CN112409484B - Multifunctional antibodies, their preparation and uses - Google Patents

Multifunctional antibodies, their preparation and uses Download PDF

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CN112409484B
CN112409484B CN202010534034.4A CN202010534034A CN112409484B CN 112409484 B CN112409484 B CN 112409484B CN 202010534034 A CN202010534034 A CN 202010534034A CN 112409484 B CN112409484 B CN 112409484B
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CN112409484A (en
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姜晓玲
姜东成
吴崇兵
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Sunho China Biopharmaceutical Co Ltd
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Abstract

The invention provides a multifunctional antibody which is obtained by a genetic engineering technology and targets PD-1 and simultaneously has the biological effect of an IL-15/IL-15R alpha compound, and discloses a nucleic acid molecule for encoding the antibody, a recombinant vector containing the nucleic acid molecule, a recombinant cell containing the recombinant vector, a preparation method of the multifunctional antibody and medical application of the multifunctional antibody. The multifunctional antibody can effectively solve the drug resistance and relapse of a single-target antibody drug, simultaneously reduce the effective dose, kill tumor cells more effectively and prolong the survival period of in-situ tumor model animals; compared with IL-15 or IL-15/IL-15 receptor compound, the serum half-life period is prolonged, the tumor targeting is improved, and the toxic and side effects are reduced.

Description

Multifunctional antibodies, their preparation and uses
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multifunctional antibody which targets PD-1 and has the biological effect of an IL-15/IL-15R alpha compound, a nucleic acid molecule for encoding the antibody, a recombinant vector containing the nucleic acid molecule, a recombinant cell containing the recombinant vector, a preparation method of the multifunctional antibody and medical application of the multifunctional antibody.
Background
Programmed cell death protein1 (PD-1) and its receptors PD-L1 and PD-L2 are important regulators of T cell activity. The binding of PD-1 on the surface of T cells to PD-L1/2 on the surface of other cells results in the suppression of T cells, which plays an important role in the process of avoiding autoimmune diseases and producing immune tolerance in humans. Tumor cells utilize an immune system self-protection regulation mechanism of a PD-1/PD-L1 check point, express PD-L1/2 in the tumor cells or a tumor microenvironment, and are combined with PD-1 on the surface of T cells to transmit negative signals, so that the function of the T cells is declined and exhausted, and the aims of inhibiting immune response and tumor escape are fulfilled. Therefore, the combination of the antibody (anti-PD-1 antibody or anti-PD-L1/2 antibody) and the antibody can be inhibited, so that the immunosuppression in a tumor microenvironment can be effectively relieved, and T cells can be reactivated to kill tumors.
Despite the breakthrough of tumor immunity in tumor therapy, studies have shown that PD-1 inhibitors have a response rate of only 20% for patients with PD-L1 overexpressing tumors, and 9% for patients with PD-L1 negatives. The killing of the PD-1 inhibitor to the tumor depends on infiltration of immune cells in a tumor microenvironment, however, the tumor microenvironment is in an immunosuppressive state for a long time, infiltration of the immune cells in the environment is reduced, and even if the immunosuppression in the tumor microenvironment is relieved by the anti-PD-1 antibody, the tumor cells are not killed by the immune cells. Furthermore, immune cell depletion in the tumor microenvironment and depletion of immune cells after drug administration are the major mechanisms of PD-1 antibody resistance and relapse.
Cytokines (CK) belong to immunoregulatory molecules and have certain activation or inhibition effects on the immune system depending on their properties, administration concentrations and sites of activity. Interleukin-15 (IL-15) is a cytokine with a molecular weight of about 12-14kD, discovered by Grabstein et al in 1994, and can play a role in normal immune responses of the body, such as promoting the proliferation of T cells, B cells and Natural Killer (NK) cells and increasing the activity of immune cells. IL-15 belongs to the gamma c cytokine, 4-alpha helix superfamily of cytokines. IL-15 is mainly expressed in monocytes or macrophages, and IL-15 is also contained in the culture supernatant of human long-term bone marrow stromal cells. IL-15 needs to exert biological activity by binding to its receptor. The IL-15 receptor is composed of three receptor subunits: IL-15 receptor alpha (IL-15R alpha), IL-2 receptor beta (IL-2R beta, also known as IL-15R beta or CD 122), and yc (also known as CD 132). IL-15 Ra contains a sushi domain, is capable of binding to IL-15, and is necessary for the bound IL-15 to perform a biological function. After binding to the receptor, IL-15 can promote the proliferation and activation of NK cells and T cells through signal transduction, and maintain the survivability of tumor-associated antigen memory T cells.
Due to the existence of IL-15 specific receptor IL-15 alpha, IL-15 does not cause apoptosis of activated T cells, does not induce the up-regulation of inhibitory T cells, more effectively activates CD8+ T cells and NK cells, and has memory function. Thus, of the most active cytokines that are immunoregulatory, IL-15 has the unique ability to gain access to many important aspects of immunity against a variety of tumor types and viral infections (including HIV, HBV, HCV, LCMV, etc.).
However, clinical use of cytokines has the disadvantage of poor targeting of single drug administration, and only high-concentration administration can achieve anti-tumor effect, while high-concentration administration can generate immunosuppressive effect and high toxicity. Moreover, the non-targeting cytokine is systemic to the activation of the immune system, which is widely activated, with fatal side effects. In addition, since cytokines are small molecular weight proteins and do not have the in vivo circulation protection mechanism of antibodies, simple cytokines often have short half-lives and require repeated high-dose administration in a short time. At present, most of clinical research medicines adopt PEG (polyethylene glycol) or Fc fusion to improve the half-life of the cytokine, and although the half-life is prolonged, the problem of poor targeting of the cytokine cannot be solved.
Tumor development and progression are accompanied by invasion of the immune system, and immunocompromised individuals often have a higher incidence of cancer and a poorer prognosis. Researchers have therefore invested a great deal of time and effort in improving immune function in cancer patients, including the administration of immunostimulatory cytokine therapies such as IFN, IL-2, and IL-15; use of dendritic cell-based vaccines to activate the immune system to enhance endogenous immune responses; adoptive cell transfer therapy with autologous cancer-specific cytotoxic T cell activation and expansion; or engineering T cells to express chimeric antigen receptors for recognition of tumor cell specific antigens, etc.
Reduction of PD-1 expression by signal inhibition can be combined with anti-PD-1 therapy, and control of the PD-1/PD-L1 pathway at low levels can significantly increase the therapeutic effect, so that immunotherapy and targeted therapy can be applied in combination. The role of NK cells and T cells, especially cytotoxic T cells, in the process of tumor immunization has been currently validated in a variety of mouse tumor models. Several clinical trials are evaluating the efficacy of certain cytokines against cancer alone or in combination with various chemotherapeutic agents and tumor-targeting monoclonal antibodies and other cytokines. However, the toxicity of cytokines such as IL-15 in high doses and the short half-life of such cytokines leading to repeated dosing over a short period of time remain problems and are a major constraint in combination strategies.
Disclosure of Invention
In order to overcome the defects of the prior art, improve the curative effect of a monoclonal antibody, improve the prognosis of tumors and have good druggability, the invention discloses a multifunctional antibody which is obtained by a genetic engineering technology and has a target PD-1 and the biological effect of an IL-15/IL-15R alpha compound, and also discloses a nucleic acid molecule for coding the antibody, a recombinant vector containing the nucleic acid molecule, a recombinant cell containing the recombinant vector, a preparation method of the multifunctional antibody and a medical application of the multifunctional antibody. The invention specifically comprises the following aspects:
the first aspect of the present invention relates to a multifunctional antibody characterized in that it comprises a first heavy chain, a second heavy chain, a first light chain and a second light chain, wherein a portion of said first heavy chain and a portion of said first light chain, a portion of said second heavy chain and a portion of said second light chain are paired, respectively, and either or both of them form a PD-1 antigen binding site, and further wherein said first heavy chain further comprises a cytokine IL-15 fragment and an immunoglobulin Fc portion, and said second heavy chain further comprises an IL-15 receptor fragment and an immunoglobulin Fc portion, and wherein said cytokine IL-15 fragment in said first heavy chain and said IL-15 receptor fragment in said second heavy chain are conjugated to each other.
Further, the immunoglobulin Fc portion of the first and second heavy chains is selected from the constant region amino acid sequences of IgG1, igG2, igG3 and/or IgG4, preferably IgG1 or IgG 4.
Further, the Fc portions of the first and second heavy chains further comprise one or more amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of: S228P, L F, L235E, P S, D356K, T366W, K392D, D K, Y407A, and K409D, preferably including S228P, T366W, Y A.
Further, the IL-15 fragment of the first heavy chain and the IL-15 receptor fragment of the second heavy chain may be chimeric within the Fc portion of said chains, respectively, or may be present outside the Fc portion, preferably between the CH1 and CH2 functional regions of said respective heavy chains.
Further, the IL-15 fragment in the first heavy chain, the IL-15 receptor fragment in the second heavy chain of the multifunctional antibody are each covalently bound in said chains, either alone or together with an additional linking peptide; the linker peptide comprises glycine (G) and serine (S) residues, preferably comprises GGGGS repeats, more preferably comprises 1-2 GGGGS repeats.
Further, the IL-15 fragment is selected from a native IL-15 or a variant thereof comprising one or more amino acid mutations selected from the group of N1D, N D, D N, E Q, N D, N D, N79A, Q E and N112A, preferably comprising one or more amino acid mutations selected from the group of N4D, N3579 zxft 3572 zxft 3525 a and N112A; the IL-15 receptor fragment is selected from the group consisting of IL-15 Ra or variants thereof, preferably IL-15 Ra variants, more preferably IL-15 Ra Sushi domains.
Further, the first heavy chain amino acid sequence of the multifunctional antibody is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: 3; the second heavy chain amino acid sequence of the multifunctional antibody is selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; the amino acid sequences of the first light chain and the second light chain of the multifunctional antibody are selected from SEQ ID NO. 7.
Further, the first heavy chain of the multifunctional antibody is SEQ ID NO. 1, the second heavy chain is SEQ ID NO. 4, and the first light chain and the second light chain (which are identical) are SEQ ID NO. 7.
Further, the multifunctional antibody has a first heavy chain of SEQ ID NO. 2, a second heavy chain of SEQ ID NO. 5, and a first light chain and a second light chain (identical) of SEQ ID NO. 7.
Further, the multifunctional antibody has a first heavy chain of SEQ ID NO. 3, a second heavy chain of SEQ ID NO. 6, and a first light chain and a second light chain (identical) of SEQ ID NO. 7.
A second aspect of the invention relates to a nucleic acid molecule encoding said multifunctional antibody, characterized in that it comprises a nucleotide sequence encoding a first light chain and/or a second light chain, or comprises a nucleotide sequence encoding a first heavy chain, or comprises a nucleotide sequence encoding a second heavy chain. The nucleotide sequence encoding the first heavy chain is selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10; the nucleotide sequence encoding the second heavy chain is selected from the group consisting of SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13; the nucleotide sequence encoding the first light chain and/or the second light chain is selected from SEQ ID NO. 14.
Further, such nucleotide sequences may be fused to polynucleotides encoding signal peptides or heterologous signal peptides native to the original antibody. Specifically, the nucleic acid molecule may further comprise a nucleotide sequence encoding a signal peptide, which may be a natural signal peptide or a heterologous signal peptide, at the 5' end of the nucleotide sequence encoding its light chain and the nucleotide sequence encoding its heavy chain, respectively; the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain further comprise a stop codon at the 3' end, respectively.
Still further, the signal peptide is selected from the amino acid sequence of SEQ ID NO. 15 and the nucleotide sequence encoding the signal peptide is selected from SEQ ID NO. 16.
The stop codon that may be included on the light chain is TGA and the stop codon that may be included on the heavy chain is TGA or TAA.
A third aspect of the invention relates to a recombinant vector, e.g. an expression vector, comprising a nucleotide sequence encoding the first heavy chain, and/or the second heavy chain, and/or the first light chain, and/or the second light chain of said multifunctional antibody. In such vectors, the nucleotide sequence of the present invention may be operably linked to one or more regulatory elements. Wherein the regulatory element is selected from expression regulatory sequences, such as promoters, enhancers, and the like.
The vector of the present invention comprises a regulatory element (e.g., a promoter or enhancer) operably linked to a nucleic acid sequence encoding the first heavy chain, the second heavy chain, the first light chain or the second light chain of the multifunctional antibody. "operably linked" refers to nucleic acid sequences that are configured so as to be arranged such that they perform their normal function. Thus, a regulatory element operably linked to a nucleotide sequence encoding the first heavy chain, the second heavy chain, the first light chain, or the second light chain is capable of directing transcription, replication, and/or translation to yield the antibody. In one embodiment, the vector encodes the amino acid sequence of the first heavy chain, the second heavy chain, the first light chain or the second light chain of said multifunctional antibody.
In the present invention, the expression vector is, for example, a prokaryotic expression vector, a eukaryotic expression vector, a phage vector, or a viral vector. Further, the vector is selected from eukaryotic vectors. Further, the vector was selected from the commercially available vectors pcDNA3.4-G418 and pcDNA3.1-G418. The heavy and light chains of the antibody can be expressed in pcDNA3.1-G418 vector and pcDNA3.4-G418, respectively. pcDNA3.4-G418 contains a promoter CMVPromoter used by a light chain, a eukaryotic selection marker G418 tag and a prokaryotic selection tag Ampicilline; the pcDNA3.1-G418 vector contains a promoter CMVPromoter used by a heavy chain, a eukaryotic screening marker G418 label and a prokaryotic screening label Ampicilline, and both can screen high-expression cell strains through neomycin pressurization.
In a specific embodiment of the present invention, hindIII restriction site, kozak sequence and signal peptide sequence are added to the 5 'end of the nucleotide sequences encoding the first light chain and the second light chain (SEQ ID NO: 14), respectively, and stop codon and XhoI restriction site are added to the 3' end, and the sequences are inserted into pcDNA3.4-G418 by restriction enzyme ligation; respectively and sequentially adding HindIII enzyme cutting sites, kozak sequences and signal peptide sequences at the 5 'ends of a nucleotide sequence (SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10) for coding a first heavy chain and a nucleotide sequence (SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO: 13) for a second heavy chain, adding a stop codon and an XhoI enzyme cutting site at the 3' ends, inserting the obtained recombinant plasmids containing the full-length first heavy chain gene of the multifunctional antibody into pcDNA3.1-G418 vectors through enzyme cutting connection, and finally naming the obtained recombinant plasmids containing the full-length first heavy chain gene of the multifunctional antibody as pcDNA3.1-G418-6-1, pcDNA3.1-G418-16-1 and pcDNA3.1-G418-17-1; the obtained recombinant plasmids containing the full-length second heavy chain gene of the multifunctional antibody are named pcDNA3.1-G418-6-2, pcDNA3.1-G418-16-2 and pcDNA3.1-G418-17-2; the obtained recombinant plasmids containing the full-length first light chain and the second light chain of the multifunctional antibody are named pcDNA3.4-G418-6-3, pcDNA3.4-G418-16-3 and pcDNA3.4-G418-17-3. Exemplary plasmid maps of pcDNA3.1-G418-16-1, pcDNA3.1-G418-16-2, and pcDNA3.4-G418-16-3 are shown in FIG. 1.
The fourth aspect of the present invention relates to a recombinant cell containing the recombinant vector of any one of the third aspects of the present invention. Further, the cells include human embryonic kidney cells HEK293 or HEK293T, HEK293E, HEK modified HEK293F, chinese hamster ovary Cells (CHO), CHO-S, CHO-dhfr - CHO/DG44, expicHO, CHO modified ExpicHO, and combinations thereof. The fifth aspect of the present invention relates to a method for preparing the multifunctional antibody, which specifically comprises: culturing the recombinant cell of the fourth aspect of the invention under conditions sufficient for expression of the multifunctional antibody of the first aspect of the invention; expressing and purifying the multifunctional antibody protein. A sixth aspect of the invention relates to the use of a nucleic acid molecule according to the second aspect of the invention, a recombinant vector according to the third aspect of the invention or a recombinant cell according to the fourth aspect of the invention for the preparation of a multifunctional antibody according to the first aspect of the invention. The seventh aspect of the present invention relates to a medicament containing the multifunctional antibody as an active ingredient, optionally containing a pharmaceutically acceptable carrier or excipient.
The invention also relates to the use of the multifunctional antibody in the preparation of a medicament for preventing or treating diseases or disorders associated with the PD-1 antigen, such as tumors; preferably, the tumor is a tumor or an advanced stage tumor that is refractory to treatment with PD-1/PD-L1 blocking monotherapy, more preferably a tumor that is resistant or refractory to treatment with anti-PD-1/PD-L1 antibody monotherapy; further preferred are B cell lymphomas, colon cancers and melanomas.
The present invention also provides a method of treating a tumor comprising administering to a cancer patient a therapeutically effective amount of the multifunctional antibody. The tumor is a tumor with pathogenesis related to PD-1/PD-L1 pathway, preferably a tumor which is ineffective to PD-1/PD-L1 blocking monotherapy or a late-stage tumor, and more preferably a tumor which is resistant or ineffective to anti-PD-1/PD-L1 antibody monotherapy; further preferred are B cell lymphomas, colon cancers and melanomas.
The present invention also relates to a pharmaceutical preparation, a pharmaceutical composition or a kit containing the multifunctional antibody as described above as an active ingredient.
Advantageous effects of the invention
The research designs and obtains a targeted PD-1 multifunctional antibody with the biological effect of an IL-15/IL-15 Ra compound through the technologies of gene recombination, codon optimization, molecular biology and the like on the basis of the existing development experience of the heterodimer, so that T cells and NK cells in PMBC can be effectively amplified and activated, the number of immune cells and the release of killer cytokines are increased, the drug resistance and relapse of a single-target antibody drug are solved, the effective dose can be reduced, tumor cells are killed more effectively, the survival period of in-situ tumor model animals is prolonged, and the in-situ tumor model animals are superior to the existing PD-1 inhibitor; compared with IL-15 or IL-15/IL-15 receptor compound, the serum half-life period is prolonged, the tumor targeting is improved, and the toxic and side effects are reduced.
In addition, the IL-15 and the receptor fragment thereof are creatively embedded in the antibody molecular chain, and the IL-15 and the receptor fragment thereof are designed to be mutually combined, so that the specific combination effect of the IL-15 and the receptor fragment is utilized, the heterodimerization of the antibody is easier, the common light chain and heavy chain mismatching problem of the common bispecific antibody is avoided, the purity of the obtained antibody is improved, the quality control is easier, and the production process and the pharmaceutical property are more certain.
Furthermore, the experiment proves that the multifunctional antibody obtained by the invention has high-efficiency PD-1 antigen affinity and IL-2R beta affinity, and has better FcRn affinity, and also has better purity, stability and bioactivity.
In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Term(s) for
The three letter and one letter codes for amino acids used in the present invention are as described in j.boil.chem.,243, p3558 (1968). The "interaction" between the Fc of a first heavy chain or a variant thereof and the Fc of a second heavy chain or a variant thereof in the present invention refers to
inter-Fc or inter-Fc variant effects. "Fc variant" refers to a change in Fc structure or function caused by the presence of one or more amino acid substitutions, insertions, or deletion mutations at the appropriate site of the Fc. "inter-Fc variant interactions" refer to the formation of space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions, etc., between mutationally designed Fc variants. The interaction between Fc variants contributes to the formation of stable heterodimers. Preferred mutation designs are those in the "Knob-in-hole" form. In addition, other mutations that result in changes in the function of the Fc of the present invention may also be present, such as glycosylation engineering mutations, mutations in the Fc γ R binding region (to modulate ADCC activity), and amino acid mutations that improve antibody stability, among others.
The "IL-15" or "IL-15 fragment" according to the invention may be any IL-15 or mutant thereof, such as human IL-15 or a non-human mammalian or non-mammalian IL-15. Exemplary non-human mammals such as pigs, rabbits, monkeys, chimpanzees, mice, etc., non-mammals such as chickens, etc.; preferably the mature molecule of human interleukin 15 (see database UniProtKB, accession number P40933, 49-162 aa). The term "IL-15 variant" refers to a mutant molecule with increased or decreased affinity for IL-15 and its receptor, or which stimulates an increase or decrease in T cell or NK cell activity, obtained by one or more amino acid substitution, addition or deletion mutation. The "IL-15 fragment" according to the invention is preferably a variant thereof, more preferably IL-15N72D (SEQ ID NO: 17).
The "IL-15 Ra" as used herein may be IL-15 Ra or a functional fragment thereof of any species, such as human IL-15 Ra or non-human mammalian IL-15 Ra or non-mammalian IL-15 Ra. Exemplary non-human mammals are, for example, pigs, rabbits, monkeys, chimpanzees, mice, etc., and non-mammals are, for example, chickens, etc. Preferably human IL-15 Ra, more preferably a fragment of the extracellular domain of human IL-15 Ra, abbreviated IL-15 Ra ECD (see database UniProtKB, accession No. Q13261, 31-205 aa). The term "IL-15 Ra variant" refers to a functional mutant formed by one or more amino acid deletion, insertion or substitution mutations in IL-15 Ra having the ability to bind to its ligand molecule, such as IL-15, preferably a shortened form of the ectodomain fragment of human IL-15 Ra, i.e.a molecule having human IL-15 receptor alpha activity obtained by one or more amino acid deletion mutations starting from the C-terminus of the ectodomain fragment, preferably a deletion mutant form retaining 65-120 amino acids, more preferably a deletion mutant shortened form retaining 65-102 amino acids, such as IL-15 Ra Sushi (65) (SEQ ID NO: 18) or IL-15 Ra Sushi (77) (SEQ ID NO: 19).
In the present invention, "covalently bound together with an additional linker peptide" means that two or more genes may be covalently bound at one or several positions between coding regions of the genes by a sequence encoding the linker peptide.
The term "immunoglobulin" refers to a globulin having the activity or chemical structure of an antibody similar to an antibody molecule, and there are five major classes of immunoglobulins: igA, igD, igE, igG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, and IgG4, igA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.
The term "immunoglobulin Fc moiety" refers to the C-terminal region of an immunoglobulin, which has no antigen binding activity, is the site of interaction of an antibody molecule with effector molecules and cells, and is a dimeric molecule comprising two disulfide-linked antibody heavy chain Fc region polypeptides. The Fc region may be produced by papain digestion or IdeS digestion to trypsin digestion of the intact (full-length) antibody or may be produced recombinantly. The "Fc portion" preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 regions of IgG.
The art of mutational design of Fc variants has relatively extensive application to the preparation of bispecific antibodies or heterodimeric Fc fusion protein forms. Representative are the "Knob-in-Hole" forms proposed by Cater et al (ProteinEngineerngvol.9no.7pp617-621,1996); the Amgen company technician uses electrostatic steering (electronic Steering) to form the Fc-containing heterodimer form (US 2010286374A 1); the heterodimeric form (SEEDbodies) formed by IgG/IgA chain exchange proposed by JonathanH.Davis et al (protein engineering, design & selectionp.1-8,2010); the bispecific molecule formed by the platform technology of DuoBody (Science, 2007.317 (5844)) of Genmab company; the technicians of Xencor company, integrating the structural calculations and the Fc amino acid mutations, integrated the different modes of action to form the heterodimeric protein form (mAbs 3:6,546-557 November/December 2011; the charge network-based Fc modification method of corning jerry, suzhou (CN 201110459100.7) yields a heterodimeric protein form; and other means based on Fc amino acid change or functional modification to form hetero-dimer functional protein. The structure of Knob-in-Hole on the Fc variant fragment of the present invention means that two Fc fragments are mutated individually, and after mutation, can be combined by means of "Knob-in-Hole". Site mutations in the Fc region are preferably engineered using the "Knob-in-Hole" model of Cater et al, so that the resulting first and second Fc variants can bind together in the form of a "Knob-in-Hole" to form a heterodimer. The selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is within the purview of those skilled in the art. Preferred are the Fc regions of human antibodies IgG1, igG2, igG3, igG4, more preferred are the Fc regions of human antibodies IgG1 and IgG 4. Optionally, one of the first Fc variant or the second Fc variant is mutated for knob and the other is mutated for hole. In embodiments, the first Fc variant is mutated for knob; the second Fc variant is mutated for hole.
The term "linker peptide" is used in the present invention to link IL-15 or IL-15R α into the corresponding heavy chain to ensure proper folding of the protein and peptide stability. The "linker peptide" of the invention is preferably (GGGGS) n, where n may be 0, 1, 2, 3, 4, 5 or more, preferably n is 1-2. If the linker peptide sequence is too short, it may interfere with the folding of the higher order structures of the two proteins; if the linker sequence is too long, problems with immunogenicity are involved, since the linker sequence itself is a new antigen.
The "heterodimer" according to the invention is preferably the product of gene co-expression. E.g., in prokaryotic cells in Escherichia coli; or co-expression in eukaryotic cells, such as 293, CHO. The "co-expression" refers to the expression of multiple genes together in one cell, with their products appearing. These genes may be present simultaneously and controlled to be expressed separately or together. In the present invention, it is preferable to co-express three genes in one eukaryotic cell. The gene expression product obtained by co-expression is beneficial to forming a compound efficiently and simply; in the present invention, the formation of heterodimers is favored.
The term "administration" as used herein refers to systemic and/or local administration. The term "systemic administration" refers to non-local administration, whereby the administered substance may affect several organs or tissues throughout the body; or so that the administered substance may traverse several organs or tissues throughout the body to reach the target site. For example, administration to the circulatory system of a subject may cause expression of a therapeutic product from the administered vector in more than one tissue or organ, or may cause expression of a therapeutic product from the administered vector at a specific site. One skilled in the art will appreciate that the systemic administration encompasses various forms of administration including, but not limited to: parenteral administration, intravenous use, intramuscular administration, subcutaneous administration, transdermal administration, intratumoral administration, oral administration, and the like. The term "topical administration" refers to administration at or around a specific site. One skilled in the art will appreciate that topical administration encompasses various forms of administration, such as injection directly to a particular site or injection around it (e.g., intratumoral administration).
The "therapeutically effective amount" of the present invention refers to the amount of the multifunctional antibody of the present invention, or the effective ingredient in the pharmaceutical preparation, pharmaceutical composition, kit, required for the disease or condition for therapeutic purposes (e.g., tumor, e.g., for causing regression or reducing the size of the tumor). The effective amount may be determined for a particular purpose by practice, in a conventional manner. In particular, the therapeutically effective amount may be that amount necessary to achieve the following: reducing the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow or stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow or stop) tumor metastasis; inhibiting tumor growth; and/or alleviating one or more symptoms associated with cancer.
The "tumor" of the present invention may be selected from B cell lymphoma, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, melanoma, uterine or endometrial cancer, oral or laryngeal cancer, liver cancer, kidney cancer, bile duct cancer, small or appendiceal cancer, salivary gland cancer, thymus cancer, adrenal cancer, osteosarcoma, chondrosarcoma, lipoma, testicular cancer, and malignant fibrous histiocytoma.
Drawings
FIG. 1 is a plasmid map of exemplary pcDNA3.1-G418-6-1, pcDNA3.1-G418-6-2, and pcDNA3.4-G418-6-3, wherein FIG. 1a is a plasmid pcDNA3.1-G418-6-1 containing a first heavy chain of antibody 6, FIG. 1b is a plasmid pcDNA3.1-G418-6-2 containing a second heavy chain of antibody 6, and FIG. 1c is a plasmid pcDNA3.1-G418-6-3 containing first/second light chains of antibody 6 (both identical).
FIGS. 2-4 are SDS-PAGE electrophoresis diagrams of antibodies 6, 16 and 17. FIG. 2a is a SDS-PAGE image under non-reducing conditions showing the bands of intact antibody 6, and FIG. 2b is a SDS-PAGE image under reducing conditions showing that antibody 6 was reduced to 3 bands, i.e., 2 different heavy chains and the same light chain; FIG. 3a is a SDS-PAGE pattern under non-reducing conditions showing the bands of intact antibody 16, and FIG. 3b is a SDS-PAGE pattern under reducing conditions showing the reduction of antibody 16 to 3 bands, i.e., 2 different heavy chains and the same light chain; FIG. 4a is a SDS-PAGE image under non-reducing conditions showing the bands of intact antibody 17, and FIG. 4b is a SDS-PAGE image under reducing conditions showing that antibody 17 was reduced to 3 bands, i.e., 2 different heavy chains and the same light chain.
FIG. 5 is a SEC-HPLC plot of the antibody 16 formulation after one week at 37 ℃.
FIG. 6 shows the ELISA results of antibody 6 binding to PD-1 antigen.
FIG. 7 shows the results of ELISA binding of antibodies 16 and 17 to PD-1 antigen.
FIG. 8 shows the results of ELISA binding of antibody 6 to the receptor IL-2R β, wherein the abscissa is concentration and the ordinate is OD value.
FIG. 9 shows the results of ELISA binding of antibodies 16 and 17 to the receptor IL-2R β, wherein the concentration is plotted on the abscissa and the OD value is plotted on the ordinate.
Figure 10 shows the results of ELISA for binding of antibodies 16 and 17 to FcRn, wherein the abscissa is concentration and the ordinate is OD value.
FIG. 11 is a tumor growth curve after mouse administration.
FIG. 12 is a graph of body weight of mice after administration.
Detailed Description
The present invention will be further described with reference to the following embodiments and drawings, and the present invention is not limited to the following embodiments. It is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It is intended that all such alterations and advantages be included in the invention, which occur to those skilled in the art, be considered as within the spirit and scope of the inventive concept, and that all such modifications and advantages be considered as within the scope of the appended claims and any equivalents thereof. In the description and claims of the present invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge of those skilled in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1 of the nucleotide sequenceObtaining and optimizing
The light chain and heavy chain amino acid sequence information of the antibodies 6, 16 and 17 is selected from published or self-developed PD-1 target monoclonal antibody sequence information, and the variable region and constant region information of the sequences is obtained through analysis. The native IL-15 sequence or IL-15 variant sequence is inserted into the amino acid sequence of one heavy chain and the IL-15 receptor sequence, preferably the IL-15 Ra Sushi sequence, is inserted at the corresponding position of the other heavy chain. If necessary, the Fc of the amino acid sequence of the antibody is adjusted to other IgG types, such as IgG1, etc., and further amino acid mutations of desired forms are designed in each heavy chain, thereby obtaining the amino acid sequences of the target antibody, which are:
antibody 6-the first heavy chain is SEQ ID NO:1, the second heavy chain is SEQ ID NO:4, and the first light chain and the second light chain (which are identical) are SEQ ID NO:7;
antibody 16-the first heavy chain is SEQ ID NO:2, the second heavy chain is SEQ ID NO:5, and the first light chain and the second light chain (which are identical) are SEQ ID NO:7;
antibody 17-the first heavy chain is SEQ ID NO:3, the second heavy chain is SEQ ID NO:6, and the first and second light chains (which are identical) are SEQ ID NO:7.
Converting each of the above target amino acid sequences into a nucleotide sequence and targeting a set of parameters that may affect the expression of the antibody in mammalian cells: optimizing codon preference, GC content (namely the ratio of guanine G and cytosine C in 4 bases of DNA), cpG islands (namely the region with higher density of CpG dinucleotides in the genome), secondary structure of mRNA, splicing sites, pre-mature PolyA sites, internal Chi sites (a short DNA fragment in the genome and the probability of homologous recombination occurring nearby the site is increased) or ribosome binding sites, RNA unstable sequences, inverted repeat sequences, restriction enzyme cutting sites which possibly interfere cloning and the like; related sequences which may improve the translation efficiency, such as Kozak sequence, SD sequence, and stop codon are added. Designing heavy chain genes and light chain genes respectively encoding the molecules of the antibodies 6, 16 and 17, and designing nucleotide sequences encoding signal peptides optimized according to amino acid sequences at the 5' ends of the heavy chain and the light chain respectively; in addition, stop codons were added to the 3' ends of the light and heavy chain nucleotide sequences, respectively.
Finally, 3 groups of optimized nucleotide sequences of the antibody are obtained through optimization, wherein the sequences are respectively as follows:
antibody 6: the first heavy chain is SEQ ID NO. 20, the second heavy chain is SEQ ID NO. 23, and the first light chain and the second light chain (which are the same) are SEQ ID NO. 26;
antibody 16: the first heavy chain is SEQ ID NO:21, the second heavy chain is SEQ ID NO:24, and the first light chain and the second light chain (which are the same) are SEQ ID NO:26;
antibody 17: the first heavy chain is SEQ ID NO. 22, the second heavy chain is SEQ ID NO. 25, and the first light chain and the second light chain (which are identical) are SEQ ID NO. 26.
Example 2 Gene Synthesis and expression vector construction
The pcDNA3.1-G418 vector is used as a special vector for expressing the light chain and the heavy chain of the multifunctional antibody. The pcDNA3.1-G418 vector contains a promoter CMVPromoter used by a heavy chain, a eukaryotic selection marker G418 tag and a prokaryotic selection tag Ampicilline. Carrying out gene synthesis to obtain nucleotide sequences of antibody expression light chains and heavy chains of antibodies 6, 16 and 17, carrying out double enzyme digestion on a vector and a target fragment by using HindIII and XhoI, carrying out enzyme ligation by using DNA ligase after recovery, transforming an escherichia coli competent cell DH5 alpha, selecting positive clones, carrying out plasmid extraction and enzyme digestion verification, and obtaining recombinant plasmids containing the full-length first heavy chain, second heavy chain, first light chain and second light chain of the antibody 6, wherein the recombinant plasmids are pcDNA3.1-G418-6-1, pcDNA3.1-G418-6-2 and pcDNA3.1-G418-6-3 (the first light chain and the second light chain are the same); recombinant plasmids containing the full-length first heavy chain, the second heavy chain, the first light chain and the second light chain of the antibody 16 are pcDNA3.1-G418-16-1, pcDNA3.1-G418-16-2 and pcDNA3.1-G418-16-3 respectively; the recombinant plasmids containing the full-length first heavy chain, the second heavy chain, the first light chain and the second light chain of the antibody 17 are pcDNA3.1-G418-17-1, pcDNA3.1-G418-17-2 and pcDNA3.1-G418-17-3 respectively. Exemplary plasmid maps of pcDNA3.1-G418-6-1, pcDNA3.1-G418-6-2, and pcDNA3.1-G418-6-3 are shown in FIGS. 1a-1c.
EXAMPLE 3 plasmid extraction
Recombinant plasmids containing the above-mentioned respective target genes were transformed into E.coli competent cells DH 5. Alpha. According to the method described in molecular cloning protocols (2002, scientific Press), transformed bacteria were plated on LB plates containing 100. Mu.g/ml ampicillin and cultured, plasmid clones were selected and cultured in liquid LB medium and shaken at 260rpm for 14 hours, and plasmids were extracted from endotoxin-free plasmid macroextraction kits, dissolved in sterile water and subjected to concentration measurement with a nucleic acid protein meter.
Example 4 plasmid transfection, transient expression and antibody purification
Culturing ExpicHO at 37 deg.C, 8% CO2, 100rpm to a cell density of 6X 10 6 cells/ml. The constructed vectors PCDNA3.1-G418-6-1, PCDNA3.1-G418-6-2 and PCDNA3.1-G418-6-3 were transfected into the above cells using liposomes at a plasmid concentration of 1. Mu.g/ml and a liposome concentration referred to ExpicHO TM Expression System kit, at 32 degrees C, 5% CO2, 100rpm under culture for 7-10 days. The feed was fed once between 18-22h and 5-8 days after transfection. The above culture product was centrifuged at 4000rpm, filtered through a 0.22 μm filter and the culture supernatant was collected, and the resulting antibody 6 protein was purified by protein A, ion column and the eluate was collected.
The concrete operation steps of the ProteinA and the ion column purification are as follows: the cell culture fluid is centrifuged at high speed, and the supernatant is taken out and subjected to affinity chromatography by using a GE protein A chromatographic column. The chromatography is performed using 1 XPBS (pH 7.4) as an equilibration buffer, and after cell supernatant is combined, the cell supernatant is washed with PBS until the UV ray returns to the baseline, and then the target protein is eluted with 0.1M glycine (pH 3.0) as an elution buffer, and the pH is adjusted to neutral with Tris. The pH of the product obtained by affinity chromatography is adjusted to be 1-2 pH units lower or higher than the pI, and the product is diluted appropriately to control the sample conductance to be below 5 ms/cm. Performing NaCl gradient elution under corresponding pH conditions by using appropriate corresponding pH buffer solution such as phosphate buffer solution, acetic acid buffer solution, etc., and ion exchange chromatography such as anion exchange or cation exchange, which is conventional in the art, and selecting collection tubes in which the target protein is located according to SDS-PAGE and storing.
Then, the eluate obtained after purification was ultrafiltered into a buffer. Proteins were detected by SDS-polyacrylamide gel electrophoresis assay.
The vectors PCDNA3.1-G418-6-1, PCDNA3.1-G418-6-2 and PCDNA3.1-G418-6-3 are respectively replaced by PCDNA3.1-G418-16-1, PCDNA3.1-G418-16-2 and PCDNA3.1-G418-16-3, and are replaced by PCDNA3.1-G418-17-1, PCDNA3.1-G418-17-2 and PCDNA3.1-G418-17-3, and the antibody 16 and the antibody 17 are obtained by transfection, expression and purification in the same way.
Results and analysis: SDS-PAGE determination proves that under non-reducing conditions, the expressed complete antibodies 6, 16 and 17 have slightly higher molecular weight than IgG1 antibody; under reducing conditions the IgG1 antibody was reduced to 2 bands, whereas antibodies 6, 16 and 17 were reduced to 3 bands, i.e. proteins of interest at positions of about 64kDa, 58kDa and 24kDa, corresponding to two different heavy chains and the same light chain of the desired antibody. Therefore, the transfection, transient expression and purification of the plasmid prove that the obtained antibody has correct structure and higher purity. The SDS-PAGE electrophoresis patterns corresponding to antibodies 6, 16 and 17 are shown in FIGS. 2-4, respectively.
The resulting antibodies 6, 16 and 17 were analyzed by mass, in vitro binding activity, cellular biological activity and in vivo pharmacodynamic assays.
Example 5 stability study
The antibody 16 was placed in the formulation of the specified formulation and after one week at 37 ℃ the purity was determined by SEC-HPLC and found to be 99.37%, indicating that the stability of the antibody was good. The corresponding SEC-HPLC profile is shown in FIG. 5.
Example 6ELISA detection of the affinity of antibodies to PD-1 antigen
PD1 antigen was diluted to 0.2. Mu.g/mL using PBS buffer, pH7.4, and 100. Mu.L per well was added to a 96-well ELISA plate and coated overnight at 4 degrees. Blocking with 2% by weight BSA blocking solution was performed for 1.5 hours. After 3 PBST washes, antibodies 6, 16 and 17 were diluted to 0.3. Mu.g/ml with 0.5% BSA sample dilutions, starting at a 3-fold gradient dilution for a total of 7 gradients, and incubated for 1h at 37 ℃ in 100. Mu.L per well, as a negative control. The plate was washed 3 times with PBST and the goat anti-human IgGFc labeled with HRP was diluted with the sample diluent at 1. After PBST washing for 4 times, 100 mu LTMB substrate is added into each hole, the plate is incubated for 10 minutes at room temperature in a dark place, and 100 mu L of 1MHCL solution is added into each hole to stop the color reaction. And (3) measuring the light absorption value of each hole in a 96-hole plate by selecting the wavelength of 450nm and the reference wavelength of 570nm on a multifunctional microplate reader, wherein the light absorption value (OD) = OD45nm-OD570nm of each hole. The concentrations of the antibodies 6, 16 and 17 were logarithmically determined and used as abscissa, the absorbance value of each well was measured and used as ordinate, and nonlinear regression was performed by using a sigmoidal-response (variable slope) method (GraphPadPrism software, graphpad software, san diego, california) to obtain binding curves of the antibodies 6, 16 and 17 with the PD1 antigen.
The ELISA results for antibody molecules 6, 16 and 17 are shown in fig. 6 and 7, respectively, and the 3 multifunctional antibodies were able to bind to PD-1 protein at each concentration and the affinity was consistent with the PD-1 parent antibody, indicating that the structure did not affect the affinity.
Example 7IL-2R beta affinity assay
IL-2R β receptor was diluted to 4 μ g/mL using PBS buffer, pH7.4, and 100 μ L per well was added to a 96-well ELISA plate and coated overnight at 4 degrees. Blocking with 1% BSA blocking solution was performed for 1 hour. After 3 PBST washes, antibodies 6, 16 and 17 were diluted to 4. Mu.g/ml with 0.5% BSA sample dilutions, starting at a 3-fold gradient dilution for a total of 7 gradients, and incubated for 1h at 37 ℃ in 100. Mu.L per well, as a negative control. The plate was washed 3 times with PBST, and the goat anti-human IgGFc labeled with HRP was diluted with 1% 10000 of sample diluent per well and incubated at room temperature for 1 hour. After PBST washing for 4 times, 100 mu LTMB substrate is added into each hole, the plate is incubated for 10 minutes at room temperature in a dark place, and 100 mu L of 1MHCL solution is added into each hole to stop the color reaction. And (3) measuring the light absorption value of each hole in a 96-hole plate by selecting the wavelength of 450nm and the reference wavelength of 570nm on a multifunctional microplate reader, wherein the light absorption value (OD) = OD450nm-OD570nm of each hole. The concentrations of antibodies 6, 16 and 17 were logarithmically determined and used as abscissa, absorbance values measured per well were used as ordinate, and nonlinear regression was performed by using a sigmoidal-response (variable slope) method (GraphPadPrism software, graphpad software, san diego, california) to obtain binding curves of antibodies 6, 16 and 17 with IL-2R β receptor.
The ELISA results for antibody molecules 6, 16 and 17 are shown in FIGS. 8 and 9, respectively, and the 3 multifunctional antibodies were able to bind to IL-2R β at each concentration and the affinity was consistent with the IL-15/IL-15R complex, indicating that the structure did not affect the affinity.
Example 8ELISA for detection of affinity of antibodies for FcRn
Antibodies 16 and 17, igG1 antibody, were diluted to 1.5. Mu.g/mL using PBS buffer pH7.4, 100. Mu.L per well was added to a 96-well ELISA plate, and coated overnight at 4 degrees. Blocking with 1% BSA blocking solution for 1 hour. After 3 PBST washes, the FcRn was diluted to 10. Mu.g/ml with 1% BSA sample dilution, starting at a concentration of 7 dilutions in 3-fold gradients, and incubated for 1h at 37 ℃ in 100. Mu.L wells, as a negative control. The plate was washed 3 more times with PBST, and HRP-labeled rabbit anti-6 × his antibody was diluted with sample diluent at 1. After PBST washing for 4 times, each well is added with 100 u LTMB substrate, and incubated for 10 minutes at room temperature in the dark, and each well is added with 100 u L of 1MHCL solution to terminate the color reaction. And (3) measuring the light absorption value of each hole in a 96-hole plate by selecting the wavelength of 450nm and the reference wavelength of 570nm on a multifunctional microplate reader, wherein the light absorption value (OD) = OD450nm-OD570nm of each hole. The concentration of the antibodies 16 and 17 was logarithmized and used as abscissa, the absorbance value of each well was measured as ordinate, and nonlinear regression was performed by using a sigmoidal-response (variblen) method (GraphPadPrism software, graphpad software, san diego, california) to obtain binding curves of the antibodies 16 and 17 and FcRn.
The ELISA results for antibody molecules 16 and 17 are shown in figure 10, and the 2 multifunctional antibodies bind FcRn at each concentration and the affinity is consistent with that of the IgG1 antibody, indicating that the half-life of the antibody may be similar to that of typical IgG 1.
Has a longer half-life similar to that of the commercially available antibody.
Example 9 evaluation of antitumor Effect in vivo
In a mouse colon cancer cell line MC38-hPD-L1 transplantation tumor C57BL/6hPD-1 mouse model, the in vivo antitumor effect evaluation of the antibody 16 was performed. A C57BL/6hPD-1 mouse model is established by using the intestinal cancer cell line MC38-hPD-L1, and the influence of the antibody 16 on the tumor growth after administration is evaluated.
Mouse colon cancer MC38-hPD-L1 cell in vitro monolayer culture with RPMI1640 medium supplemented with 10% fetal bovine serum, 2mm glutamine, 37 deg.C 5% 2 And (4) culturing. Passage was performed twice a week with conventional digestion treatment with pancreatin-EDTA. When the saturation degree of the cells is 80% -90%, collecting the cells, counting and inoculating. 0.1ml (3x10 ^5 cells) MC38-hPD-L1 cells were inoculated subcutaneously into the right hind dorsal aspect of each mouse, with the mean tumor volume reaching 64mm 3 The grouped administration is started.
The tumor-bearing mice were administered with the antibody 16, keytruda, administered once every 3 days at 0.5mg/kg, and a total of 8 times with the same volume of PBS as a control.
Animals are monitored daily for health and mortality, and routine examinations include observations of the effects of tumor growth and drug treatment on the animal's daily performance such as behavioral activity, food intake (visual only), weight changes (three weekly weight measurements), appearance signs, or other abnormalities. The number of deaths and side effects of animals within each group were recorded based on the number of animals in each group.
The experimental criteria were to investigate whether tumor growth was inhibited, retarded or cured. Tumor diameters were measured three times a week with a vernier caliper. The formula for tumor volume is: v =0.5a × b 2 And a and b represent the major and minor diameters of the tumor, respectively. The tumor suppressor therapeutic effect of the compound was evaluated as TGI (%) or relative tumor proliferation rate T/C (%). TGI (%), reflecting the rate of tumor growth inhibition. Calculation of TGI (%): TGI (%) = [ 1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group))/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group) × 100%. Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C% = T RTV /C RTV ×100％(T RTV : treatment group RTV; c RTV : negative control group RTV). Calculating Relative Tumor Volume (RTV) according to the tumor measurement result, wherein the calculation formula is RTV = V t /V 0 In which V is 0 When administered in groups (i.e. d) 0 ) Measurement of the mean tumor volume, V t Mean tumor volume at a certain measurement, T RTV And C RTV The same day data was taken.
Mouse colon cancer cell line MC38-hPD-L1 transplantable tumor model tumor-bearing mice were given PBS control, antibody 16 and Keytruda drugs, respectively, with tumor growth curves as shown in FIG. 11, where the abscissa represents the number of days after the start of treatment and the ordinate represents the tumor volume. 23 days after the start of the administration, the tumor volume of the PBS control group tumor-bearing mice reached 1052mm 3 . Compared with a PBS control group, the antibody 16 has obvious tumor inhibition effect at the dose of 0.5mg/kg, and the tumor volume is 149mm 3 (T/C =14.01%, TGI =91.37%, p = 0.011), and complete remission was achieved in 7 animals tumors. The 0.5mg/kg Keytruda group had tumor inhibiting effect, and the average tumor volume was 1075mm 3 (T/C =100.42%, TGI = -2.20%, p = 1.000), and complete remission was achieved for 2 animal tumors. At a dose of 0.5mg/kg, the anti-tumor effect of the antibody 16 is better than that of the marketed drug Keytruda, a significant difference exists (p = 0.022), and the complete remission rate of the tumor is improved by 50%.
The effect of antibody 16 on body weight changes in tumor-bearing mice is depicted in FIG. 12, where the abscissa represents the number of days after the start of treatment and the ordinate represents the body weight of the mice after administration. In the experimental process, all mice in the administration groups do not show remarkable weight loss, do not cause diseases, do not die, indirectly show that the current administration dosage has no obvious toxic or side effect and has good tolerance.
Although specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that, based upon the overall teachings of the disclosure, various modifications and alternatives to those details could be developed and still be encompassed by the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Sequence listing
<110> Shenghe (China) biopharmaceutical Co., ltd
<120> multifunctional antibodies, their preparation and use
<130> 2019
<141> 2020-06-12
<150> 2019107768486
<151> 2019-08-22
<160> 26
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Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala
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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
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Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Ile Val Ser
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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly
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Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser
225 230 235 240
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala
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Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala
260 265 270
Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly
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Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn
290 295 300
Asp Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu
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Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe
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Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Gly Gly Ser
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Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
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<210> 2
<211> 581
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Val Gln Leu Val Gln Ser Gly Val 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 Asn Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile
225 230 235 240
Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu
245 250 255
Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
260 265 270
Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His
275 280 285
Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser
290 295 300
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu
305 310 315 320
Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln
325 330 335
Met Phe Ile Asn Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
340 345 350
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
370 375 380
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
420 425 430
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
435 440 445
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
450 455 460
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
465 470 475 480
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
485 490 495
Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
500 505 510
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
515 520 525
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
530 535 540
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
545 550 555 560
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
565 570 575
Leu Ser Leu Gly Lys
580
<210> 3
<211> 589
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gln Val Gln Leu Val Gln Ser Gly Val 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 Asn Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Ile Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser
225 230 235 240
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala
245 250 255
Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala
260 265 270
Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly
275 280 285
Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn
290 295 300
Asp Ser Leu Ser Ser Asn Gly Ala Val Thr Glu Ser Gly Cys Lys Glu
305 310 315 320
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe
325 330 335
Val His Ile Val Gln Met Phe Ile Ala Thr Ser Gly Gly Gly Gly Ser
340 345 350
Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
355 360 365
Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu
370 375 380
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
385 390 395 400
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
405 410 415
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
420 425 430
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
435 440 445
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
450 455 460
Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys
465 470 475 480
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
485 490 495
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
500 505 510
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
515 520 525
Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
530 535 540
Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln
545 550 555 560
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
565 570 575
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
580 585
<210> 4
<211> 535
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gln Val Gln Leu Val Gln Ser Gly Val 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 Asn Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Ile Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met
225 230 235 240
Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser
245 250 255
Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr
260 265 270
Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His
275 280 285
Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu
290 295 300
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
305 310 315 320
Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
325 330 335
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
340 345 350
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
355 360 365
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
370 375 380
Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
385 390 395 400
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
405 410 415
Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
420 425 430
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys
435 440 445
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
450 455 460
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
465 470 475 480
Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Ala Ser
485 490 495
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
500 505 510
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
515 520 525
Leu Ser Leu Ser Pro Gly Lys
530 535
<210> 5
<211> 527
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gln Val Gln Leu Val Gln Ser Gly Val 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 Asn Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala
225 230 235 240
Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile
245 250 255
Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu
260 265 270
Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser
275 280 285
Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro
290 295 300
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
305 310 315 320
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
325 330 335
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
340 345 350
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
355 360 365
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
370 375 380
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
385 390 395 400
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
405 410 415
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
420 425 430
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
435 440 445
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
450 455 460
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
465 470 475 480
Asp Gly Ser Phe Phe Leu Ala Ser Arg Leu Thr Val Asp Lys Ser Arg
485 490 495
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
500 505 510
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
515 520 525
<210> 6
<211> 535
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gln Val Gln Leu Val Gln Ser Gly Val 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 Asn Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Ile Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met
225 230 235 240
Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser
245 250 255
Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr
260 265 270
Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His
275 280 285
Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu
290 295 300
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
305 310 315 320
Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
325 330 335
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
340 345 350
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
355 360 365
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
370 375 380
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
385 390 395 400
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
405 410 415
Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
420 425 430
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys
435 440 445
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
450 455 460
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
465 470 475 480
Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Ala Ser
485 490 495
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
500 505 510
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
515 520 525
Leu Ser Leu Ser Pro Gly Lys
530 535
<210> 7
<211> 218
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 8
<211> 1767
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caggtgcagc tggtgcagag cggcgtggag gtgaagaagc caggcgcctc cgtgaaggtg 60
agctgcaagg cttctggcta cacattcacc aactactata tgtattgggt gagacaggct 120
ccaggacagg gactggagtg gatgggaggc atcaacccct ccaatggcgg cacaaacttc 180
aatgagaagt ttaagaatcg cgtgaccctg accacagatt ccagcaccac aaccgcttac 240
atggagctga agagcctgca gttcgacgat accgccgtgt actattgcgc taggcgggac 300
taccggttcg atatgggctt tgactattgg ggccagggca caaccgtgac agtgtcttcc 360
gcctctacca agggcccttc cgtgtttccc ctggctccta gctctaagtc cacaagcgga 420
ggaaccgccg ctctgggatg tctggtgaag gattatttcc cagagcccgt gatcgtgagc 480
tggaactctg gcgccctgac aagcggcgtg cacacctttc cagctgtgct gcagtccagc 540
ggcctgtact ctctgtcttc cgtggtgaca gtgcccagct cttccctggg cacacagacc 600
tatatctgca acgtgaatca taagccctcc aacaccaagg tggacaagaa ggtggagcct 660
aagagctgtg gaggaggagg aagcggcgga ggaggctcta actgggtgaa tgtgatcagc 720
gatctgaaga agatcgagga cctgatccag tctatgcaca tcgatgccac actgtacacc 780
gagtccgacg tgcatccaag ctgcaaggtg acagctatga agtgtttcct gctggagctg 840
caggtcatct ccctggagag cggcgatgcc tctatccacg acacagtgga gaacctgatc 900
atcctggcta atgattccct gagctctaac ggcaatgtga ccgagagcgg ctgcaaggag 960
tgtgaggagc tggaggagaa gaacatcaag gagttcctgc agtcctttgt gcacatcgtg 1020
cagatgttca tcaatacctc tggaggagga ggatccggag gaggaggatc cgagcccaag 1080
agctgcgata agacacatac ctgcccccct tgtcctgccc cagagtttga gggcggccct 1140
agcgtgttcc tgtttccacc caagccaaag gacacactga tgatctctag gacacccgag 1200
gtgacctgcg tggtggtgga cgtgtcccac gaggaccctg aggtgaagtt taactggtac 1260
gtggatggcg tggaggtgca taatgccaag accaagccca gagaggagca gtacgctagc 1320
acatatcgcg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1380
tataagtgca aggtgtctaa taaggccctg cctgcttcca tcgagaagac catcagcaag 1440
gctaagggac agccaaggga gccacaggtg tacacactgc ctccatctcg ggacgagctg 1500
accaagaacc aggtgtccct gtggtgtctg gtgaagggct tctatcctag cgatatcgct 1560
gtggagtggg agtctaatgg ccagccagag aacaattacg acacaacccc ccctgtgctg 1620
gactctgatg gctccttctt tctgtattct gatctgacag tggacaagtc caggtggcag 1680
cagggcaacg tgttttcttg ttccgtgatg catgaggctc tgcacaatca ttacacccag 1740
aagagcctgt ctctgtcccc tggcaag 1767
<210> 9
<211> 1743
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caggtgcagc tggtgcagag cggagtggag gtgaagaagc caggcgccag cgtgaaggtg 60
tcttgcaagg cttccggcta cacattcacc aactactata tgtattgggt gagacaggct 120
ccaggacagg gactggagtg gatgggaggc atcaacccct ccaatggcgg caccaacttc 180
aatgagaagt ttaagaatcg cgtgacactg accacagatt ccagcaccac aaccgcttac 240
atggagctga agagcctgca gttcgacgat accgccgtgt actattgtgc taggcgggac 300
tacaggttcg atatgggctt tgactattgg ggccagggca caaccgtgac cgtgtcttcc 360
gccagcacaa agggcccatc cgtgttccca ctggctccat gctcccggag cacctctgag 420
tccacagccg ctctgggctg tctggtgaag gactatttcc ctgagccagt gacagtgtct 480
tggaactccg gcgccctgac cagcggagtg cacacatttc ccgctgtgct gcagagctct 540
ggcctgtact ctctgtccag cgtggtgacc gtgccatctt ccagcctggg cacaaagacc 600
tatacatgca acgtggatca taagcccagc aatacaaagg tggacaagag ggtgggagga 660
ggaggatccg gaggaggagg aagcaactgg gtgaatgtga tcagcgatct gaagaagatc 720
gaggacctga tccagtctat gcacatcgat gccaccctgt acacagagtc tgacgtgcat 780
ccttcctgca aggtgaccgc tatgaagtgt tttctgctgg agctgcaggt catctccctg 840
gagtctggcg atgcctctat ccacgacacc gtggagaacc tgatcatcct ggctaatgat 900
tccctgtctt ccaacggcaa tgtgacagag agcggctgca aggagtgtga ggagctggag 960
gagaagaaca tcaaggagtt cctgcagtct tttgtgcata tcgtgcagat gttcatcaat 1020
accagcggag gaggaggatc tggaggagga ggaagcgagt ctaagtacgg accaccttgc 1080
ccaccatgtc cagctcctga gtttctggga ggaccatccg tgttcctgtt tcctccaaag 1140
cctaaggata ccctgatgat ctccagaacc cccgaggtga catgcgtggt ggtggatgtg 1200
agccaggagg accctgaggt gcagttcaac tggtacgtgg acggcgtgga ggtgcacaat 1260
gctaagacaa agcccaggga ggagcagttt aactccacct accgggtggt gagcgtgctg 1320
acagtgctgc atcaggactg gctgaacggc aaggagtata agtgcaaggt gtctaataag 1380
ggcctgccta gctctatcga gaagaccatc tccaaggcta agggacagcc tcgcgagcca 1440
caggtgtaca ccctgccccc ttctcaggag gagatgacaa agaaccaggt gtccctgtgg 1500
tgtctggtga agggcttcta tcctagcgat atcgctgtgg agtgggagtc taatggccag 1560
ccagagaaca attacaagac aaccccaccc gtgctggact ccgatggcag cttctttctg 1620
tattctagac tgaccgtgga caagtcccgc tggcaggagg gcaacgtgtt ttcctgtagc 1680
gtgatgcacg aggctctgca caatcattac acacagaagt ctctgtccct gagcctgggc 1740
aag 1743
<210> 10
<211> 1767
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
caggtgcagc tggtgcagag cggcgtggag gtgaagaagc caggcgcctc cgtgaaggtg 60
agctgcaagg cttctggcta cacattcacc aattactata tgtattgggt gagacaggct 120
ccaggacagg gactggagtg gatgggaggc atcaacccct ccaatggcgg cacaaacttc 180
aatgagaagt ttaagaaccg cgtgaccctg accacagatt ccagcaccac aaccgcttac 240
atggagctga agagcctgca gttcgacgat accgccgtgt actattgcgc taggcgggac 300
taccggttcg atatgggctt tgactattgg ggccagggca caaccgtgac agtgtcttcc 360
gcctctacca agggcccttc cgtgtttccc ctggctccta gctctaagtc cacaagcgga 420
ggaaccgccg ctctgggatg tctggtgaag gattatttcc cagagcccgt gatcgtgagc 480
tggaattctg gcgccctgac aagcggcgtg cacacctttc cagctgtgct gcagtccagc 540
ggcctgtact ctctgtcttc cgtggtgaca gtgcccagct cttccctggg cacacagacc 600
tatatctgca acgtgaatca taagccctcc aacaccaagg tggacaagaa ggtggagcct 660
aagagctgtg gaggaggagg aagcggcgga ggaggctcta actgggtgaa tgtgatcagc 720
gatctgaaga agatcgagga cctgatccag tctatgcaca tcgatgccac actgtacacc 780
gagtccgacg tgcatccaag ctgcaaggtg acagctatga agtgtttcct gctggagctg 840
caggtcatct ccctggagag cggcgatgcc tctatccacg acacagtgga gaacctgatc 900
atcctggcca atgattccct gagctctaac ggcgctgtga ccgagagcgg ctgcaaggag 960
tgtgaggagc tggaggagaa gaatatcaag gagttcctgc agtcctttgt gcacatcgtg 1020
cagatgttca tcgccacctc tggaggagga ggatccggag gaggaggatc cgagcccaag 1080
agctgcgata agacacatac ctgcccccct tgtcctgctc cagagtttga gggcggccct 1140
agcgtgttcc tgtttccacc caagccaaag gacacactga tgatctctag gacacccgag 1200
gtgacctgcg tggtggtgga cgtgtcccac gaggaccctg aggtgaagtt taattggtac 1260
gtggatggcg tggaggtgca taacgctaag accaagccca gagaggagca gtacaacagc 1320
acatatcgcg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa tggcaaggag 1380
tataagtgca aggtgtctaa caaggccctg cctgcttcca tcgagaagac catcagcaag 1440
gctaagggac agccaaggga gccacaggtg tacacactgc ctccatctcg ggacgagctg 1500
accaagaatc aggtgtccct gtggtgtctg gtgaagggct tctatcctag cgatatcgct 1560
gtggagtggg agtctaacgg ccagccagag aacaattacg acacaacccc ccctgtgctg 1620
gactctgatg gctccttctt tctgtattct gatctgacag tggacaagtc caggtggcag 1680
cagggcaacg tgttctcctg ttccgtgatg catgaggctc tgcacaacca ttacacccag 1740
aagagcctgt ctctgtcccc tggcaag 1767
<210> 11
<211> 1605
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ctggcgcctc cgtgaaggtg 60
agctgcaagg cttctggcta cacattcacc aactactata tgtattgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggaggc atcaacccct ccaatggcgg cacaaacttc 180
aatgagaagt ttaagaatcg ggtgaccctg accacagatt ccagcaccac aaccgcttac 240
atggagctga agagcctgca gttcgacgat accgccgtgt actattgcgc taggcgggac 300
tacaggttcg atatgggctt tgactattgg ggccagggca caaccgtgac agtgtcttcc 360
gcctctacca agggcccttc cgtgtttccc ctggctccta gctctaagtc cacaagcgga 420
ggaaccgccg ctctgggatg tctggtgaag gactatttcc cagagcccgt gatcgtgtcc 480
tggaacagcg gcgccctgac aagcggagtg cacacctttc ctgctgtgct gcagtccagc 540
ggcctgtact ctctgtcttc cgtggtcaca gtgccaagct cttccctggg cacacagacc 600
tatatctgca acgtgaatca taagccctcc aataccaagg tggataagaa ggtggagcct 660
aagagctgcg gaggaggagg aagcggcgga ggaggctcta tcacctgtcc ccctccaatg 720
agcgtggagc acgccgacat ctgggtgaag tcttactccc tgtactccag ggagaggtac 780
atctgcaaca gcggctttaa gagaaaggct ggcacaagct ctctgaccga gtgcgtgctg 840
aacaaggcca ccaatgtggc tcactggaca accccttctc tgaagtgcat caggggagga 900
ggaggatccg agccaaagag ctgtgataag acacatacct gccccccttg tcctgctcca 960
gagttcgagg gcggcccttc cgtgttcctg tttccaccca agccaaagga cacactgatg 1020
atctctagga caccagaggt gacctgcgtg gtggtggacg tgtcccacga ggaccccgag 1080
gtgaagttta actggtacgt ggatggcgtg gaggtgcata atgccaagac caagccaagg 1140
gaggagcagt acgcttccac atatcgggtg gtgagcgtgc tgaccgtgct gcaccaggac 1200
tggctgaacg gcaaggagta caagtgcaag gtgtctaata aggccctgcc cgcttctatc 1260
gagaagacaa tctccaaggc caagggccag ccaagagagc cccaggtgta taccctgcct 1320
ccaagccgca aggagctgac aaagaaccag gtgtctctga cctgtctggt gaagggcttc 1380
tacccctctg atatcgctgt ggagtgggag tccaatggcc agcctgagaa caattataag 1440
acaacccccc ctgtgctgaa gtctgatggc tccttctttc tggccagcaa gctgacagtg 1500
gacaagtctc ggtggcagca gggcaacgtg tttagctgtt ctgtgatgca tgaggctctg 1560
cacaatcatt acacccagaa gtccctgagc ctgtctcctg gcaag 1605
<210> 12
<211> 1581
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
caggtgcagc tggtgcagag cggagtggag gtgaagaagc caggcgccag cgtgaaggtg 60
tcttgcaagg cttccggcta cacattcacc aactactata tgtattgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggaggc atcaacccct ctaatggcgg caccaacttc 180
aatgagaagt ttaagaatcg ggtgacactg accacagatt ccagcaccac aaccgcttac 240
atggagctga agtccctgca gttcgacgat accgccgtgt actattgtgc taggcgggac 300
tacaggttcg atatgggctt tgactattgg ggccagggca caaccgtgac cgtgtcttcc 360
gccagcacaa agggcccatc cgtgttccca ctggctccat gctcccggag cacctctgag 420
tccacagccg ctctgggctg tctggtgaag gactatttcc ctgagccagt gaccgtgagc 480
tggaactctg gcgccctgac cagcggagtg cacacatttc ccgctgtgct gcagagctct 540
ggcctgtact ctctgtccag cgtggtgaca gtgccatctt ccagcctggg cacaaagacc 600
tatacatgca acgtggatca caagccctcc aataccaagg tggacaagag ggtgggagga 660
ggaggatccg gaggaggcgg cagcatcaca tgtccccctc caatgagcgt ggagcatgcc 720
gatatctggg tgaagagcta ctctctgtac tccagggaga ggtacatctg caatagcggc 780
ttcaagagaa aggctggcac ctcttccctg acagagtgcg tgctgaacaa ggccaccaat 840
gtggctcatt ggacaacccc tagcctgaag tgcatcaggg gaggaggagg atccgagagc 900
aagtatggac caccttgccc accatgtcca gctcctgagt ttctgggagg accatccgtg 960
ttcctgtttc ctccaaagcc taaggatacc ctgatgatct ccagaacccc cgaggtgaca 1020
tgcgtggtgg tggatgtgag ccaggaggac cctgaggtgc agttcaactg gtacgtggac 1080
ggcgtggagg tgcacaatgc taagaccaag cccagagagg agcagtttaa ctctacctac 1140
cgcgtggtgt ccgtgctgac agtgctgcat caggactggc tgaacggcaa ggagtataag 1200
tgcaaggtgt ctaataaggg cctgcctagc tctatcgaga agaccatctc caaggctaag 1260
ggacagcctc gcgagccaca ggtgtataca ctgcccccta gccaggagga gatgaccaag 1320
aaccaggtgt ctctgacatg tctggtgaag ggcttctacc cttctgatat cgctgtggag 1380
tgggagtcca atggccagcc agagaacaat tataagacaa ccccacccgt gctggactcc 1440
gatggcagct tctttctggc cagcaggctg accgtggaca agtctcggtg gcaggagggc 1500
aacgtgtttt cttgctccgt gatgcacgag gctctgcaca atcattacac acagaagagc 1560
ctgtctctgt ccctgggcaa g 1581
<210> 13
<211> 1605
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ctggcgcctc cgtgaaggtg 60
agctgcaagg cttctggcta cacattcacc aactactata tgtattgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggaggc atcaacccct ccaatggcgg cacaaacttc 180
aatgagaagt ttaagaatcg ggtgaccctg accacagatt ccagcaccac aaccgcttac 240
atggagctga agagcctgca gttcgacgat accgccgtgt actattgcgc taggcgggac 300
tacaggttcg atatgggctt tgactattgg ggccagggca caaccgtgac agtgtcttcc 360
gcctctacca agggcccttc cgtgtttccc ctggctccta gctctaagtc cacaagcgga 420
ggaaccgccg ctctgggatg tctggtgaag gactatttcc cagagcccgt gatcgtgtcc 480
tggaacagcg gcgccctgac aagcggagtg cacacctttc ctgctgtgct gcagtccagc 540
ggcctgtact ctctgtcttc cgtggtcaca gtgccaagct cttccctggg cacacagacc 600
tatatctgca acgtgaatca taagccctcc aataccaagg tggataagaa ggtggagcct 660
aagagctgcg gaggaggagg aagcggcgga ggaggctcta tcacctgtcc ccctccaatg 720
agcgtggagc acgccgacat ctgggtgaag tcttactccc tgtactccag ggagaggtac 780
atctgcaaca gcggctttaa gagaaaggct ggcacaagct ctctgaccga gtgcgtgctg 840
aacaaggcca ccaatgtggc tcactggaca accccttctc tgaagtgcat caggggagga 900
ggaggatccg agccaaagag ctgtgataag acacatacct gccccccttg tcctgctcca 960
gagttcgagg gcggcccttc cgtgttcctg tttccaccca agccaaagga cacactgatg 1020
atctctagga caccagaggt gacctgcgtg gtggtggacg tgtcccacga ggaccccgag 1080
gtgaagttta actggtacgt ggatggcgtg gaggtgcata atgccaagac caagccaagg 1140
gaggagcagt acaactccac atatcgggtg gtgagcgtgc tgaccgtgct gcaccaggac 1200
tggctgaacg gcaaggagta caagtgcaag gtgtctaata aggccctgcc cgcttctatc 1260
gagaagacaa tctccaaggc caagggccag ccaagagagc cccaggtgta taccctgcct 1320
ccaagccgca aggagctgac aaagaaccag gtgtctctga cctgtctggt gaagggcttc 1380
tacccctctg atatcgctgt ggagtgggag tccaatggcc agcctgagaa caattataag 1440
acaacccccc ctgtgctgaa gtctgatggc tccttctttc tggccagcaa gctgacagtg 1500
gacaagtctc ggtggcagca gggcaacgtg tttagctgtt ctgtgatgca tgaggctctg 1560
cacaatcatt acacccagaa gtccctgagc ctgtctcctg gcaag 1605
<210> 14
<211> 654
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gagatcgtgc tgacccagtc tcctgctaca ctgtccctga gcccaggaga gagggccacc 60
ctgagctgtc gggcttctaa gggcgtgtct acatccggct actcctatct gcactggtac 120
cagcagaagc caggccaggc ccccagactg ctgatctacc tggcttccta tctggagagc 180
ggagtgccag ctcgcttcag cggctctggc tccggcaccg actttaccct gacaatctcc 240
agcctggagc cagaggactt cgccgtgtac tattgccagc atagcaggga tctgcccctg 300
acctttggcg gcggcacaaa ggtggagatc aagcggaccg tggccgctcc tagcgtgttc 360
atctttcccc cttctgacga gcagctgaag tctggcacag cttccgtggt gtgcctgctg 420
aacaatttct acccaagaga ggccaaggtg cagtggaagg tggataacgc tctgcagagc 480
ggcaattctc aggagtccgt gaccgagcag gacagcaagg attctacata ttccctgtct 540
tccaccctga cactgtccaa ggccgattac gagaagcaca aggtgtatgc ttgcgaggtg 600
acccatcagg gcctgagctc tcctgtgaca aagagcttta accgcggcga gtgt 654
<210> 15
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Met Ala Val Leu Gly Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys
1 5 10 15
Val Leu Ser
<210> 16
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atggccgtgc tgggcctgct gttctgcctg gtgacctttc ctagctgcgt gctgtct 57
<210> 17
<211> 114
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser Asn Gly Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser
<210> 18
<211> 65
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg
65
<210> 19
<211> 77
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro
65 70 75
<210> 20
<211> 1845
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
aagcttgcca ccatggccgt gctgggcctg ctgttctgcc tggtgacctt tcctagctgc 60
gtgctgtctc aggtgcagct ggtgcagagc ggcgtggagg tgaagaagcc aggcgcctcc 120
gtgaaggtga gctgcaaggc ttctggctac acattcacca actactatat gtattgggtg 180
agacaggctc caggacaggg actggagtgg atgggaggca tcaacccctc caatggcggc 240
acaaacttca atgagaagtt taagaatcgc gtgaccctga ccacagattc cagcaccaca 300
accgcttaca tggagctgaa gagcctgcag ttcgacgata ccgccgtgta ctattgcgct 360
aggcgggact accggttcga tatgggcttt gactattggg gccagggcac aaccgtgaca 420
gtgtcttccg cctctaccaa gggcccttcc gtgtttcccc tggctcctag ctctaagtcc 480
acaagcggag gaaccgccgc tctgggatgt ctggtgaagg attatttccc agagcccgtg 540
atcgtgagct ggaactctgg cgccctgaca agcggcgtgc acacctttcc agctgtgctg 600
cagtccagcg gcctgtactc tctgtcttcc gtggtgacag tgcccagctc ttccctgggc 660
acacagacct atatctgcaa cgtgaatcat aagccctcca acaccaaggt ggacaagaag 720
gtggagccta agagctgtgg aggaggagga agcggcggag gaggctctaa ctgggtgaat 780
gtgatcagcg atctgaagaa gatcgaggac ctgatccagt ctatgcacat cgatgccaca 840
ctgtacaccg agtccgacgt gcatccaagc tgcaaggtga cagctatgaa gtgtttcctg 900
ctggagctgc aggtcatctc cctggagagc ggcgatgcct ctatccacga cacagtggag 960
aacctgatca tcctggctaa tgattccctg agctctaacg gcaatgtgac cgagagcggc 1020
tgcaaggagt gtgaggagct ggaggagaag aacatcaagg agttcctgca gtcctttgtg 1080
cacatcgtgc agatgttcat caatacctct ggaggaggag gatccggagg aggaggatcc 1140
gagcccaaga gctgcgataa gacacatacc tgcccccctt gtcctgcccc agagtttgag 1200
ggcggcccta gcgtgttcct gtttccaccc aagccaaagg acacactgat gatctctagg 1260
acacccgagg tgacctgcgt ggtggtggac gtgtcccacg aggaccctga ggtgaagttt 1320
aactggtacg tggatggcgt ggaggtgcat aatgccaaga ccaagcccag agaggagcag 1380
tacgctagca catatcgcgt ggtgtctgtg ctgaccgtgc tgcaccagga ctggctgaac 1440
ggcaaggagt ataagtgcaa ggtgtctaat aaggccctgc ctgcttccat cgagaagacc 1500
atcagcaagg ctaagggaca gccaagggag ccacaggtgt acacactgcc tccatctcgg 1560
gacgagctga ccaagaacca ggtgtccctg tggtgtctgg tgaagggctt ctatcctagc 1620
gatatcgctg tggagtggga gtctaatggc cagccagaga acaattacga cacaaccccc 1680
cctgtgctgg actctgatgg ctccttcttt ctgtattctg atctgacagt ggacaagtcc 1740
aggtggcagc agggcaacgt gttttcttgt tccgtgatgc atgaggctct gcacaatcat 1800
tacacccaga agagcctgtc tctgtcccct ggcaagtgac tcgag 1845
<210> 21
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
aagcttgcca ccatggccgt gctgggcctg ctgttctgcc tggtgacctt tccttcttgc 60
gtgctgtccc aggtgcagct ggtgcagagc ggagtggagg tgaagaagcc aggcgccagc 120
gtgaaggtgt cttgcaaggc ttccggctac acattcacca actactatat gtattgggtg 180
agacaggctc caggacaggg actggagtgg atgggaggca tcaacccctc caatggcggc 240
accaacttca atgagaagtt taagaatcgc gtgacactga ccacagattc cagcaccaca 300
accgcttaca tggagctgaa gagcctgcag ttcgacgata ccgccgtgta ctattgtgct 360
aggcgggact acaggttcga tatgggcttt gactattggg gccagggcac aaccgtgacc 420
gtgtcttccg ccagcacaaa gggcccatcc gtgttcccac tggctccatg ctcccggagc 480
acctctgagt ccacagccgc tctgggctgt ctggtgaagg actatttccc tgagccagtg 540
acagtgtctt ggaactccgg cgccctgacc agcggagtgc acacatttcc cgctgtgctg 600
cagagctctg gcctgtactc tctgtccagc gtggtgaccg tgccatcttc cagcctgggc 660
acaaagacct atacatgcaa cgtggatcat aagcccagca atacaaaggt ggacaagagg 720
gtgggaggag gaggatccgg aggaggagga agcaactggg tgaatgtgat cagcgatctg 780
aagaagatcg aggacctgat ccagtctatg cacatcgatg ccaccctgta cacagagtct 840
gacgtgcatc cttcctgcaa ggtgaccgct atgaagtgtt ttctgctgga gctgcaggtc 900
atctccctgg agtctggcga tgcctctatc cacgacaccg tggagaacct gatcatcctg 960
gctaatgatt ccctgtcttc caacggcaat gtgacagaga gcggctgcaa ggagtgtgag 1020
gagctggagg agaagaacat caaggagttc ctgcagtctt ttgtgcatat cgtgcagatg 1080
ttcatcaata ccagcggagg aggaggatct ggaggaggag gaagcgagtc taagtacgga 1140
ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 1200
cctccaaagc ctaaggatac cctgatgatc tccagaaccc ccgaggtgac atgcgtggtg 1260
gtggatgtga gccaggagga ccctgaggtg cagttcaact ggtacgtgga cggcgtggag 1320
gtgcacaatg ctaagacaaa gcccagggag gagcagttta actccaccta ccgggtggtg 1380
agcgtgctga cagtgctgca tcaggactgg ctgaacggca aggagtataa gtgcaaggtg 1440
tctaataagg gcctgcctag ctctatcgag aagaccatct ccaaggctaa gggacagcct 1500
cgcgagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1560
tccctgtggt gtctggtgaa gggcttctat cctagcgata tcgctgtgga gtgggagtct 1620
aatggccagc cagagaacaa ttacaagaca accccacccg tgctggactc cgatggcagc 1680
ttctttctgt attctagact gaccgtggac aagtcccgct ggcaggaggg caacgtgttt 1740
tcctgtagcg tgatgcacga ggctctgcac aatcattaca cacagaagtc tctgtccctg 1800
agcctgggca agtgactcga g 1821
<210> 22
<211> 1845
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
aagcttgcca ccatggccgt gctgggcctg ctgttctgcc tggtgacctt tcctagctgc 60
gtgctgtctc aggtgcagct ggtgcagagc ggcgtggagg tgaagaagcc aggcgcctcc 120
gtgaaggtga gctgcaaggc ttctggctac acattcacca attactatat gtattgggtg 180
agacaggctc caggacaggg actggagtgg atgggaggca tcaacccctc caatggcggc 240
acaaacttca atgagaagtt taagaaccgc gtgaccctga ccacagattc cagcaccaca 300
accgcttaca tggagctgaa gagcctgcag ttcgacgata ccgccgtgta ctattgcgct 360
aggcgggact accggttcga tatgggcttt gactattggg gccagggcac aaccgtgaca 420
gtgtcttccg cctctaccaa gggcccttcc gtgtttcccc tggctcctag ctctaagtcc 480
acaagcggag gaaccgccgc tctgggatgt ctggtgaagg attatttccc agagcccgtg 540
atcgtgagct ggaattctgg cgccctgaca agcggcgtgc acacctttcc agctgtgctg 600
cagtccagcg gcctgtactc tctgtcttcc gtggtgacag tgcccagctc ttccctgggc 660
acacagacct atatctgcaa cgtgaatcat aagccctcca acaccaaggt ggacaagaag 720
gtggagccta agagctgtgg aggaggagga agcggcggag gaggctctaa ctgggtgaat 780
gtgatcagcg atctgaagaa gatcgaggac ctgatccagt ctatgcacat cgatgccaca 840
ctgtacaccg agtccgacgt gcatccaagc tgcaaggtga cagctatgaa gtgtttcctg 900
ctggagctgc aggtcatctc cctggagagc ggcgatgcct ctatccacga cacagtggag 960
aacctgatca tcctggccaa tgattccctg agctctaacg gcgctgtgac cgagagcggc 1020
tgcaaggagt gtgaggagct ggaggagaag aatatcaagg agttcctgca gtcctttgtg 1080
cacatcgtgc agatgttcat cgccacctct ggaggaggag gatccggagg aggaggatcc 1140
gagcccaaga gctgcgataa gacacatacc tgcccccctt gtcctgctcc agagtttgag 1200
ggcggcccta gcgtgttcct gtttccaccc aagccaaagg acacactgat gatctctagg 1260
acacccgagg tgacctgcgt ggtggtggac gtgtcccacg aggaccctga ggtgaagttt 1320
aattggtacg tggatggcgt ggaggtgcat aacgctaaga ccaagcccag agaggagcag 1380
tacaacagca catatcgcgt ggtgtctgtg ctgaccgtgc tgcaccagga ctggctgaat 1440
ggcaaggagt ataagtgcaa ggtgtctaac aaggccctgc ctgcttccat cgagaagacc 1500
atcagcaagg ctaagggaca gccaagggag ccacaggtgt acacactgcc tccatctcgg 1560
gacgagctga ccaagaatca ggtgtccctg tggtgtctgg tgaagggctt ctatcctagc 1620
gatatcgctg tggagtggga gtctaacggc cagccagaga acaattacga cacaaccccc 1680
cctgtgctgg actctgatgg ctccttcttt ctgtattctg atctgacagt ggacaagtcc 1740
aggtggcagc agggcaacgt gttctcctgt tccgtgatgc atgaggctct gcacaaccat 1800
tacacccaga agagcctgtc tctgtcccct ggcaagtgac tcgag 1845
<210> 23
<211> 1683
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
aagcttgcca ccatggccgt gctgggcctg ctgttctgcc tggtgacctt tcccagctgc 60
gtgctgtctc aggtgcagct ggtgcagagc ggcgtggagg tgaagaagcc tggcgcctcc 120
gtgaaggtga gctgcaaggc ttctggctac acattcacca actactatat gtattgggtg 180
aggcaggctc caggacaggg actggagtgg atgggaggca tcaacccctc caatggcggc 240
acaaacttca atgagaagtt taagaatcgg gtgaccctga ccacagattc cagcaccaca 300
accgcttaca tggagctgaa gagcctgcag ttcgacgata ccgccgtgta ctattgcgct 360
aggcgggact acaggttcga tatgggcttt gactattggg gccagggcac aaccgtgaca 420
gtgtcttccg cctctaccaa gggcccttcc gtgtttcccc tggctcctag ctctaagtcc 480
acaagcggag gaaccgccgc tctgggatgt ctggtgaagg actatttccc agagcccgtg 540
atcgtgtcct ggaacagcgg cgccctgaca agcggagtgc acacctttcc tgctgtgctg 600
cagtccagcg gcctgtactc tctgtcttcc gtggtcacag tgccaagctc ttccctgggc 660
acacagacct atatctgcaa cgtgaatcat aagccctcca ataccaaggt ggataagaag 720
gtggagccta agagctgcgg aggaggagga agcggcggag gaggctctat cacctgtccc 780
cctccaatga gcgtggagca cgccgacatc tgggtgaagt cttactccct gtactccagg 840
gagaggtaca tctgcaacag cggctttaag agaaaggctg gcacaagctc tctgaccgag 900
tgcgtgctga acaaggccac caatgtggct cactggacaa ccccttctct gaagtgcatc 960
aggggaggag gaggatccga gccaaagagc tgtgataaga cacatacctg ccccccttgt 1020
cctgctccag agttcgaggg cggcccttcc gtgttcctgt ttccacccaa gccaaaggac 1080
acactgatga tctctaggac accagaggtg acctgcgtgg tggtggacgt gtcccacgag 1140
gaccccgagg tgaagtttaa ctggtacgtg gatggcgtgg aggtgcataa tgccaagacc 1200
aagccaaggg aggagcagta cgcttccaca tatcgggtgg tgagcgtgct gaccgtgctg 1260
caccaggact ggctgaacgg caaggagtac aagtgcaagg tgtctaataa ggccctgccc 1320
gcttctatcg agaagacaat ctccaaggcc aagggccagc caagagagcc ccaggtgtat 1380
accctgcctc caagccgcaa ggagctgaca aagaaccagg tgtctctgac ctgtctggtg 1440
aagggcttct acccctctga tatcgctgtg gagtgggagt ccaatggcca gcctgagaac 1500
aattataaga caaccccccc tgtgctgaag tctgatggct ccttctttct ggccagcaag 1560
ctgacagtgg acaagtctcg gtggcagcag ggcaacgtgt ttagctgttc tgtgatgcat 1620
gaggctctgc acaatcatta cacccagaag tccctgagcc tgtctcctgg caagtgactc 1680
gag 1683
<210> 24
<211> 1659
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aagcttgcca ccatggccgt gctgggcctg ctgttctgcc tggtgacctt tccttcttgc 60
gtgctgtccc aggtgcagct ggtgcagagc ggagtggagg tgaagaagcc aggcgccagc 120
gtgaaggtgt cttgcaaggc ttccggctac acattcacca actactatat gtattgggtg 180
aggcaggctc caggacaggg actggagtgg atgggaggca tcaacccctc taatggcggc 240
accaacttca atgagaagtt taagaatcgg gtgacactga ccacagattc cagcaccaca 300
accgcttaca tggagctgaa gtccctgcag ttcgacgata ccgccgtgta ctattgtgct 360
aggcgggact acaggttcga tatgggcttt gactattggg gccagggcac aaccgtgacc 420
gtgtcttccg ccagcacaaa gggcccatcc gtgttcccac tggctccatg ctcccggagc 480
acctctgagt ccacagccgc tctgggctgt ctggtgaagg actatttccc tgagccagtg 540
accgtgagct ggaactctgg cgccctgacc agcggagtgc acacatttcc cgctgtgctg 600
cagagctctg gcctgtactc tctgtccagc gtggtgacag tgccatcttc cagcctgggc 660
acaaagacct atacatgcaa cgtggatcac aagccctcca ataccaaggt ggacaagagg 720
gtgggaggag gaggatccgg aggaggcggc agcatcacat gtccccctcc aatgagcgtg 780
gagcatgccg atatctgggt gaagagctac tctctgtact ccagggagag gtacatctgc 840
aatagcggct tcaagagaaa ggctggcacc tcttccctga cagagtgcgt gctgaacaag 900
gccaccaatg tggctcattg gacaacccct agcctgaagt gcatcagggg aggaggagga 960
tccgagagca agtatggacc accttgccca ccatgtccag ctcctgagtt tctgggagga 1020
ccatccgtgt tcctgtttcc tccaaagcct aaggataccc tgatgatctc cagaaccccc 1080
gaggtgacat gcgtggtggt ggatgtgagc caggaggacc ctgaggtgca gttcaactgg 1140
tacgtggacg gcgtggaggt gcacaatgct aagaccaagc ccagagagga gcagtttaac 1200
tctacctacc gcgtggtgtc cgtgctgaca gtgctgcatc aggactggct gaacggcaag 1260
gagtataagt gcaaggtgtc taataagggc ctgcctagct ctatcgagaa gaccatctcc 1320
aaggctaagg gacagcctcg cgagccacag gtgtatacac tgccccctag ccaggaggag 1380
atgaccaaga accaggtgtc tctgacatgt ctggtgaagg gcttctaccc ttctgatatc 1440
gctgtggagt gggagtccaa tggccagcca gagaacaatt ataagacaac cccacccgtg 1500
ctggactccg atggcagctt ctttctggcc agcaggctga ccgtggacaa gtctcggtgg 1560
caggagggca acgtgttttc ttgctccgtg atgcacgagg ctctgcacaa tcattacaca 1620
cagaagagcc tgtctctgtc cctgggcaag tgactcgag 1659
<210> 25
<211> 1683
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
aagcttgcca ccatggccgt gctgggcctg ctgttctgcc tggtgacctt tcccagctgc 60
gtgctgtctc aggtgcagct ggtgcagagc ggcgtggagg tgaagaagcc tggcgcctcc 120
gtgaaggtga gctgcaaggc ttctggctac acattcacca actactatat gtattgggtg 180
aggcaggctc caggacaggg actggagtgg atgggaggca tcaacccctc caatggcggc 240
acaaacttca atgagaagtt taagaatcgg gtgaccctga ccacagattc cagcaccaca 300
accgcttaca tggagctgaa gagcctgcag ttcgacgata ccgccgtgta ctattgcgct 360
aggcgggact acaggttcga tatgggcttt gactattggg gccagggcac aaccgtgaca 420
gtgtcttccg cctctaccaa gggcccttcc gtgtttcccc tggctcctag ctctaagtcc 480
acaagcggag gaaccgccgc tctgggatgt ctggtgaagg actatttccc agagcccgtg 540
atcgtgtcct ggaacagcgg cgccctgaca agcggagtgc acacctttcc tgctgtgctg 600
cagtccagcg gcctgtactc tctgtcttcc gtggtcacag tgccaagctc ttccctgggc 660
acacagacct atatctgcaa cgtgaatcat aagccctcca ataccaaggt ggataagaag 720
gtggagccta agagctgcgg aggaggagga agcggcggag gaggctctat cacctgtccc 780
cctccaatga gcgtggagca cgccgacatc tgggtgaagt cttactccct gtactccagg 840
gagaggtaca tctgcaacag cggctttaag agaaaggctg gcacaagctc tctgaccgag 900
tgcgtgctga acaaggccac caatgtggct cactggacaa ccccttctct gaagtgcatc 960
aggggaggag gaggatccga gccaaagagc tgtgataaga cacatacctg ccccccttgt 1020
cctgctccag agttcgaggg cggcccttcc gtgttcctgt ttccacccaa gccaaaggac 1080
acactgatga tctctaggac accagaggtg acctgcgtgg tggtggacgt gtcccacgag 1140
gaccccgagg tgaagtttaa ctggtacgtg gatggcgtgg aggtgcataa tgccaagacc 1200
aagccaaggg aggagcagta caactccaca tatcgggtgg tgagcgtgct gaccgtgctg 1260
caccaggact ggctgaacgg caaggagtac aagtgcaagg tgtctaataa ggccctgccc 1320
gcttctatcg agaagacaat ctccaaggcc aagggccagc caagagagcc ccaggtgtat 1380
accctgcctc caagccgcaa ggagctgaca aagaaccagg tgtctctgac ctgtctggtg 1440
aagggcttct acccctctga tatcgctgtg gagtgggagt ccaatggcca gcctgagaac 1500
aattataaga caaccccccc tgtgctgaag tctgatggct ccttctttct ggccagcaag 1560
ctgacagtgg acaagtctcg gtggcagcag ggcaacgtgt ttagctgttc tgtgatgcat 1620
gaggctctgc acaatcatta cacccagaag tccctgagcc tgtctcctgg caagtgactc 1680
gag 1683
<210> 26
<211> 732
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aagcttgcca ccatggctcc agtgcagctg ctgggactgc tggtgctgtt cctgcctgcc 60
atgaggtgcg agatcgtgct gacccagtct cctgctacac tgtccctgag cccaggagag 120
agggccaccc tgagctgtcg ggcttctaag ggcgtgtcta catccggcta ctcctatctg 180
cactggtacc agcagaagcc aggccaggcc cccagactgc tgatctacct ggcttcctat 240
ctggagagcg gagtgccagc tcgcttcagc ggctctggct ccggcaccga ctttaccctg 300
acaatctcca gcctggagcc agaggacttc gccgtgtact attgccagca tagcagggat 360
ctgcccctga cctttggcgg cggcacaaag gtggagatca agcggaccgt ggccgctcct 420
agcgtgttca tctttccccc ttctgacgag cagctgaagt ctggcacagc ttccgtggtg 480
tgcctgctga acaatttcta cccaagagag gccaaggtgc agtggaaggt ggataacgct 540
ctgcagagcg gcaattctca ggagtccgtg accgagcagg acagcaagga ttctacatat 600
tccctgtctt ccaccctgac actgtccaag gccgattacg agaagcacaa ggtgtatgct 660
tgcgaggtga cccatcaggg cctgagctct cctgtgacaa agagctttaa ccgcggcgag 720
tgttgactcg ag 732

Claims (11)

1. A multifunctional antibody comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain, wherein a portion of the first heavy chain and a portion of the first light chain, a portion of the second heavy chain and a portion of the second light chain are each paired and one or both form a PD-1 antigen binding site, wherein the first heavy chain further comprises a cytokine IL-15 fragment and an immunoglobulin Fc portion, wherein the second heavy chain further comprises an IL-15 receptor fragment and an immunoglobulin Fc portion, and wherein the cytokine IL-15 fragment of the first heavy chain and the IL-15 receptor fragment of the second heavy chain bind to each other; wherein the first heavy chain amino acid sequence of the multifunctional antibody is selected from the group consisting of SEQ ID NO 2; the second heavy chain amino acid sequence of the multifunctional antibody is selected from the group consisting of SEQ ID NO 5; the amino acid sequences of the first and second light chains of the multifunctional antibody are selected from the group consisting of SEQ ID NO 7.
2. A nucleic acid molecule encoding the multifunctional antibody of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence encoding the first heavy chain is selected from the group consisting of SEQ ID NO 9; the nucleotide sequence encoding the second heavy chain is selected from the group consisting of SEQ ID NO 12; the nucleotide sequences encoding the first and second light chains are selected from SEQ ID NO 14.
4. The nucleic acid molecule according to claim 3, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a signal peptide at the 5 'end of the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain, and a stop codon at the 3' end of the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain, respectively.
5. The nucleic acid molecule of claim 4, wherein the signal peptide is selected from the amino acid sequence of SEQ ID NO. 15 and the nucleotide sequence encoding the signal peptide is selected from the group consisting of SEQ ID NO. 16.
6. A recombinant vector comprising a nucleotide sequence encoding the multifunctional antibody of claim 1.
7. A recombinant cell comprising the recombinant vector of claim 6.
8. A method of making the multifunctional antibody of claim 1, said method comprising in particular: culturing the recombinant cell of claim 7 under conditions sufficient for expression of the multifunctional antibody of claim 1; expressing and purifying the multifunctional antibody protein.
9. Use of the nucleic acid molecule of claim 5, the recombinant vector of claim 6 or the recombinant cell of claim 7 for the preparation of the multifunctional antibody of claim 1.
10. Use of a multifunctional antibody according to claim 1 for the preparation of a medicament for the treatment of a disease associated with the PD-1 antigen, such as B-cell lymphoma, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, cancer of the central nervous system, cancer of the esophagus, cervical cancer, melanoma, endometrial cancer, oral or laryngeal cancer, liver cancer, kidney cancer, bile duct cancer, small or appendiceal cancer, salivary gland carcinoma, thymus carcinoma, adrenal gland carcinoma, osteosarcoma, chondrosarcoma, lipoma, testicular cancer, or malignant fibrous histiocytoma.
11. A pharmaceutical formulation or kit comprising as an active ingredient the multifunctional antibody according to claim 1.
CN202010534034.4A 2019-08-22 2020-06-12 Multifunctional antibodies, their preparation and uses Active CN112409484B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103951754A (en) * 2014-03-26 2014-07-30 中国药科大学 Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection
WO2018071918A1 (en) * 2016-10-14 2018-04-19 Xencor, Inc. Bispecific heterodimeric fusion proteins containing il-15/il-15ralpha fc-fusion proteins and pd-1 antibody fragments
WO2018209194A2 (en) * 2017-05-12 2018-11-15 Icahn School Of Medicine At Mount Sinai Newcastle disease viruses and uses thereof
CN109562162A (en) * 2016-01-13 2019-04-02 指南针制药有限责任公司 Multispecific immune modulability antigen-binding constructs

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI680138B (en) * 2014-01-23 2019-12-21 美商再生元醫藥公司 Human antibodies to pd-l1

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103951754A (en) * 2014-03-26 2014-07-30 中国药科大学 Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection
CN109562162A (en) * 2016-01-13 2019-04-02 指南针制药有限责任公司 Multispecific immune modulability antigen-binding constructs
WO2018071918A1 (en) * 2016-10-14 2018-04-19 Xencor, Inc. Bispecific heterodimeric fusion proteins containing il-15/il-15ralpha fc-fusion proteins and pd-1 antibody fragments
WO2018209194A2 (en) * 2017-05-12 2018-11-15 Icahn School Of Medicine At Mount Sinai Newcastle disease viruses and uses thereof

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