) Positions 460-588 of SEQ ID NO. 7;
(2) Positions 460-588 of SEQ ID NO. 8;
(3) Positions 460-588 of SEQ ID NO. 9;
(4) Positions 460-596 of SEQ ID NO. 10;
(5) Positions 460-587 of SEQ ID NO. 11.
In another preferred embodiment, said D1 and said D2 are connected by a linker.
In another preferred embodiment, the joint is a flexible joint.
In another preferred embodiment, the flexible linker comprises 5-30 amino acids, preferably 10-25 amino acids.
In another preferred embodiment, the linker is (G 4 S) n Or (G) 4 S) n G, wherein n is 1, 2, 3 or 4, preferably n is 3.
In another preferred embodiment, D1 is an anti-CD 73 monoclonal antibody and D2 is attached to D1 at a position selected from the group consisting of: the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, the C-terminus of the light chain, or a combination thereof.
In another preferred embodiment, D1 is an anti-CD 73 monoclonal antibody and D2 is attached to the C-terminal end of the heavy chain constant region of D1 by a linker.
In another preferred embodiment, D1 is an anti-CD 73 monoclonal antibody and D2 is linked to the N-terminus of the heavy chain variable region of D1 by a linker.
In another preferred embodiment, D1 is an anti-CD 73 monoclonal antibody and D2 is attached to the C-terminus of the light chain constant region of D1 by a linker.
In another preferred embodiment, D1 is an anti-CD 73 monoclonal antibody and D2 is linked N-terminal to the light chain variable region of D1 by a linker.
In another preferred embodiment, the bifunctional fusion protein is a homodimer.
In another preferred embodiment, each monomer in the bifunctional fusion protein has a structure from N-terminus to C-terminus as shown in formula I:
Figure BDA0003418230160000041
wherein, the liquid crystal display device comprises a liquid crystal display device,
t1, T2, T3, T4 are each independently absent or tgfβrii extracellular domain elements, and at least one is not absent;
l1, L2, L3, L4 are each independently absent or a bond or linker;
VL represents the light chain variable region of an anti-CD 73 antibody;
CL represents the light chain constant region of an anti-CD 73 antibody;
VH represents the heavy chain variable region of an anti-CD 73 antibody;
CH represents the heavy chain constant region of an anti-CD 73 antibody;
"-" represents disulfide or covalent bonds;
"-" represents a peptide bond;
wherein the bifunctional fusion protein has an activity of simultaneously binding TGF-beta and binding CD 73.
In another preferred embodiment, the term "to" is used to refer to one or more interchain disulfide bonds between heavy and light chains.
In another preferred embodiment, each of L1, L2, L3 and L4 is independently (G) 4 S) n Or (G) 4 S) n G, n is 1, 2, 3 or 4.
In another preferred embodiment, T2, T3, T4, L2, L3 and L4 are none and L1 is (G 4 S) 3 Or (G) 4 S) 3 G。
In another preferred embodiment, T1 is a TGF-beta RII ectodomain element selected from one or more of the following groups:
(1) Positions 460-588 of SEQ ID NO. 7;
(2) Positions 460-588 of SEQ ID NO. 8;
(3) Positions 460-588 of SEQ ID NO. 9;
(4) Positions 460-596 of SEQ ID NO. 10;
(5) Positions 460-587 of SEQ ID NO. 11.
In another preferred embodiment, the TGF-beta protein is selected from the group consisting of: tgfβ1 protein, tgfβ2 protein, tgfβ3 protein, or a combination thereof; preferably a TGF-beta 1 protein or a TGF-beta 3 protein.
In another preferred embodiment, the TGF-beta protein is derived from human, mouse, rat or cynomolgus monkey.
In another preferred embodiment, the TGF-beta protein is a human TGF-beta protein.
In another preferred embodiment, the anti-CD 73 antibody is a monoclonal antibody.
In another preferred embodiment, the anti-CD 73 antibody is a humanized antibody.
In another preferred embodiment, the anti-CD 73 antibody is an IgG class antibody.
In another preferred embodiment, the heavy chain amino acid sequence of the anti-CD 73 antibody is as set forth in SEQ ID NO:14, the light chain amino acid sequence of the anti-CD 73 antibody is shown in SEQ ID NO: 15.
In another preferred embodiment, the bifunctional fusion protein is selected from the group consisting of:
(1) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:7, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15;
(2) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:8, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15;
(3) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:9, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15;
(4) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:10, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15;
(5) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:11, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15; or (b)
(6) Polypeptides derived from (1) through (5) that are formed by substitution, deletion, or addition of one or more amino acid residues to the amino acid sequences of (1) through (5) and that have the activity of binding CD73 as well as binding tgfβ.
In a second aspect of the invention there is provided a polynucleotide molecule encoding a fusion protein according to the first aspect of the invention.
In another preferred embodiment, the heavy chain nucleotide sequence of the fusion protein is set forth in SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO:21 or SEQ ID NO:22, the light chain nucleotide sequence of the fusion protein is shown as SEQ ID NO: shown at 26.
In a third aspect of the invention there is provided an expression vector comprising a polynucleotide molecule according to the second aspect of the invention.
In another preferred embodiment, the expression vector comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In another preferred embodiment, the expression vector comprises: pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), pDHFR, pcDNA4, pDHFF, pGM-CSF or pCHO 1.0.
In a fourth aspect of the invention there is provided a host cell comprising an expression vector according to the third aspect of the invention.
In a fifth aspect of the invention, there is provided a method of preparing a fusion protein according to the first aspect of the invention, the method comprising the steps of:
a) Culturing the host cell according to the fourth aspect of the invention under expression conditions, thereby expressing the bifunctional fusion protein;
b) Isolating and purifying the fusion protein of step a).
In a sixth aspect of the invention there is provided a pharmaceutical composition comprising an effective amount of a fusion protein according to the first aspect of the invention and one or more pharmaceutically acceptable carriers, diluents or excipients.
In another preferred embodiment, the pharmaceutical composition is in unit dosage form.
In another preferred embodiment, the pharmaceutical composition further comprises an anti-tumor agent.
In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a gastrointestinal dosage form or a parenteral dosage form.
In another preferred embodiment, the parenteral administration comprises intravitreal injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.
In a seventh aspect of the invention, there is provided an immunoconjugate comprising:
(a) A fusion protein according to the first aspect of the invention; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another preferred embodiment, the conjugate moiety is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.) capable of producing a detectable product.
In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate (ADC).
In another preferred embodiment, the immunoconjugate is used for preparing a pharmaceutical composition for treating a tumor.
In an eighth aspect of the invention there is provided the use of a bifunctional fusion protein as described in the first aspect of the invention, a pharmaceutical composition as described in the sixth aspect of the invention or an immunoconjugate as described in the seventh aspect of the invention, for (a) preparing a detection reagent or kit; and/or (b) preparing a medicament for preventing and/or treating CD73 and/or TGF beta related diseases.
In another preferred embodiment, the detection reagent or kit is for:
(1) Detecting CD73 protein and/or tgfβ protein in the sample or tumor cells; and/or
(2) Diagnosing CD73 and/or TGF beta related diseases.
In another preferred embodiment, the CD73 and/or tgfβ related disease is a cancer selected from the group consisting of: colorectal cancer, bile duct cancer, gall bladder cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, renal cancer, bladder cancer, head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma.
In a ninth aspect of the invention there is provided a method of treating a CD73 and/or tgfβ related disease, the method comprising administering to a subject in need thereof a fusion protein according to the first aspect of the invention, a pharmaceutical composition according to the sixth aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the structural general formula of an antibody fusion protein, anti-CD73 is 48A11-HuV33 or other Anti-CD73 antibodies, TGF beta RII-m is each mutant protein of TGF beta RII, linker is a Linker (G) 4 S) x Other flexible polypeptide sequences are also possible, and x may be 1, 2, 3 or 4.
FIG. 2 shows ELISA assay for binding activity of antibody fusion proteins to human TGF-beta 1.
FIG. 3 shows ELISA assay for binding activity of antibody fusion proteins to human TGF-beta 1.
FIG. 4 shows ELISA assay for binding activity of antibody fusion proteins to human CD 73.
FIG. 5 shows the blocking effect of an antibody fusion protein on TGF-beta signaling pathway.
FIG. 6 shows the blocking effect of antibody fusion proteins on CD73 protein level enzyme activity.
FIG. 7 shows the blocking effect of the antibody fusion protein on CD73 enzyme activity on the surface of MDA-MB-231 cell membrane.
FIG. 8 shows the blocking effect of the antibody fusion protein on CD73 enzyme activity on the surface of H1975 cell membrane.
FIG. 9 shows the blocking effect of the antibody fusion protein on CD73 enzyme activity on the surface of H292 cell membrane.
FIG. 10 shows ELISA assay for binding activity of 48A 11-TGF-beta RII to human TGF-beta family proteins.
FIG. 11 shows ELISA assay for TGF-beta RII-Fc binding activity to human TGF-beta family proteins.
Detailed Description
The inventors have studied extensively and intensively to construct an anti-CD 73/TGF-beta fusion protein for the first time. Wherein the anti-human CD73 antibody moiety is capable of binding highly specifically to CD73 antigen; the constructed bifunctional fusion protein can effectively bind to human TGF-beta protein (especially TGF-beta 1 or TGF-beta 3 protein) and human CD73 protein, effectively inhibit TGF-beta signaling pathway and inhibit CD73 enzyme activity on the cell membrane surface of various cancer cells. The double-function fusion protein has the advantages of high binding affinity with double targets, strong specificity and the like, and has important clinical application prospect. The present invention has been completed on the basis of this finding.
Terminology
In the present invention, the term "fusion protein" refers to a novel polypeptide sequence obtained by fusing two or more identical or different polypeptide sequences. The term "fusion" refers to a direct linkage by a peptide bond or an operative linkage via one or more linking peptides (peptide linkers). The term "linker" refers to a short peptide, typically 2-30 amino acids in length, that can join two polypeptide sequences.
As used herein, the term "linker" refers to the insertion of one or more amino acid residues into an immunoglobulin domain that provides sufficient mobility for the domains of the light and heavy chains to fold into an exchanged double variable region immunoglobulin. In the present invention, a preferred peptide linker refers to peptide linker L, wherein L connects the N-terminus of the extracellular domain of TGF-beta RII to the C-terminus of the heavy chain of an anti-CD 73 antibody.
Examples of suitable peptide linkers include mono glycine (Gly), or serine (Ser) residues, the identity and sequence of the amino acid residues in the peptide linker may vary with the type of secondary structural element that needs to be achieved in the peptide linker. In the present invention, the peptide linker is selected from (G) 4 S) n Preferably (G) 4 S) 3 G or (G) 4 S) 3 。
In the present invention, the terms "Antibody (abbreviated Ab)" and "Immunoglobulin G (abbreviated IgG)" are isotetralin proteins having the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes (isotype). Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a constant region, the heavy chain constant region consisting of three domains CH1, CH2, and CH 3. One end of each light chain has a variable region (VL) and the other end has a constant region, the light chain constant region comprising a domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region of the heavy chain and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC, anti-independent cell-mediated cytotoxicity), and the like. Heavy chain constant regions include the IgG1, igG2, igG3, igG4 subtypes; the light chain constant region includes Kappa (Kappa) or Lambda (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of an antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
The term "antigen-binding fragment" as used herein refers to a fragment of an antibody that is capable of specifically binding to human CD 73. Examples of antigen binding fragments of the invention include Fab fragments, F (ab') 2 Fragments, fv fragments, and the like. Fab fragments are fragments produced by digestion of antibodies with papain. F (ab') 2 Fragments are fragments produced by digestion of antibodies with pepsin. Fv fragments are composed of dimers of the antibody in which the heavy and light chain variable regions are closely non-covalently associated.
In the present invention, the terms "Fab" and "Fc" refer to papain that cleaves antibodies into two identical Fab fragments and one Fc fragment. The Fab fragment consists of VH and CH1 of the heavy chain and VL and CL domains of the light chain of the antibody. The Fc fragment, i.e., the crystallisable fragment (fragment crystallizable, fc), consists of the CH2 and CH3 domains of the antibody. The Fc segment has no antigen binding activity and is the site where an antibody interacts with an effector molecule or cell.
In the present invention, the term "scFv" is a single chain antibody (single chain antibody fragment, scFv) comprising an antibody heavy chain variable region and a light chain variable region, which are usually linked by a linking short peptide (linker) of 15 to 25 amino acids.
The "murine antibody" of the present invention refers to an antibody derived from a rat or mouse, preferably a mouse. The murine antibody is obtained by immunizing a mouse with human CD73 as an antigen and screening hybridoma cells.
The "chimeric antibody" of the present invention refers to an antibody comprising heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.
In the present invention, the term "variable" means that some portion of the variable region in an antibody differs in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the heavy and light chain variable regions, known as complementarity-determining region (CDR) or hypervariable regions. The more conserved parts of the variable region are called the Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)).
The term "humanized antibody" as used herein refers to antibodies whose CDRs are derived from a non-human species (preferably a mouse) and the remainder of the antibody molecule (including the framework and constant regions) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity.
As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, i.e., refers to those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR-L and FR1-H, FR2-H, FR3-H, FR-H, respectively. Accordingly, the light chain variable domain may thus be referred to as (FR 1-L) - (CDR 1-L) - (FR 2-L) - (CDR 2-L) - (FR 3-L) - (CDR 3-L) - (FR 4-L) and the heavy chain variable domain may thus be denoted as (FR 1-H) - (CDR 1-H) - (FR 2-H) - (CDR 2-H) - (FR 3-H) - (CDR 3-H) - (FR 4-H). Preferably, the FR of the invention is a human antibody FR or a derivative thereof which is substantially identical to a naturally occurring human antibody FR, i.e. has a sequence identity of up to 85%, 90%, 95%, 96%, 97%, 98% or 99%. Knowing the amino acid sequence of the CDRs, one skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR-H.
As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.
In the present invention, the terms "anti", "binding", "specific binding" refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. Typically, the antibody is present at less than about 10 -7 M, e.g. less than about 10 -8 M、10 -9 M、10 -10 M、10 -11 An equilibrium dissociation constant (KD) of M or less binds to the antigen. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. For example, the binding affinity of an antibody to an antigen is determined in a BIACORE instrument using surface plasmon resonance (Surface Plasmon Resonance, abbreviated SPR) or the relative affinity of an antibody to antigen binding is determined using ELISA.
In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. An epitope of the invention is a region of an antigen to which an antibody binds.
Bifunctional fusion proteins
The bifunctional fusion protein of the present invention is a bifunctional fusion protein against CD73 and TGF-beta, comprising an anti-CD 73 antibody portion, an extracellular domain of TGF-beta receptor II responsible for capturing TGF-beta (TGF-beta RII), or a mutant portion thereof.
In another preferred embodiment, the anti-CD 73 antibody comprises a heavy chain variable region comprising the following complementarity determining region CDRs:
HCDR1 shown in SEQ ID NO.1,
HCDR2 shown in SEQ ID NO.2, and
HCDR3 shown in SEQ ID No. 3; and
the light chain variable region comprises the following complementarity determining region CDRs:
LCDR1 shown in SEQ ID NO.4,
LCDR2 as shown in SEQ ID NO.5, and
LCDR3 as shown in SEQ ID NO. 6.
Further, the anti-CD 73 antibody is 48A11-Huv33, comprising a heavy chain variable region as shown in SEQ ID NO.16 and a light chain variable region as shown in SEQ ID NO. 17.
As used herein, "bifunctional fusion proteins of the invention," "antibody fusion proteins of the invention," "bifunctional fusion proteins of anti-CD 73 and tgfβ," or "bifunctional fusion proteins targeting CD73 and tgfβ" are used interchangeably and refer to the fusion proteins of the first aspect of the invention. In specific embodiments, the bifunctional fusion proteins of the invention comprise 48A 11-TGF-beta RII, 48A 11-TGF-beta RII WT, 48A 11-TGF-beta RII WD, 48A 11-TGF-beta RIIM2 and 48A 11-TGF-beta RIIM2 delT. Preferably, the bifunctional fusion protein of the present invention is 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIM2 or 48A 11-TGF-beta RIIM2 delT.
The anti-CD 73 antibodies of the invention may also be modified or engineered by techniques well known in the art, such as by adding, deleting and/or substituting one or more amino acid residues, to further increase the affinity or structural stability of the anti-CD 73 and to obtain modified or engineered results by conventional assay methods.
The bifunctional fusion proteins of the invention comprise proteins that specifically bind to the target molecule TGF-beta, such as binding to human TGF-beta family proteins (e.g., TGF-beta 1 protein, TGF-beta 2 protein, and TGF-beta 3 protein). Preferably, the protein that specifically binds to the target molecule TGF-beta is the extracellular domain of TGF-beta receptor II (TGF-beta RII) or a mutant portion thereof.
Wherein the tgfβrii extracellular domain or mutant portion thereof is selected from one or more of the group consisting of:
(1) Full length tgfβrii extracellular domain;
(2) A 6-10 amino acid C-terminally truncated TGF-beta RII extracellular domain, more preferably an 8 amino acid C-terminally truncated TGF-beta RII extracellular domain;
(3) A tgfβrii extracellular domain N-terminally truncated by 18-22 amino acids;
(4) A full-length or truncated tgfβrii extracellular domain comprising at least 1 glycosylation site mutation selected from the group consisting of S9P, T17P, N Q.
Specifically, the sequence of the full-length TGF-beta RII extracellular domain is shown in 460-596 of SEQ ID NO.10, and the sequence of the mutant part thereof is selected from one or more of the following groups:
(1) Positions 460-588 of SEQ ID NO. 7;
(2) Positions 460-588 of SEQ ID NO. 8;
(3) Positions 460-588 of SEQ ID NO. 9;
(4) Positions 460-587 of SEQ ID NO. 11.
In the present invention, the bifunctional fusion proteins of the present invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the bifunctional fusion protein of the present invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
Figure BDA0003418230160000121
Figure BDA0003418230160000131
The bifunctional fusion proteins of the present invention may be used alone, or may be conjugated or coupled to a detectable label (for diagnostic purposes), a therapeutic agent, or a combination of any of the above.
Coding nucleic acids and expression vectors
The invention also provides polynucleotide molecules encoding the antibodies or fragments or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
The sequence of the DNA molecule of the bifunctional fusion protein of the present invention or a fragment thereof can be obtained by a conventional technique such as a method using PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, it is already possible to obtain the DNA sequences encoding the antibodies of the invention (or fragments or derivatives thereof) described, entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
Wherein the vector is a conventional expression vector in the art, and refers to an expression vector comprising appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate sequences. The expression vector may be a virus or plasmid, such as a suitable phage or phagemid, see, e.g., sambrook et al Molecular Cloning for further technical details: a Laboratory Manual, second edition, cold Spring Harbor Laboratory Press,1989. A number of known techniques and protocols for nucleic acid manipulation are described in Current Protocols in Molecular Biology, second edition, ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), pDHFR, pcDNA4, pDHFF, pGM-CSF or pCHO 1.0.
In the present invention, the term "host cell" is a variety of host cells conventional in the art, as long as the vector is stably self-replicating and the polynucleotide molecule carried can be efficiently expressed. Wherein the host cell comprises a prokaryotic expression cell and a eukaryotic expression cell, preferably the host cell comprises: COS, CHO, NS0, sf9, sf21, DH5 a, BL21 (DE 3), TG1, BL21 (DE 3), 293F or 293E cells.
Preparation of fusion proteins
Typically, the transformed host cell is cultured under conditions suitable for expression of the bifunctional fusion monoproteins of the invention. The antibodies of the invention are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. Biochem.,107:220 (1980).
The antibodies of the invention may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
Detection application and kit
The fusion proteins of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include, for example, resected samples of tumors, tissue samples prepared by endoscopic methods or puncture of organs or needle biopsies.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit containing the fusion protein, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
Pharmaceutical composition and application
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 4 to 8, preferably about 5 to 7, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection.
In the present invention, the term "pharmaceutical composition" means that the bifunctional fusion proteins of the present invention can be combined with pharmaceutically acceptable carriers to form pharmaceutical formulation compositions for more stable therapeutic effects, which can ensure the conformational integrity of the amino acid core sequences of the bifunctional fusion proteins disclosed herein, while also protecting the multifunctional groups of the proteins from degradation (including, but not limited to, aggregation, deamination or oxidation).
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the bifunctional fusion protein of the invention (or a conjugate thereof) as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the bifunctional fusion proteins of the present invention may also be used with other therapeutic agents.
In using the pharmaceutical composition, a safe and effective amount of the bifunctional fusion protein or an immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight and in most cases no more than about 50 milligrams/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 milligrams/kg body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
In the present invention, the term "effective amount" refers to an amount or dose that produces a desired effect in a treated individual, including an improvement in the condition of the individual, following administration of the pharmaceutical composition of the present invention to a subject. The term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, rats, mice, and the like.
The invention also provides uses of the bifunctional fusion proteins of the invention, or pharmaceutical compositions thereof, or immunoconjugates thereof, including (but not limited to):
diagnosing, preventing and/or treating tumorigenesis, growth and/or metastasis, for example, diseases associated with CD73 and/or tgfβ or a tumor or cancer with high expression of CD 73: colorectal cancer, bile duct cancer, gall bladder cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, renal cancer, bladder cancer, head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma.
The main advantages of the invention include
(1) The double-function fusion protein has high binding affinity with double targets and strong specificity, and can effectively bind human TGF beta and human CD73 protein; in particular human TGF-beta 1 and human CD73 proteins.
(2) The bifunctional fusion protein provided by the invention can inhibit the CD73 enzyme activity on the cell membrane surface of various cancer cells.
(3) The bifunctional fusion protein provided by the invention can effectively inhibit the activation of TGF beta/SMAD signal channels.
(4) The double-function fusion protein can effectively improve tumor microenvironment immunity and has obvious synergistic anti-tumor activity.
The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Experimental materials and reagents:
293SBE cells: purchased from BPS bioscience under the trade designation 60653
MDA-MB-231 cells: purchased from cell bank of China academy of sciences, cat No. SCSP-5043
H1975 cells: purchased from cell bank of China academy of sciences, cat No. SCSP-597
H292 cells: purchased from cell bank of China academy of sciences, cat No. SCSP-582
Human CD73-his: the mature human CD73 sequence (Met 1-Lys547, NCBI accession No. NP-002517.1) was cloned into the expression vector pcDNA3.4 (available from Thermo Fisher, cat. A14697) by transfection of HEK293F (available from Thermo Fisher, cat. A14527) and after 5d, the cell supernatant was collected and purified to obtain the CD73-His protein.
TGF-beta RII-Fc protein: the Thr23-Glu161 (Uniprot registration number P37173) of the human TGF-beta RII sequence is fused with the Fc region sequence of human IgG4, the signal peptide sequence is added at the N-end, and then the mixture is cloned into an expression vector pcDNA3.4, and the mixture is expressed by transfecting HEK293F cells, and after 5d, the supernatant of the cells is collected and purified to obtain the TGF-beta RII-Fc protein.
Human tgfβ1-his: purchased from R & D systems under the designation 240-B-010.
Human tgfβ2: purchased from Biosystems ACRO under the designation TG2-H4215.
Human tgfβ3: purchased from Biosystems ACRO under the designation TG3-H5213.
CD73-his-biotin protein: CD73-his protein was biotinylated according to the instructions of EZ-Link NHS-Biotin Reagent (available from Thermo Fisher, cat. No. 20217).
HRP-anti human IgG Fc secondary antibody: purchased from Sigma, cat No. a0170.
TMB: purchased from BD company under the accession number 555214.
Adenosine-5-monophosphate sodium salt (AMP): purchased from sigma company under the product number A1752-5G
Adenosine 5' -triphosphate (ATP) disodium salt hydrate: purchased from sigma company under the product number a7699-1G.
Bio-GloTMLuciferase Assay System: purchased from promega company under the trade designation G7940.
CellTiter-Glo Luminescent Cell Viability Assay: purchased from promega company under the trade designation G7572.
RPMI1640 basal medium, MEM basal medium, 1% glutine (glutamine), 1%Sodium pyruvate (sodium pyruvate), 1% MEM-NEAA (minimal basal medium-non-essential amino acid solution), 1% penicillin-streptomycin (penicillin-streptomycin), beta-mercaptoethanol, FBS (fetal bovine serum): all purchased from Gibco company.
SA protein: streptavidin, available from Sigma under accession number 85878-1MG.
SFM medium: purchased from life technologies company under the trade designation 12045-076.
Tris MgCl 2 Buffer: laboratory preparation, which comprises the following components: 25mM Tris,5mM MgCl 2 ，pH7.5。
Example 1 preparation of antibody fusion proteins
1.1 preparation and screening of antigen immunized animals and hybridomas
1. Antigen expression
The extracellular region gene of CD73 (SEQ ID NO: P21589 from UniProt) was constructed into pcDNA 3.4 expression vector by conventional gene synthesis and molecular cloning method, and the N-terminal thereof was added with a signal peptide sequence, the C-terminal thereof was added with a 6 XHis tag, HEK-293F cells were transfected, expressed for 5d, and cell culture supernatant was collected and purified to obtain CD73-His protein. Similarly, HEK-293F cells were transfected after the 6 XHis tag was replaced with the Fc sequence of human IgG1, and the CD73-Fc protein was obtained after expression and purification.
2 antigen immunized mice
Balb/c mice were routinely immunized with CD73-His protein. Balb/c mice were subcutaneously injected at multiple points (CD 73-His protein, 100. Mu.g/mouse/0.5 mL) after emulsification of soluble human CD73-His protein with Freund's complete adjuvant, at day 14, after emulsification of soluble CD73-His protein with Freund's incomplete adjuvant, balb/c mice were subcutaneously injected (CD 73-His protein, 50. Mu.g/mouse/0.5 mL), after emulsification of soluble CD73-His protein with Freund's incomplete adjuvant, at day 28, balb/c mice were subcutaneously injected (CD 73-His protein, 50. Mu.g/mouse/0.5 mL), three weeks later, and mice spleen was taken for fusion experiments after 3-4 days.
Preparation and screening of hybridomas
The spleen cells of the mice were PEG-fused with myeloma cells SP2/0 of the mice 3-4 days after the last immunization of the mice using conventional hybridoma protocols. The fused cells were suspended uniformly in complete medium composed of RPMI1640-GLUMAX added to 1% penicillin-streptomycin,20% fbs,1 x hat. Cell mass after fusion was 3×10 4 A total of 62 96-well cell culture plates were plated at 200. Mu.l/well and incubated in an incubator. After 7-12 days, the supernatant was harvested and hybridoma wells positive for human CD73 binding activity were screened by ELISA.
Wherein, the ELISA method is used for screening hybridoma holes positive for human CD73 binding activity, and the method is as follows: CD73-Fc was diluted to 1. Mu.g/ml with PBS buffer, 100. Mu.l/well was added to ELISA plates and coated overnight at 4 ℃. The supernatant was thrown off the next day, PBST plates were washed 1 time, 5% skim milk powder prepared with PBS was added, blocking was performed at 37℃for 2 hours, and PBST plates were washed 3 times for use. The collected hybridoma supernatants were sequentially added to the blocked ELISA plates at 100. Mu.l/well and left at 37℃for 1 hour. PBST washing the plate for 3 times, adding HRP-marked goat anti-mouse IgG secondary antibody, and standing at 37 ℃ for 30min; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu l of TMB is added into each hole, and the color development is carried out at room temperature and in a dark place for 5min; mu.l of 2M H are added to each well 2 SO 4 The termination solution terminates the substrate reaction, the OD value is read at the 450nm position of the multifunctional enzyme-labeled instrument, and the binding capacity of the antibody to be detected and the target antigen CD73 is analyzed. A total of 30 hybridoma cell lines were obtained by screening. Amplifying and screening 30 Hybridoma cell strains in a serum-containing complete culture medium, centrifuging and changing the liquid to a serum-free Hybridoma-SFM culture medium to ensure that the cell density is 1-2 multiplied by 10 7 Per ml, at 8% CO 2 Culturing at 37deg.C for 1 week, centrifuging to obtain culture supernatant, purifying by Protein G affinity chromatography to obtain mouse monoclonal antibody proteins against human CD73, and naming.
1.2 binding ability of murine antibody to human CD73-His protein
Indirect enzyme-linked immunosorbent assay (ELISA) measures the binding capacity of murine antibodies to human CD73-His protein. The specific method comprises the following steps:
SA protein was diluted with coating solution (50 mM carbonate coating buffer, pH 9.6) to 1.5. Mu.g/mL coated ELISA plate, 4℃overnight; discarding the supernatant, washing the plate 3 times by PBST, preparing 5% skimmed milk powder for sealing by PBS, and incubating for 2h at 37 ℃; after washing the plates 1 time with PBST, the CD73-Biotin protein (obtained by biotinylating CD73-His protein according to EZ-Link NHS-Biotin Reagent instructions) was diluted to 0.5. Mu.g/mL, 100. Mu.L/well and incubated for 1h at room temperature; PBST plate was washed 3 times, and the prepared anti-human CD73 murine monoclonal antibodies were subjected to gradient dilution with 1% BSA buffer prepared from PBSTRelease, add 100 μl/well to the ELISA plate above, incubate for 1h at 37deg.C; PBST washing the plate for 3 times, adding HRP-labeled goat anti-mouse IgG secondary antibody, and incubating for 30min at 37 ℃; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu l of TMB developing solution is added into each hole, the color development is carried out for 5min at room temperature and in a dark place, 50 mu l of 2M H is added into each hole 2 SO 4 The termination solution terminates the substrate reaction, the OD value is read at the 450nm position of the multifunctional enzyme-labeled instrument, and the binding capacity of the antibody to be detected and the target antigen human CD73-His is analyzed.
As shown in Table 1, the binding activity of murine antibodies 48A11, 56B10 to CD73 protein was relatively strong.
Table 1: EC of each murine monoclonal antibody to CD73-Fc binding 50
Sample of 59D6 41A11 48A11 32G2 56B10 4H1
EC 50 (ng/mL) 6114 148673 19.68 35.64 26.87 1235
1.3 ability of murine antibodies to inhibit CD73 enzymatic Activity
CD73 is an enzyme that catalyzes the dephosphorylation of Adenosine Monophosphate (AMP) to adenosine. The inhibition of the activity of the CD73 protease on the surface of H1975 cells by the murine antibodies was determined here by means of ATP detection. The specific method comprises the following steps:
h1975 cells in the logarithmic growth phase were collected, the cell culture solution was removed by centrifugation, and the cells were washed 1 time with PBS buffer; counts were taken and diluted to 3 x 10 with RPMI-1640 medium containing 10% fbs 4 Well, spreading the cells into 96-well cell culture plates, 100 mu L/well, and culturing overnight at 37 ℃ in a cell culture box; the next day, the cell culture supernatant was discarded, the antibody to be tested was diluted to 10. Mu.g/ml with RPMI-1640 medium, diluted 5-fold in a gradient, then added to the above cell culture plate at 50. Mu.L/well, and incubated at 37℃for 30min; then, 800. Mu.M AMP was added thereto, 50. Mu.L/well was incubated at 37℃for 3 hours, 25. Mu.L of the culture supernatant was mixed with 25. Mu.L of 80. Mu.M ATP in a 96-well white opaque assay plate, 50. Mu.L of Cell Titer-Glo assay reagent was added thereto, and incubated at room temperature for 5 minutes, and fluorescence intensity was read and analyzed in a multifunctional microplate reader.
Representative experimental results are shown in Table 2, in which 48A11, 56B10 inhibited the activity of the H1975 cell surface CD73 protease most strongly than other murine monoclonal antibodies.
Table 2: inhibition of H1975 cell surface CD73 protease activity by various murine monoclonal antibodies
Sample of 59D6 41A11 48A11 32G2 56B10 4H1
IC 50 (ng/mL) 164.7 1.041 17.01 130.1 20.74 2507
Sequence analysis was performed on the preferred murine antibody 48A11 to obtain the antigen Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs), and humanization was performed to construct and prepare humanized anti-CD 73 monoclonal antibodies 48A11-Huv33, wherein the heavy chain variable regions of the obtained anti-CD 73 monoclonal antibodies 48A11-Huv are shown in SEQ ID NO.16 and the light chain variable regions are shown in SEQ ID NO. 17. The heavy chain variable region is recombined with the human IgG4 (S228P) constant region, and the light chain variable region is recombined with the human kappa chain constant region to obtain heavy chains (SEQ ID NO. 14) and light chains (SEQ ID NO. 15) of 48A11-Huv33 respectively.
1.4 preparation of fusion proteins
The invention adopts gene synthesis and conventional molecular cloning means, the heavy chain C-terminal of the anti-CD 73 monoclonal antibody 48A11-Huv33 is connected in series with the TGF beta RII extracellular region partial fragments and mutants thereof through a polypeptide Linker (Linker), the heavy chain of fusion protein is obtained and constructed to an expression vector pcDNA 3.4, HEK293F cells are co-transfected with light chain expression vectors of the 48A11-Huv33 monoclonal antibodies respectively, after 5d expression, cell culture supernatants are collected and purified to obtain each sample.
The antibody fusion proteins were designated 48A 11-TGF-beta RII, 48A 11-TGF-beta RII WT, 48A 11-TGF-beta RII WD, 48A 11-TGF-beta RIIM2 and 48A 11-TGF-beta RIIM2 delT, respectively, the control monoclonal antibody was 48A11-Huv33, and the control fusion protein was TGF-beta RII-Fc (SEQ ID NO: 12).
The structural general formula of the antibody fusion protein is shown in figure 1, the Anti-CD73 is 48A11-HuV33 or other Anti-CD73 antibodies, TGF beta RII-m is each mutant protein of TGF beta RII, and Linker is a connecting sequence (G) 4 S) n Other flexible polypeptide sequences are also possible, and n may be 1, 2, 3 or 4.
The relevant sequence information is as follows:
table 3: sequence listing of each fusion protein
Figure BDA0003418230160000201
Figure BDA0003418230160000211
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EXAMPLE 2ELISA assay of binding Activity of antibody fusion proteins to human TGF-beta 1
Human TGF-beta 1-his was diluted to 0.1. Mu.g/ml with coating solution, 100. Mu.l/well, 4℃overnight; then, the mixture was blocked with 5% nonfat milk powder prepared with PBS and incubated at room temperature for 2 hours. PBST plate was washed 3 times, and samples to be tested, which were gradient diluted with PBS containing 1% BSA, were sequentially added to the blocked ELISA plate, 100. Mu.l/well, and incubated for 1h at room temperature. PBST is washed for 3 times, HRP-marked goat anti-human IgG secondary antibody is added, and the mixture is incubated for 30 minutes at room temperature; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu l of TMB is added into each hole, and the color development is carried out at room temperature and in a dark place for 5min; mu.l of 2M H are added to each well 2 SO 4 The stopping solution stops the substrate reaction, the OD value is read at 450nm of the enzyme-labeled instrument, and the binding capacity of the sample to be tested and the target antigen human TGF beta 1-his is analyzed.
As a result, as shown in FIG. 2, each sample showed a good binding activity to TGF-beta 1-his, and the EC of 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIPT, 48A 11-TGF-beta RIIDWD, 48A 11-TGF-beta RIIM2 50 0.171nM, 0.22 respectively2nM, 0.168nM, 0.176nM, which is not less active than the control fusion protein TGF-beta RII-Fc, its EC 50 0.132nM.
As shown in FIG. 3, the binding activities of 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIM2 and 48A 11-TGF-beta RIIM2 delT fusion proteins to TGF-beta 1-his were again compared, and the results indicate that 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIM2 delT bind to the EC of TGF-beta 1-his 50 The differences between the three were not significant, namely 0.089nM, 0.085nM and 0.088nM, respectively, and the EC of the control fusion protein TGFβRII-Fc in this experiment 50 0.074nM.
Example 3 ELISA method for determining binding Activity of antibody fusion protein to human CD73
Biotin-avidin (SA) was diluted to 1. Mu.g/ml with coating solution (50 mM carbonate coating buffer, pH 9.6), 100. Mu.l/well, coated ELISA plate, 4℃overnight; then, 5% nonfat dry milk formulated with PBS was blocked at room temperature for 2 hours. Biotinylated recombinant human CD73-his protein was diluted to 0.2. Mu.g/ml with coating solution, 100. Mu.l/well, added to plates of pre-coated SA and incubated for 1h at room temperature. The PBST plate was washed 3 times, and samples to be tested, which were diluted in PBS containing 1% BSA, were sequentially added to the blocked ELISA plate, 100. Mu.l/well, and incubated at room temperature for 1h. PBST is washed for 3 times, HRP-marked goat anti-human IgG secondary antibody is added, and the mixture is incubated for 30 minutes at room temperature; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu l of TMB is added into each hole, and the color development is carried out at room temperature and in a dark place for 5min; mu.l of 2M H are added to each well 2 SO 4 The stopping solution stops the substrate reaction, the OD value is read at 450nm of the enzyme labeling instrument, and the binding capacity of the sample to be tested and the target antigen human CD73-his is analyzed.
As a result, as shown in FIG. 4, each sample was able to bind to CD73 protein, EC of 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIM2 delT 50 0.166nM, 0.145nM, 0.223nM, corresponding to the affinity of control antibody 48A11-HuV33 for CD73, EC 50 0.17nM.
EXAMPLE 4 blocking of TGF-beta Signal pathway by antibody fusion proteins
The blocking activity of the antibody fusion proteins on tgfβ signaling pathways was determined here using 293SBE cells. The specific method comprises the following steps: 293SBE cells in the logarithmic growth phase were digested, counted, plated in 96 Kong Baiban, 30000 cells were plated per well at 100. Mu.L/well, and cultured overnight for about 24 hours. The plate supernatant was discarded, and 100. Mu.L of a mixture of TGF-beta.1 (final working concentration 10 ng/ml) and antibody fusion protein (highest working concentration 1.5nM for diabody, diluted in 1.5-fold gradient) diluted in MEM medium containing 0.5% FBS was added to the cell wells. The culture was performed in a carbon dioxide incubator at 37℃for 22 hours. 100 mu L of Bio-GloTMLuciferase Assay System is added to each well, the mixture is incubated for 10min at room temperature, the fluorescence intensity value of each experimental well is read by a multifunctional enzyme-labeled instrument, and the inhibition effect of each sample on a TGF beta signal channel is analyzed.
As a result, as shown in FIG. 5, each antibody fusion protein was effective in inhibiting TGF-beta signaling pathway, 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIM2 delT IC 50 IC of control fusion protein TGF-beta RII-Fc at 0.534nM, 0.587nM, 0.619nM, respectively 50 0.636nM.
EXAMPLE 5 blocking of enzyme Activity of antibody fusion proteins against CD73 protein levels
CD73 catalyzes the conversion of AMP to adenosine, and thus this example uses the consumption of AMP by CD73 to determine the blocking of CD73 protease activity by the antibody fusion protein. The specific method comprises the following steps: CD73-his was treated with Tris MgCl 2 Buffer was added to a 96-well plate at a concentration of 1. Mu.g/mL, 25. Mu.L per well. The respective Tris MgCl 2 buffer diluted antibody fusion protein was added to the 96-well plate described above, and 25. Mu.L of each well was added. Incubate at 37℃for 1h. Addition of Tris MgCl to 96 well plates 2 Buffer diluted AMP/ATP mix (final concentrations 300. Mu.M and 100. Mu.M, respectively) at 25. Mu.L per well. Incubate at 37℃for 1.5h. 50 mu L of Cell Titer-Glo reagent is added into each hole, and the mixture is mixed for 2min under shaking, then the mixture is placed at room temperature for reaction for 8min, each fluorescence value is read by a multifunctional enzyme-labeling instrument, and the inhibition effect of each antibody fusion protein on the activity of CD73 protease is analyzed.
As shown in FIG. 6, each antibody fusion protein was effective in inhibiting the activity of CD73 protein in catalyzing the conversion of AMP to adenosine. IC of 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIM2 delT 50 17.855nM, 13.890nM, 20.285nM, control monoclonal antibody 48A11-HuV33 IC 50 12.82nM.
EXAMPLE 6 blocking of CD73 enzymatic Activity by antibody fusion proteins against MDA-MB-231 cell membrane surface
MDA-MB-231 is a triple negative breast cancer cell with high expression of CD73 protein on the surface of the cell membrane, and is used as a source of CD73 protein in the embodiment to determine the activity of each antibody fusion protein.
MDA-MB-231 cells in the logarithmic growth phase were digested and washed once, counted, and plated in 96-well cell culture plates at 100. Mu.L, 4000 cells per well. After overnight adherent culture, the cell culture supernatant was discarded and the cells were washed once with 200. Mu.L of basal medium per well (RPMI 1640), 50. Mu.L of antibody fusion protein diluted with basal medium was added per well, and incubated at 37℃for 0.5h. 50. Mu.L of AMP diluted in basal medium with a final concentration of 300. Mu.M was then added to each well and incubated at 37℃for 3h. From each well 50. Mu.L of the incubation supernatant was removed to a 96 well white plate, and an equal volume of ATP diluted in basal medium with a final concentration of 100. Mu.M was added and incubated at 37℃for 15min. 100 mu L of Cell Titer-Glo reagent is added into each hole, and the mixture is mixed with shaking for 2min and reacts for 8min at room temperature, a multifunctional enzyme label instrument is used for reading fluorescence values, and the inhibition effect of the antibody fusion protein on the activity of the CD73 protease on the Cell surface of the MDA-MB-231 is analyzed.
As shown in FIG. 7, each antibody fusion protein can effectively inhibit the activity of MDA-MB-231 cell surface CD73 protein for catalyzing AMP to be converted into adenosine, and the IC of 48A11-TGF beta RII, 48A11-TGF beta RIIM2 and 48A11-TGF beta RIIM2 delT 50 IC of control monoclonal antibody 48A11-HuV33 at 0.425nM, 0.298nM, 0.300nM, respectively 50 0.246nM.
Example 7 blocking action of antibody fusion proteins on CD73 enzymatic Activity on H1975 cell membrane surface
H1975 is a non-small cell lung cancer cell with a high expression of CD73 protein on the surface of the cell membrane, and is used as a source of CD73 protein in this example to determine the activity of each antibody fusion protein.
H1975 cells in the logarithmic growth phase were lysed and washed once, counted, and plated at 100. Mu.L, 10000 cells per well in 96 well cell culture plates. After overnight adherent culture, the cell culture supernatant was discarded and the cells were washed once with 200. Mu.L of basal medium per well (RPMI 1640), 50. Mu.L of antibody fusion protein diluted with basal medium was added per well, and incubated at 37℃for 0.5h. 50. Mu.L of AMP diluted in basal medium with a final concentration of 300. Mu.M was then added to each well and incubated at 37℃for 3h. From each well 50. Mu.L of incubation supernatant was removed to a 96 well white plate and an equal volume of ATP diluted in basal medium with a final concentration of 60. Mu.M was added. Incubate at 37℃for 15min. 100 mu L of Cell Titer-Glo reagent is added into each hole, and the mixture is mixed with shaking for 2min and reacted at room temperature for 8min, the fluorescence value is read by a multifunctional enzyme-labeling instrument, and the inhibition effect of the antibody fusion protein on the activity of the CD73 on the surface of H1975 cells is analyzed.
As a result, as shown in FIG. 8, each antibody fusion protein was effective in inhibiting the activity of H1975 cell surface CD73 protein in catalyzing AMP to adenosine, 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIM2 delT 50 IC of control monoclonal antibody 48A11-HuV at 0.658nM, 0.730nM, 0.716nM, respectively 50 0.5nM.
Example 8 blocking action of antibody fusion proteins on CD73 enzymatic Activity on H292 cell membrane surface
The activity of each antibody fusion protein was determined using H292 cell membrane surface overexpressing CD73 protein lymph node-metastatic human lung cancer cells whose CD73 expression level was lower than MDA-MB-231 and H1975, as well as the source of CD73 protein in this example.
H292 cells in the logarithmic growth phase were digested and washed once, counted, and plated in 96-well cell culture plates at 100 μl, 8000 cells per well. After overnight adherent culture, the culture supernatant was discarded, and cells were washed once with 200. Mu.L of basal medium (RPMI 1640) per well, 50. Mu.L of antibody fusion protein diluted with basal medium was added per well, and incubated at 37℃for 0.5h. 50. Mu.L of AMP diluted in basal medium with a final concentration of 300. Mu.M was then added to each well and incubated at 37℃for 3h. After 50. Mu.L of incubation supernatant was removed from each well and placed in 96-well white plates, and an equal volume of ATP diluted in basal medium with a final concentration of 60. Mu.M was added. Incubating at 37 ℃ for 15min, adding 100 mu L of Cell Titer-Glo reagent into each hole, shaking and uniformly mixing for 2min, standing at room temperature for reacting for 8min, reading fluorescence value by a multifunctional enzyme-labeling instrument, and analyzing the inhibition effect of the antibody fusion protein on the activity of the CD73 on the surface of the H292 Cell.
As shown in FIG. 9, each antibody fusion protein was effective in inhibiting the activity of H292 cell surface CD73 protein in catalyzing AMP to adenosine, and the IC of 48A 11-TGF-beta RII, 48A 11-TGF-beta RIIM2 delT 50 IC of control monoclonal antibody 48A11-HuV33 with 2.109nM, 1.838nM, 2.326nM, respectively 50 1.103nM.
EXAMPLE 9 ELISA assay of binding Activity of antibody fusion proteins to human TGF-beta family proteins
Referring to example 2, human TGF-beta 1, TGF-beta 2 and TGF-beta 3 were diluted to 0.5 μg/ml each with coating solution to coat ELISA plates and the affinities of 48A 11-TGF-beta RII and TGF-beta RII-Fc were measured, respectively.
As shown in FIG. 10, the results of the experiment are shown in FIG. 10, which shows that 48A 11-TGF-beta RII binds to TGF-beta 1, TGF-beta 2 and TGF-beta 3 50 0.512nM,1.387nM and 0.631nM, respectively; as shown in FIG. 11, TGF-beta RII-Fc binds TGF-beta 1 and TGF-beta 3 ECs 50 0.4nM and 0.449nM, respectively. It can be seen that 48A 11-TGF-beta RII and TGF-beta RII-Fc bind to TGF-beta 1, TGF-beta 3 at the same level, and bind to TGF-beta 2 much less.
Sequence list (red underlined part is CDR region, kabat rule)
48A11 heavy chain complementarity determining region HCDR1 SEQ ID NO.1
SYWMH
48A11 heavy chain complementarity determining region HCDR2 SEQ ID NO.2
EINPSIGRTNYNEKFKS
48A11 heavy chain complementarity determining region HCDR3 SEQ ID NO.3
RVYGTMDY
48A11 light chain complementarity determining region LCDR1 SEQ ID NO.4
KASQDINSYLS
48A11 light chain complementarity determining region LCDR2 SEQ ID NO.5
RANIWVD
48A11 light chain complementarity determining region LCDR3 SEQ ID NO.6
LQYDELYT
48A 11-TGF-beta RII fusion protein heavy chain amino acid sequence of SEQ ID NO.7
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPSIGRTNYNEKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARRVYGTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEQITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE
48A 11-TGF-beta RII fusion protein heavy chain nucleotide sequence of SEQ ID NO.18
CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCTCCTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATTGGCGAGATCAACCCCAGCATCGGCAGGACCAACTACAACGAGAAGTTCAAGAGCAGGGTGACCTTGACCAGAGACACGTCCACCAGCACCGCTTACATGGAGCTGAGCTCTCTGAGGTCTGAGGACACCGCCGTGTACTACTGCGCCAGGCGGGTGTACGGCACCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCCGCCAGCACCAAGGGCCCATCCGTGTTCCCCCTGGCCCCTTGCTCTCGGTCTACCTCCGAGAGCACCGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGATAAGAGGGTGGAGAGCAAGTACGGCCCCCCCTGCCCTCCTTGCCCCGCCCCTGAGTTCCTGGGCGGCCCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCAGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACATACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCTTCCAGCATTGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCACGGGAGCCTCAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCAAGGGCGGAGGCGGCTCCGGCGGCGGGGGCTCCGGCGGCGGCGGGTCCACCATCCCTCCTCATGTGCAGAAGTCTGTGAACAACGATATGATCGTGACAGATAATAATGGCGCCGTGAAGTTCCCACAGCTGTGTAAGTTCTGCGATGTGAGGTTCTCTACCTGCGACAATCAGAAGTCATGCATGAGTAATTGTAGCATAACTTCTATCTGTGAGAAGCCTCAGGAGGTGTGTGTGGCCGTGTGGAGAAAGAATGATGAGCAGATCACACTGGAGACAGTGTGTCATGATCCTAAGCTGCCTTATCATGACTTTATCCTGGAGGACGCCGCCTCCCCTAAGTGTATCATGAAGGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGTTCTTGTAGCTCTGATGAGTGTAATGATAACATCATCTTCTCTGAG
48A 11-TGF-beta RIIBWD fusion protein heavy chain amino acid sequence of SEQ ID NO.8
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPSIGRTNYNEKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARRVYGTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE
48A 11-TGF-beta RIIDWD fusion protein heavy chain nucleotide sequence of SEQ ID NO.19
CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCTCCTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATTGGCGAGATCAACCCCAGCATCGGCAGGACCAACTACAACGAGAAGTTCAAGAGCAGGGTGACCTTGACCAGAGACACGTCCACCAGCACCGCTTACATGGAGCTGAGCTCTCTGAGGTCTGAGGACACCGCCGTGTACTACTGCGCCAGGCGGGTGTACGGCACCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCCGCCAGCACCAAGGGCCCATCCGTGTTCCCCCTGGCCCCTTGCTCTCGGTCTACCTCCGAGAGCACCGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGATAAGAGGGTGGAGAGCAAGTACGGCCCCCCCTGCCCTCCTTGCCCCGCCCCTGAGTTCCTGGGCGGCCCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCAGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACATACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCTTCCAGCATTGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCACGGGAGCCTCAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCAAGGGCGGAGGCGGCTCCGGCGGCGGGGGCTCCGGCGGCGGCGGGTCCACCATCCCTCCTCATGTGCAGAAGTCTGTGAACAACGATATGATCGTGACAGATAATAATGGCGCCGTGAAGTTCCCACAGCTGTGTAAGTTCTGCGATGTGAGGTTCTCTACCTGCGACAATCAGAAGTCATGCATGAGTAATTGTAGCATAACTTCTATCTGTGAGAAGCCTCAGGAGGTGTGTGTGGCCGTGTGGAGAAAGAATGATGAGAACATCACACTGGAGACAGTGTGTCATGATCCTAAGCTGCCTTATCATGACTTTATCCTGGAGGACGCCGCCTCCCCTAAGTGTATCATGAAGGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGTTCTTGTAGCTCTGATGAGTGTAATGATAACATCATCTTCTCTGAG
The amino acid sequence of the heavy chain of the 48A 11-TGF-beta RIIM2 fusion protein is SEQ ID NO.9
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPSIGRTNYNEKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARRVYGTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSTIPPHVQKPVNNDMIVPDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEQITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE
Nucleotide sequence of heavy chain of 48A 11-TGF-beta RIIM2 fusion protein SEQ ID NO.20
CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCTCCTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATTGGCGAGATCAACCCCAGCATCGGCAGGACCAACTACAACGAGAAGTTCAAGAGCAGGGTGACCTTGACCAGAGACACGTCCACCAGCACCGCTTACATGGAGCTGAGCTCTCTGAGGTCTGAGGACACCGCCGTGTACTACTGCGCCAGGCGGGTGTACGGCACCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCCGCCAGCACCAAGGGCCCATCCGTGTTCCCCCTGGCCCCTTGCTCTCGGTCTACCTCCGAGAGCACCGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGATAAGAGGGTGGAGAGCAAGTACGGCCCCCCCTGCCCTCCTTGCCCCGCCCCTGAGTTCCTGGGCGGCCCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCAGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACATACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCTTCCAGCATTGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCACGGGAGCCTCAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCAAGGGCGGAGGCGGCTCCGGCGGCGGGGGCTCCGGCGGCGGCGGGTCCACCATCCCTCCTCATGTGCAGAAGCCTGTGAACAACGATATGATCGTGCCAGATAATAATGGCGCCGTGAAGTTCCCACAGCTGTGTAAGTTCTGCGATGTGAGGTTCTCTACCTGCGACAATCAGAAGTCATGCATGAGTAATTGTAGCATAACTTCTATCTGTGAGAAGCCTCAGGAGGTGTGTGTGGCCGTGTGGAGAAAGAATGATGAGCAGATCACACTGGAGACAGTGTGTCATGATCCTAAGCTGCCTTATCATGACTTTATCCTGGAGGACGCCGCCTCCCCTAAGTGTATCATGAAGGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGTTCTTGTAGCTCTGATGAGTGTAATGATAACATCATCTTCTCTGAG
48A 11-TGF-beta RIIBWT fusion protein heavy chain amino acid sequence of SEQ ID NO.10
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPSIGRTNYNEKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARRVYGTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
48A 11-TGF-beta RIIPWT fusion protein heavy chain nucleotide sequence of SEQ ID NO.21
CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCTCCTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATTGGCGAGATCAACCCCAGCATCGGCAGGACCAACTACAACGAGAAGTTCAAGAGCAGGGTGACCTTGACCAGAGACACGTCCACCAGCACCGCTTACATGGAGCTGAGCTCTCTGAGGTCTGAGGACACCGCCGTGTACTACTGCGCCAGGCGGGTGTACGGCACCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCCGCCAGCACCAAGGGCCCATCCGTGTTCCCCCTGGCCCCTTGCTCTCGGTCTACCTCCGAGAGCACCGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGATAAGAGGGTGGAGAGCAAGTACGGCCCCCCCTGCCCTCCTTGCCCCGCCCCTGAGTTCCTGGGCGGCCCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCAGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACATACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCTTCCAGCATTGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCACGGGAGCCTCAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCAAGGGCGGAGGCGGCTCCGGCGGCGGGGGCTCCGGCGGCGGCGGGTCCACCATCCCTCCTCATGTGCAGAAGTCTGTGAACAACGATATGATCGTGACAGATAATAATGGCGCCGTGAAGTTCCCACAGCTGTGTAAGTTCTGCGATGTGAGGTTCTCTACCTGCGACAATCAGAAGTCATGCATGAGTAATTGTAGCATAACTTCTATCTGTGAGAAGCCTCAGGAGGTGTGTGTGGCCGTGTGGAGAAAGAATGATGAGAACATCACACTGGAGACAGTGTGTCATGATCCTAAGCTGCCTTATCATGACTTTATCCTGGAGGACGCCGCCTCCCCTAAGTGTATCATGAAGGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGTTCTTGTAGCTCTGATGAGTGTAATGATAACATCATCTTCTCTGAGGAATATAACACCAGCAATCCTGAC
48A 11-TGF-beta RIIM2delT fusion protein heavy chain amino acid sequence of SEQ ID NO.11
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPSIGRTNYNEKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARRVYGTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSIPPHVQKPVNNDMIVPDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEQITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE
48A 11-TGF-beta RIIM2delT fusion protein heavy chain nucleotide sequence of SEQ ID NO.22
CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCTCCTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATTGGCGAGATCAACCCCAGCATCGGCAGGACCAACTACAACGAGAAGTTCAAGAGCAGGGTGACCTTGACCAGAGACACGTCCACCAGCACCGCTTACATGGAGCTGAGCTCTCTGAGGTCTGAGGACACCGCCGTGTACTACTGCGCCAGGCGGGTGTACGGCACCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCCGCCAGCACCAAGGGCCCATCCGTGTTCCCCCTGGCCCCTTGCTCTCGGTCTACCTCCGAGAGCACCGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGATAAGAGGGTGGAGAGCAAGTACGGCCCCCCCTGCCCTCCTTGCCCCGCCCCTGAGTTCCTGGGCGGCCCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCAGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACATACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCTTCCAGCATTGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCACGGGAGCCTCAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCAAGGGCGGAGGCGGCTCCGGCGGCGGGGGCTCCGGCGGCGGCGGGTCCATCCCTCCTCATGTGCAGAAGCCTGTGAACAACGATATGATCGTGCCAGATAATAATGGCGCCGTGAAGTTCCCACAGCTGTGTAAGTTCTGCGATGTGAGGTTCTCTACCTGCGACAATCAGAAGTCATGCATGAGTAATTGTAGCATAACTTCTATCTGTGAGAAGCCTCAGGAGGTGTGTGTGGCCGTGTGGAGAAAGAATGATGAGCAGATCACACTGGAGACAGTGTGTCATGATCCTAAGCTGCCTTATCATGACTTTATCCTGGAGGACGCCGCCTCCCCTAAGTGTATCATGAAGGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGTTCTTGTAGCTCTGATGAGTGTAATGATAACATCATCTTCTCTGAG
TGF-beta RII-Fc fusion protein amino acid sequence of SEQ ID NO.12
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEQITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE
TGF-beta RII-Fc fusion protein nucleotide sequence of SEQ ID NO.23
GACAAGACCCACACATGTCCCCCCTGTCCCGCTCCTGAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCTAAGCCCAAGGACACCCTGATGATTTCCAGGACACCCGAGGTGACCTGTGTGGTGGTGGACGTCAGCCACGAGGACCCCGAGGTGAAATTCAACTGGTACGTCGATGGCGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTACAATTCCACCTACAGGGTGGTGTCCGTGCTGACCGTCCTCCATCAGGACTGGCTGAACGGCAAAGAGTATAAGTGCAAGGTGAGCAACAAGGCCCTCCCTGCTCCCATCGAGAAGACCATCAGCAAAGCCAAGGGCCAGCCCAGGGAACCTCAAGTCTATACCCTGCCTCCCAGCAGGGAGGAGATGACCAAGAACCAAGTGAGCCTCACATGCCTCGTCAAGGGCTTCTATCCTTCCGATATTGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAACAACTACAAGACAACACCCCCCGTGCTCGATTCCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGATGGCAACAAGGCAACGTCTTCAGTTGCAGCGTCATGCATGAGGCCCTCCACAACCACTACACCCAGAAGAGCCTCTCCCTGAGCCCTGGAAAGGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGCAGATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAA
Amino acid sequence of CD73-his SEQ ID NO.13
MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKHHHHHH
Nucleotide sequence of CD73-his SEQ ID NO.24
ATGTGCCCTAGAGCCGCCAGAGCCCCCGCCACCCTGCTGCTGGCCCTGGGCGCCGTGCTGTGGCCCGCCGCCGGCGCCTGGGAGCTGACAATCCTGCACACCAACGACGTGCACTCCAGACTGGAACAGACCAGCGAGGATAGCTCTAAGTGCGTGAATGCCAGCAGGTGCATGGGCGGCGTGGCCCGGCTGTTTACCAAGGTGCAGCAGATCCGGAGAGCCGAGCCCAACGTGCTGCTGCTGGACGCCGGCGACCAGTACCAGGGCACCATCTGGTTCACCGTGTACAAGGGCGCCGAGGTGGCCCACTTTATGAATGCCCTGAGGTATGACGCCATGGCCCTGGGCAACCACGAGTTTGATAACGGCGTGGAGGGCCTGATTGAGCCTCTGCTGAAGGAGGCCAAGTTCCCAATCCTGAGCGCCAACATCAAGGCCAAGGGCCCCCTGGCCTCTCAGATCAGCGGCCTGTACCTGCCCTACAAGGTGCTGCCCGTGGGAGACGAGGTGGTGGGCATCGTGGGCTACACCTCCAAGGAGACCCCCTTCCTGAGCAACCCCGGCACAAATCTGGTGTTCGAGGACGAGATCACCGCTCTGCAGCCCGAGGTGGATAAGCTGAAGACCCTGAACGTGAATAAGATCATCGCCCTGGGCCACAGCGGCTTCGAGATGGACAAGCTGATCGCCCAGAAGGTGCGGGGCGTGGACGTGGTGGTGGGCGGCCACAGCAACACCTTTCTGTACACCGGCAACCCACCCAGCAAGGAGGTGCCCGCCGGCAAGTACCCTTTCATCGTGACTAGCGACGACGGGCGGAAGGTGCCCGTGGTGCAGGCCTACGCCTTCGGCAAGTACCTGGGCTACCTGAAGATCGAGTTTGATGAGAGAGGCAACGTGATCAGCTCCCACGGCAACCCTATCCTGCTGAATAGCTCCATCCCTGAGGACCCCTCCATCAAGGCCGACATCAACAAGTGGCGCATCAAGCTGGACAACTACTCTACCCAGGAGCTGGGCAAGACCATCGTGTACCTGGATGGCAGCTCCCAGTCCTGCAGGTTCCGGGAGTGTAACATGGGCAATCTGATCTGCGACGCCATGATCAATAACAACCTGAGGCACACAGACGAGATGTTCTGGAATCACGTGAGCATGTGCATTCTGAACGGCGGGGGCATCCGCAGCCCCATCGACGAGAGGAACAACGGGACCATCACCTGGGAGAACCTGGCCGCCGTGCTGCCCTTCGGCGGCACCTTTGACCTGGTGCAGCTGAAGGGCAGCACCCTGAAGAAGGCCTTTGAACACAGCGTGCACCGCTACGGGCAGAGCACCGGCGAGTTTCTGCAGGTGGGCGGCATCCACGTGGTGTACGACCTGAGCAGGAAGCCCGGCGACCGGGTGGTGAAGCTGGACGTGCTGTGCACCAAGTGCCGCGTGCCAAGCTACGACCCTCTGAAGATGGACGAGGTGTATAAAGTGATCCTGCCTAACTTCCTGGCCAACGGCGGCGACGGCTTCCAGATGATCAAGGACGAGCTGCTGCGCCACGACAGCGGCGACCAGGACATCAACGTGGTGAGCACCTACATCAGCAAGATGAAAGTGATCTACCCCGCCGTGGAGGGCAGAATCAAGCACCACCACCATCACCAC
48A11-Huv33 of the heavy chain of SEQ ID NO.14
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPSIGRTNYNEKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARRVYGTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
48A11-Huv33 nucleotide sequence of SEQ ID NO.25
CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCTCCTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATTGGCGAGATCAACCCCAGCATCGGCAGGACCAACTACAACGAGAAGTTCAAGAGCAGGGTGACCTTGACCAGAGACACGTCCACCAGCACCGCTTACATGGAGCTGAGCTCTCTGAGGTCTGAGGACACCGCCGTGTACTACTGCGCCAGGCGGGTGTACGGCACCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCCGCCAGCACCAAGGGCCCATCCGTGTTCCCCCTGGCCCCTTGCTCTCGGTCTACCTCCGAGAGCACCGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGATAAGAGGGTGGAGAGCAAGTACGGCCCCCCCTGCCCTCCTTGCCCCGCCCCTGAGTTCCTGGGCGGCCCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCAGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACATACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCTTCCAGCATTGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCACGGGAGCCTCAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCAAG
48A11-Huv33 light chain amino acid sequence SEQ ID NO.15
DIQMTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKSPKLLIYRANIWVDGVPSRFSGSGSGQDYTFTISSLQPEDIATYYCLQYDELYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE
48A11-Huv33 nucleotide sequence of SEQ ID NO.26
GACATCCAGATGACCCAGTCCCCCAGCTCCCTGAGCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCAAGGCCTCCCAGGACATCAACTCCTACCTGTCCTGGTTCCAGCAGAAGCCCGGCAAGTCCCCTAAGCTACTGATCTACAGGGCCAACATCTGGGTGGACGGCGTGCCCTCCAGGTTCAGCGGCTCCGGCAGCGGCCAGGACTACACCTTCACCATCTCCTCCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCTGCAGTACGACGAGCTGTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCCTCCGTGTTCATCTTCCCCCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGCCTGCTGAACAACTTCTACCCTAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACTCCACCTACTCCCTGAGCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCTCCCCCGTGACCAAGTCCTTCAACAGGGGCGAGTGC
48A11-Huv33 heavy chain variable region SEQ ID NO.16
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPSIGRTNYNEKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARRVYGTMDYWGQGTLVTVSS
48A11-Huv33 light chain variable region SEQ ID NO.17
DIQMTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKSPKLLIYRANIWVDGVPSRFSGSGSGQDYTFTISSLQPEDIATYYCLQYDELYTFGQGTKVEIK
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Sequence listing
<110> Sansheng national health pharmaceutical Co., ltd
<120> bifunctional fusion protein simultaneously targeting human CD73 and human TGF beta, preparation method and application thereof
<130> P2021-2745
<160> 26
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Ser Tyr Trp Met His
1 5
<210> 2
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Glu Ile Asn Pro Ser Ile Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Ser
<210> 3
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Arg Val Tyr Gly Thr Met Asp Tyr
1 5
<210> 4
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Arg Ala Asn Ile Trp Val Asp
1 5
<210> 6
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Leu Gln Tyr Asp Glu Leu Tyr Thr
1 5
<210> 7
<211> 588
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Ile Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Tyr Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro Pro His
450 455 460
Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
465 470 475 480
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
485 490 495
Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser
500 505 510
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn
515 520 525
Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
530 535 540
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
545 550 555 560
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
565 570 575
Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
580 585
<210> 8
<211> 588
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Ile Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Tyr Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro Pro His
450 455 460
Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
465 470 475 480
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
485 490 495
Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser
500 505 510
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn
515 520 525
Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
530 535 540
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
545 550 555 560
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
565 570 575
Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
580 585
<210> 9
<211> 588
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Ile Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Tyr Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro Pro His
450 455 460
Val Gln Lys Pro Val Asn Asn Asp Met Ile Val Pro Asp Asn Asn Gly
465 470 475 480
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
485 490 495
Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser
500 505 510
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn
515 520 525
Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
530 535 540
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
545 550 555 560
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
565 570 575
Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
580 585
<210> 10
<211> 596
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Ile Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Tyr Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro Pro His
450 455 460
Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
465 470 475 480
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
485 490 495
Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser
500 505 510
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn
515 520 525
Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
530 535 540
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
545 550 555 560
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
565 570 575
Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr
580 585 590
Ser Asn Pro Asp
595
<210> 11
<211> 587
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Ile Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Tyr Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val
450 455 460
Gln Lys Pro Val Asn Asn Asp Met Ile Val Pro Asp Asn Asn Gly Ala
465 470 475 480
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr
485 490 495
Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile
500 505 510
Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp
515 520 525
Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr
530 535 540
His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys
545 550 555 560
Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser
565 570 575
Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
580 585
<210> 12
<211> 371
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met
245 250 255
Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys
260 265 270
Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met
275 280 285
Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys
290 295 300
Val Ala Val Trp Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val
305 310 315 320
Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala
325 330 335
Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr
340 345 350
Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile
355 360 365
Phe Ser Glu
370
<210> 13
<211> 553
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu
1 5 10 15
Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu
20 25 30
His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser
35 40 45
Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu
50 55 60
Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu
65 70 75 80
Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr
85 90 95
Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala
100 105 110
Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile
115 120 125
Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro Ile Leu Ser Ala Asn Ile
130 135 140
Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro
145 150 155 160
Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr
165 170 175
Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val
180 185 190
Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu Val Asp Lys Leu Lys
195 200 205
Thr Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu
210 215 220
Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val
225 230 235 240
Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys
245 250 255
Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile Val Thr Ser Asp Asp Gly
260 265 270
Arg Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly
275 280 285
Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His
290 295 300
Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile
305 310 315 320
Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr
325 330 335
Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser
340 345 350
Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met
355 360 365
Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val
370 375 380
Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu
385 390 395 400
Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro
405 410 415
Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys
420 425 430
Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu
435 440 445
Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys
450 455 460
Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg
465 470 475 480
Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile
485 490 495
Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys
500 505 510
Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val
515 520 525
Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly
530 535 540
Arg Ile Lys His His His His His His
545 550
<210> 14
<211> 444
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Ile Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Tyr Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 15
<211> 212
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Arg Ala Asn Ile Trp Val Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Leu Tyr Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val 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
210
<210> 16
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Ile Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Tyr Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 17
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Arg Ala Asn Ile Trp Val Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Leu Tyr Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 18
<211> 1764
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
tcctgcaagg ccagcggcta caccttcacc tcctactgga tgcactgggt gaggcaggcc 120
cccggccagg gcctggagtg gattggcgag atcaacccca gcatcggcag gaccaactac 180
aacgagaagt tcaagagcag ggtgaccttg accagagaca cgtccaccag caccgcttac 240
atggagctga gctctctgag gtctgaggac accgccgtgt actactgcgc caggcgggtg 300
tacggcacca tggactactg gggccagggc accctggtga ccgtgagctc cgccagcacc 360
aagggcccat ccgtgttccc cctggcccct tgctctcggt ctacctccga gagcaccgct 420
gccctgggct gcctggtgaa ggactacttc cccgagcctg tgacagtgtc ctggaactcc 480
ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540
tccctgtcta gcgtggtgac cgtgcccagc tcctccctgg gcaccaagac ctacacctgt 600
aacgtggacc acaagcccag caacaccaag gtggataaga gggtggagag caagtacggc 660
cccccctgcc ctccttgccc cgcccctgag ttcctgggcg gcccctccgt gttcctgttc 720
cctcctaagc ctaaggacac cctgatgatc tcccgcaccc ccgaggtgac ctgcgtggtg 780
gtggacgtgt cccaggagga cccagaggtg cagttcaact ggtacgtgga cggcgtggag 840
gtgcacaacg ccaagaccaa gcccagagag gagcagttca acagcacata cagggtggtg 900
tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960
agcaacaagg gcctgccttc cagcattgag aaaaccatct ccaaggccaa gggccagcca 1020
cgggagcctc aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtg 1080
agcctgacct gcctggtgaa gggattctac ccctccgaca tcgccgtgga gtgggagagc 1140
aacggccagc ccgagaacaa ctacaagacc acccctcccg tgctggacag cgacggcagc 1200
ttcttcctgt actcccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttt 1260
agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320
agcctgggca agggcggagg cggctccggc ggcgggggct ccggcggcgg cgggtccacc 1380
atccctcctc atgtgcagaa gtctgtgaac aacgatatga tcgtgacaga taataatggc 1440
gccgtgaagt tcccacagct gtgtaagttc tgcgatgtga ggttctctac ctgcgacaat 1500
cagaagtcat gcatgagtaa ttgtagcata acttctatct gtgagaagcc tcaggaggtg 1560
tgtgtggccg tgtggagaaa gaatgatgag cagatcacac tggagacagt gtgtcatgat 1620
cctaagctgc cttatcatga ctttatcctg gaggacgccg cctcccctaa gtgtatcatg 1680
aaggagaaaa agaagcctgg cgagaccttt ttcatgtgtt cttgtagctc tgatgagtgt 1740
aatgataaca tcatcttctc tgag 1764
<210> 19
<211> 1764
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
tcctgcaagg ccagcggcta caccttcacc tcctactgga tgcactgggt gaggcaggcc 120
cccggccagg gcctggagtg gattggcgag atcaacccca gcatcggcag gaccaactac 180
aacgagaagt tcaagagcag ggtgaccttg accagagaca cgtccaccag caccgcttac 240
atggagctga gctctctgag gtctgaggac accgccgtgt actactgcgc caggcgggtg 300
tacggcacca tggactactg gggccagggc accctggtga ccgtgagctc cgccagcacc 360
aagggcccat ccgtgttccc cctggcccct tgctctcggt ctacctccga gagcaccgct 420
gccctgggct gcctggtgaa ggactacttc cccgagcctg tgacagtgtc ctggaactcc 480
ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540
tccctgtcta gcgtggtgac cgtgcccagc tcctccctgg gcaccaagac ctacacctgt 600
aacgtggacc acaagcccag caacaccaag gtggataaga gggtggagag caagtacggc 660
cccccctgcc ctccttgccc cgcccctgag ttcctgggcg gcccctccgt gttcctgttc 720
cctcctaagc ctaaggacac cctgatgatc tcccgcaccc ccgaggtgac ctgcgtggtg 780
gtggacgtgt cccaggagga cccagaggtg cagttcaact ggtacgtgga cggcgtggag 840
gtgcacaacg ccaagaccaa gcccagagag gagcagttca acagcacata cagggtggtg 900
tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960
agcaacaagg gcctgccttc cagcattgag aaaaccatct ccaaggccaa gggccagcca 1020
cgggagcctc aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtg 1080
agcctgacct gcctggtgaa gggattctac ccctccgaca tcgccgtgga gtgggagagc 1140
aacggccagc ccgagaacaa ctacaagacc acccctcccg tgctggacag cgacggcagc 1200
ttcttcctgt actcccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttt 1260
agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320
agcctgggca agggcggagg cggctccggc ggcgggggct ccggcggcgg cgggtccacc 1380
atccctcctc atgtgcagaa gtctgtgaac aacgatatga tcgtgacaga taataatggc 1440
gccgtgaagt tcccacagct gtgtaagttc tgcgatgtga ggttctctac ctgcgacaat 1500
cagaagtcat gcatgagtaa ttgtagcata acttctatct gtgagaagcc tcaggaggtg 1560
tgtgtggccg tgtggagaaa gaatgatgag aacatcacac tggagacagt gtgtcatgat 1620
cctaagctgc cttatcatga ctttatcctg gaggacgccg cctcccctaa gtgtatcatg 1680
aaggagaaaa agaagcctgg cgagaccttt ttcatgtgtt cttgtagctc tgatgagtgt 1740
aatgataaca tcatcttctc tgag 1764
<210> 20
<211> 1764
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
tcctgcaagg ccagcggcta caccttcacc tcctactgga tgcactgggt gaggcaggcc 120
cccggccagg gcctggagtg gattggcgag atcaacccca gcatcggcag gaccaactac 180
aacgagaagt tcaagagcag ggtgaccttg accagagaca cgtccaccag caccgcttac 240
atggagctga gctctctgag gtctgaggac accgccgtgt actactgcgc caggcgggtg 300
tacggcacca tggactactg gggccagggc accctggtga ccgtgagctc cgccagcacc 360
aagggcccat ccgtgttccc cctggcccct tgctctcggt ctacctccga gagcaccgct 420
gccctgggct gcctggtgaa ggactacttc cccgagcctg tgacagtgtc ctggaactcc 480
ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540
tccctgtcta gcgtggtgac cgtgcccagc tcctccctgg gcaccaagac ctacacctgt 600
aacgtggacc acaagcccag caacaccaag gtggataaga gggtggagag caagtacggc 660
cccccctgcc ctccttgccc cgcccctgag ttcctgggcg gcccctccgt gttcctgttc 720
cctcctaagc ctaaggacac cctgatgatc tcccgcaccc ccgaggtgac ctgcgtggtg 780
gtggacgtgt cccaggagga cccagaggtg cagttcaact ggtacgtgga cggcgtggag 840
gtgcacaacg ccaagaccaa gcccagagag gagcagttca acagcacata cagggtggtg 900
tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960
agcaacaagg gcctgccttc cagcattgag aaaaccatct ccaaggccaa gggccagcca 1020
cgggagcctc aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtg 1080
agcctgacct gcctggtgaa gggattctac ccctccgaca tcgccgtgga gtgggagagc 1140
aacggccagc ccgagaacaa ctacaagacc acccctcccg tgctggacag cgacggcagc 1200
ttcttcctgt actcccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttt 1260
agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320
agcctgggca agggcggagg cggctccggc ggcgggggct ccggcggcgg cgggtccacc 1380
atccctcctc atgtgcagaa gcctgtgaac aacgatatga tcgtgccaga taataatggc 1440
gccgtgaagt tcccacagct gtgtaagttc tgcgatgtga ggttctctac ctgcgacaat 1500
cagaagtcat gcatgagtaa ttgtagcata acttctatct gtgagaagcc tcaggaggtg 1560
tgtgtggccg tgtggagaaa gaatgatgag cagatcacac tggagacagt gtgtcatgat 1620
cctaagctgc cttatcatga ctttatcctg gaggacgccg cctcccctaa gtgtatcatg 1680
aaggagaaaa agaagcctgg cgagaccttt ttcatgtgtt cttgtagctc tgatgagtgt 1740
aatgataaca tcatcttctc tgag 1764
<210> 21
<211> 1788
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
tcctgcaagg ccagcggcta caccttcacc tcctactgga tgcactgggt gaggcaggcc 120
cccggccagg gcctggagtg gattggcgag atcaacccca gcatcggcag gaccaactac 180
aacgagaagt tcaagagcag ggtgaccttg accagagaca cgtccaccag caccgcttac 240
atggagctga gctctctgag gtctgaggac accgccgtgt actactgcgc caggcgggtg 300
tacggcacca tggactactg gggccagggc accctggtga ccgtgagctc cgccagcacc 360
aagggcccat ccgtgttccc cctggcccct tgctctcggt ctacctccga gagcaccgct 420
gccctgggct gcctggtgaa ggactacttc cccgagcctg tgacagtgtc ctggaactcc 480
ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540
tccctgtcta gcgtggtgac cgtgcccagc tcctccctgg gcaccaagac ctacacctgt 600
aacgtggacc acaagcccag caacaccaag gtggataaga gggtggagag caagtacggc 660
cccccctgcc ctccttgccc cgcccctgag ttcctgggcg gcccctccgt gttcctgttc 720
cctcctaagc ctaaggacac cctgatgatc tcccgcaccc ccgaggtgac ctgcgtggtg 780
gtggacgtgt cccaggagga cccagaggtg cagttcaact ggtacgtgga cggcgtggag 840
gtgcacaacg ccaagaccaa gcccagagag gagcagttca acagcacata cagggtggtg 900
tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960
agcaacaagg gcctgccttc cagcattgag aaaaccatct ccaaggccaa gggccagcca 1020
cgggagcctc aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtg 1080
agcctgacct gcctggtgaa gggattctac ccctccgaca tcgccgtgga gtgggagagc 1140
aacggccagc ccgagaacaa ctacaagacc acccctcccg tgctggacag cgacggcagc 1200
ttcttcctgt actcccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttt 1260
agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320
agcctgggca agggcggagg cggctccggc ggcgggggct ccggcggcgg cgggtccacc 1380
atccctcctc atgtgcagaa gtctgtgaac aacgatatga tcgtgacaga taataatggc 1440
gccgtgaagt tcccacagct gtgtaagttc tgcgatgtga ggttctctac ctgcgacaat 1500
cagaagtcat gcatgagtaa ttgtagcata acttctatct gtgagaagcc tcaggaggtg 1560
tgtgtggccg tgtggagaaa gaatgatgag aacatcacac tggagacagt gtgtcatgat 1620
cctaagctgc cttatcatga ctttatcctg gaggacgccg cctcccctaa gtgtatcatg 1680
aaggagaaaa agaagcctgg cgagaccttt ttcatgtgtt cttgtagctc tgatgagtgt 1740
aatgataaca tcatcttctc tgaggaatat aacaccagca atcctgac 1788
<210> 22
<211> 1761
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
tcctgcaagg ccagcggcta caccttcacc tcctactgga tgcactgggt gaggcaggcc 120
cccggccagg gcctggagtg gattggcgag atcaacccca gcatcggcag gaccaactac 180
aacgagaagt tcaagagcag ggtgaccttg accagagaca cgtccaccag caccgcttac 240
atggagctga gctctctgag gtctgaggac accgccgtgt actactgcgc caggcgggtg 300
tacggcacca tggactactg gggccagggc accctggtga ccgtgagctc cgccagcacc 360
aagggcccat ccgtgttccc cctggcccct tgctctcggt ctacctccga gagcaccgct 420
gccctgggct gcctggtgaa ggactacttc cccgagcctg tgacagtgtc ctggaactcc 480
ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540
tccctgtcta gcgtggtgac cgtgcccagc tcctccctgg gcaccaagac ctacacctgt 600
aacgtggacc acaagcccag caacaccaag gtggataaga gggtggagag caagtacggc 660
cccccctgcc ctccttgccc cgcccctgag ttcctgggcg gcccctccgt gttcctgttc 720
cctcctaagc ctaaggacac cctgatgatc tcccgcaccc ccgaggtgac ctgcgtggtg 780
gtggacgtgt cccaggagga cccagaggtg cagttcaact ggtacgtgga cggcgtggag 840
gtgcacaacg ccaagaccaa gcccagagag gagcagttca acagcacata cagggtggtg 900
tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960
agcaacaagg gcctgccttc cagcattgag aaaaccatct ccaaggccaa gggccagcca 1020
cgggagcctc aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtg 1080
agcctgacct gcctggtgaa gggattctac ccctccgaca tcgccgtgga gtgggagagc 1140
aacggccagc ccgagaacaa ctacaagacc acccctcccg tgctggacag cgacggcagc 1200
ttcttcctgt actcccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttt 1260
agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320
agcctgggca agggcggagg cggctccggc ggcgggggct ccggcggcgg cgggtccatc 1380
cctcctcatg tgcagaagcc tgtgaacaac gatatgatcg tgccagataa taatggcgcc 1440
gtgaagttcc cacagctgtg taagttctgc gatgtgaggt tctctacctg cgacaatcag 1500
aagtcatgca tgagtaattg tagcataact tctatctgtg agaagcctca ggaggtgtgt 1560
gtggccgtgt ggagaaagaa tgatgagcag atcacactgg agacagtgtg tcatgatcct 1620
aagctgcctt atcatgactt tatcctggag gacgccgcct cccctaagtg tatcatgaag 1680
gagaaaaaga agcctggcga gacctttttc atgtgttctt gtagctctga tgagtgtaat 1740
gataacatca tcttctctga g 1761
<210> 23
<211> 1113
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gacaagaccc acacatgtcc cccctgtccc gctcctgaac tgctgggagg cccttccgtg 60
ttcctgttcc cccctaagcc caaggacacc ctgatgattt ccaggacacc cgaggtgacc 120
tgtgtggtgg tggacgtcag ccacgaggac cccgaggtga aattcaactg gtacgtcgat 180
ggcgtggagg tgcacaacgc taagaccaag cccagggagg agcagtacaa ttccacctac 240
agggtggtgt ccgtgctgac cgtcctccat caggactggc tgaacggcaa agagtataag 300
tgcaaggtga gcaacaaggc cctccctgct cccatcgaga agaccatcag caaagccaag 360
ggccagccca gggaacctca agtctatacc ctgcctccca gcagggagga gatgaccaag 420
aaccaagtga gcctcacatg cctcgtcaag ggcttctatc cttccgatat tgccgtcgag 480
tgggagtcca acggacagcc cgagaacaac tacaagacaa caccccccgt gctcgattcc 540
gatggcagct tcttcctgta ctccaagctg accgtggaca agtccagatg gcaacaaggc 600
aacgtcttca gttgcagcgt catgcatgag gccctccaca accactacac ccagaagagc 660
ctctccctga gccctggaaa gggtggaggc ggttcaggcg gaggtggcag cggcggtggc 720
gggtcgacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 780
aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 840
tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 900
caggaagtct gtgtggctgt atggagaaag aatgacgagc agataacact agagacagtt 960
tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 1020
tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 1080
gatgagtgca atgacaacat catcttctca gaa 1113
<210> 24
<211> 1659
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
atgtgcccta gagccgccag agcccccgcc accctgctgc tggccctggg cgccgtgctg 60
tggcccgccg ccggcgcctg ggagctgaca atcctgcaca ccaacgacgt gcactccaga 120
ctggaacaga ccagcgagga tagctctaag tgcgtgaatg ccagcaggtg catgggcggc 180
gtggcccggc tgtttaccaa ggtgcagcag atccggagag ccgagcccaa cgtgctgctg 240
ctggacgccg gcgaccagta ccagggcacc atctggttca ccgtgtacaa gggcgccgag 300
gtggcccact ttatgaatgc cctgaggtat gacgccatgg ccctgggcaa ccacgagttt 360
gataacggcg tggagggcct gattgagcct ctgctgaagg aggccaagtt cccaatcctg 420
agcgccaaca tcaaggccaa gggccccctg gcctctcaga tcagcggcct gtacctgccc 480
tacaaggtgc tgcccgtggg agacgaggtg gtgggcatcg tgggctacac ctccaaggag 540
acccccttcc tgagcaaccc cggcacaaat ctggtgttcg aggacgagat caccgctctg 600
cagcccgagg tggataagct gaagaccctg aacgtgaata agatcatcgc cctgggccac 660
agcggcttcg agatggacaa gctgatcgcc cagaaggtgc ggggcgtgga cgtggtggtg 720
ggcggccaca gcaacacctt tctgtacacc ggcaacccac ccagcaagga ggtgcccgcc 780
ggcaagtacc ctttcatcgt gactagcgac gacgggcgga aggtgcccgt ggtgcaggcc 840
tacgccttcg gcaagtacct gggctacctg aagatcgagt ttgatgagag aggcaacgtg 900
atcagctccc acggcaaccc tatcctgctg aatagctcca tccctgagga cccctccatc 960
aaggccgaca tcaacaagtg gcgcatcaag ctggacaact actctaccca ggagctgggc 1020
aagaccatcg tgtacctgga tggcagctcc cagtcctgca ggttccggga gtgtaacatg 1080
ggcaatctga tctgcgacgc catgatcaat aacaacctga ggcacacaga cgagatgttc 1140
tggaatcacg tgagcatgtg cattctgaac ggcgggggca tccgcagccc catcgacgag 1200
aggaacaacg ggaccatcac ctgggagaac ctggccgccg tgctgccctt cggcggcacc 1260
tttgacctgg tgcagctgaa gggcagcacc ctgaagaagg cctttgaaca cagcgtgcac 1320
cgctacgggc agagcaccgg cgagtttctg caggtgggcg gcatccacgt ggtgtacgac 1380
ctgagcagga agcccggcga ccgggtggtg aagctggacg tgctgtgcac caagtgccgc 1440
gtgccaagct acgaccctct gaagatggac gaggtgtata aagtgatcct gcctaacttc 1500
ctggccaacg gcggcgacgg cttccagatg atcaaggacg agctgctgcg ccacgacagc 1560
ggcgaccagg acatcaacgt ggtgagcacc tacatcagca agatgaaagt gatctacccc 1620
gccgtggagg gcagaatcaa gcaccaccac catcaccac 1659
<210> 25
<211> 1332
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
tcctgcaagg ccagcggcta caccttcacc tcctactgga tgcactgggt gaggcaggcc 120
cccggccagg gcctggagtg gattggcgag atcaacccca gcatcggcag gaccaactac 180
aacgagaagt tcaagagcag ggtgaccttg accagagaca cgtccaccag caccgcttac 240
atggagctga gctctctgag gtctgaggac accgccgtgt actactgcgc caggcgggtg 300
tacggcacca tggactactg gggccagggc accctggtga ccgtgagctc cgccagcacc 360
aagggcccat ccgtgttccc cctggcccct tgctctcggt ctacctccga gagcaccgct 420
gccctgggct gcctggtgaa ggactacttc cccgagcctg tgacagtgtc ctggaactcc 480
ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540
tccctgtcta gcgtggtgac cgtgcccagc tcctccctgg gcaccaagac ctacacctgt 600
aacgtggacc acaagcccag caacaccaag gtggataaga gggtggagag caagtacggc 660
cccccctgcc ctccttgccc cgcccctgag ttcctgggcg gcccctccgt gttcctgttc 720
cctcctaagc ctaaggacac cctgatgatc tcccgcaccc ccgaggtgac ctgcgtggtg 780
gtggacgtgt cccaggagga cccagaggtg cagttcaact ggtacgtgga cggcgtggag 840
gtgcacaacg ccaagaccaa gcccagagag gagcagttca acagcacata cagggtggtg 900
tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960
agcaacaagg gcctgccttc cagcattgag aaaaccatct ccaaggccaa gggccagcca 1020
cgggagcctc aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtg 1080
agcctgacct gcctggtgaa gggattctac ccctccgaca tcgccgtgga gtgggagagc 1140
aacggccagc ccgagaacaa ctacaagacc acccctcccg tgctggacag cgacggcagc 1200
ttcttcctgt actcccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttt 1260
agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320
agcctgggca ag 1332
<210> 26
<211> 639
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
gacatccaga tgacccagtc ccccagctcc ctgagcgcct ccgtgggcga cagggtgacc 60
atcacctgca aggcctccca ggacatcaac tcctacctgt cctggttcca gcagaagccc 120
ggcaagtccc ctaagctact gatctacagg gccaacatct gggtggacgg cgtgccctcc 180
aggttcagcg gctccggcag cggccaggac tacaccttca ccatctcctc cctgcagccc 240
gaggacatcg ccacctacta ctgcctgcag tacgacgagc tgtacacctt cggccagggc 300
accaaggtgg agatcaagcg gaccgtggcc gccccctccg tgttcatctt ccccccctcc 360
gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaacaa cttctaccct 420
agggaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa ctcccaggag 480
tccgtgaccg agcaggactc caaggactcc acctactccc tgagctccac cctgaccctg 540
tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600
agctcccccg tgaccaagtc cttcaacagg ggcgagtgc 639

Claims (15)

1. A bifunctional fusion protein comprising:
a first binding domain D1; and
a second binding domain D2;
wherein D1 is an anti-CD 73 antibody or antigen-binding fragment thereof;
d2 protein that specifically binds to the target molecule tgfβ;
the anti-CD 73 antibody comprises a heavy chain variable region comprising the complementarity determining region CDRs:
HCDR1 shown in SEQ ID NO.1,
HCDR2 shown in SEQ ID NO.2, and
HCDR3 shown in SEQ ID No. 3; and
the light chain variable region comprises the following complementarity determining region CDRs:
LCDR1 shown in SEQ ID NO.4,
LCDR2 as shown in SEQ ID NO.5, and
LCDR3 as shown in SEQ ID NO. 6.
2. The bifunctional fusion protein of claim 1, wherein the heavy chain variable region of the anti-CD 73 antibody comprises the amino acid sequence of SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.
3. The bifunctional fusion protein of claim 1, wherein the light chain variable region of the anti-CD 73 antibody comprises the amino acid sequence depicted in SEQ ID No. 17.
4. The bifunctional fusion protein of claim 1, wherein D2 is a tgfβrii extracellular domain element.
5. The bifunctional fusion protein of claim 4, wherein the tgfβrii extracellular domain element is selected from one or more of the group consisting of:
(1) Positions 460-588 of SEQ ID NO. 7;
(2) Positions 460-588 of SEQ ID NO. 8;
(3) Positions 460-588 of SEQ ID NO. 9;
(4) Positions 460-596 of SEQ ID NO. 10;
(5) Positions 460-587 of SEQ ID NO. 11.
6. The bifunctional fusion protein of claim 1, wherein each monomer in the bifunctional fusion protein has a structure from N-terminus to C-terminus as shown in formula I:
Figure FDA0003418230150000021
Wherein, the liquid crystal display device comprises a liquid crystal display device,
t1, T2, T3, T4 are each independently absent or tgfβrii extracellular domain elements, and at least one is not absent;
l1, L2, L3, L4 are each independently absent or a bond or linker;
VL represents the light chain variable region of an anti-CD 73 antibody;
CL represents the light chain constant region of an anti-CD 73 antibody;
VH represents the heavy chain variable region of an anti-CD 73 antibody;
CH represents the heavy chain constant region of an anti-CD 73 antibody;
"-" represents disulfide or covalent bonds;
"-" represents a peptide bond;
wherein the bifunctional fusion protein has an activity of simultaneously binding TGF-beta and binding CD 73.
7. The bifunctional fusion protein of claim 1, wherein the bifunctional fusion protein is selected from the group consisting of:
(1) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:7, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15;
(2) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:8, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15;
(3) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:9, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15;
(4) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:10, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15;
(5) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:11, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 15; or (b)
(6) Polypeptides derived from (1) through (5) that are formed by substitution, deletion, or addition of one or more amino acid residues to the amino acid sequences of (1) through (5) and that have the activity of binding CD73 as well as binding tgfβ.
8. A polynucleotide molecule encoding the bifunctional fusion protein of any one of claims 1-7.
9. An expression vector comprising the polynucleotide molecule of claim 8.
10. A host cell comprising the expression vector of claim 9.
11. A method for preparing the bifunctional fusion protein of any one of claims 1-7, comprising the steps of:
a) Culturing the host cell of claim 10 under expression conditions, thereby expressing the bifunctional fusion protein;
b) Isolating and purifying the fusion protein of step a).
12. A pharmaceutical composition comprising an effective amount of the bifunctional fusion protein of any one of claims 1-7 and one or more pharmaceutically acceptable carriers, diluents, or excipients.
13. An immunoconjugate, the immunoconjugate comprising:
(a) The bifunctional fusion protein of any one of claims 1-7; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
14. Use of a bifunctional fusion protein of any one of claims 1-7, a pharmaceutical composition of claim 12 or an immunoconjugate of claim 13, for
(a) Preparing a detection reagent or a kit; and/or
(b) Preparing the medicine for preventing and/or treating CD73 and/or TGF beta related diseases.
15. Use according to claim 14, wherein the CD73 and/or tgfβ related disease is a cancer selected from the group consisting of: colorectal cancer, bile duct cancer, gall bladder cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, renal cancer, bladder cancer, head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma.
CN202111552939.5A 2021-12-17 2021-12-17 Bifunctional fusion protein for simultaneously targeting human CD73 and human TGF beta, preparation method and application thereof Pending CN116265489A (en)

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