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CN114437228A - Bifunctional fusion protein formed by IL-2 and antibody subunit - Google Patents

Bifunctional fusion protein formed by IL-2 and antibody subunit Download PDF

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CN114437228A
CN114437228A CN202011189127.4A CN202011189127A CN114437228A CN 114437228 A CN114437228 A CN 114437228A CN 202011189127 A CN202011189127 A CN 202011189127A CN 114437228 A CN114437228 A CN 114437228A
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CN114437228B (en
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彭华
曹帅帅
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Abstract

本发明涉及一种IL‑2与抗体亚单位构成的双功能融合蛋白，其包括：由如下第一单体和第二单体构成的异源二聚体：(1)白介素2(IL‑2)与免疫球蛋白Fc单链连接而成的第一单体；(2)TNF家族共刺激或共抑制性分子或CTLA4分子的抗体的Fab/ScFv与Fc单链连接而成的第二单体。所述的双功能融合蛋白还包括同源二聚体，所述同源二聚体单体包括：(1)IL‑2功能嵌段；和(2)抗体的单体或抗体的Fab/ScFv与Fc单链连接而成的单体，所述的抗体为TNF家族共刺激或共抑制性分子或CTLA4分子的抗体。本发明还涉及所述的双功能融合蛋白在制备抗肿瘤药物中的应用。

Figure 202011189127

The present invention relates to a bifunctional fusion protein composed of IL-2 and antibody subunits, comprising: a heterodimer composed of the following first monomers and second monomers: (1) interleukin 2 (IL-2 ) the first monomer formed by connecting with the immunoglobulin Fc single chain; (2) the second monomer formed by connecting the Fab/ScFv of the antibody of the TNF family costimulatory or costinhibitory molecule or CTLA4 molecule to the Fc single chain . Described bifunctional fusion protein also includes homodimer, and described homodimer monomer includes: (1) IL-2 functional block; and (2) antibody monomer or antibody Fab/ScFv A monomer linked to an Fc single chain, and the antibody is a co-stimulatory or co-inhibitory molecule of the TNF family or an antibody of a CTLA4 molecule. The invention also relates to the application of the bifunctional fusion protein in the preparation of antitumor drugs.

Figure 202011189127

Description

Bifunctional fusion protein formed by IL-2 and antibody subunit
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a bifunctional fusion protein formed by IL-2 and an antibody subunit.
Background
Interleukin 2(IL2), also known as T cell growth factor, was found in 1976 to stimulate clonal expansion of T cells in vitro experiments [1 ]. IL2 is a glycoprotein with four alpha helices, has a molecular weight of 15.5Kd [2], is secreted mainly by activated CD 4T cells, and other cells such as activated CD8 cells, NK cells, NKT cells and ILCs cells also produce small amounts of IL2[3,4 ]. IL2 functions by binding to receptor cells in autocrine and paracrine pathways and has important regulatory effects on T, NK cells.
The receptor for IL2 consists of three subunits, IL2R α (CD25), IL2R β (CD122) and IL2R γ c (CD132), with differences in the affinity between IL2 and the three subunits [5,6 ]. IL2 binds with low affinity to the alpha subunit (Kd-10-8M), with moderate affinity to the beta-gamma dimer (Kd-10-9M), and with high affinity to the alpha-beta-gamma dimer (Kd-10-11M) [7,8 ].
The expression levels of different affinity receptors differ on different cells: the beta gamma receptor is mainly expressed on T cells in a resting state, CD8T memory cells and NK cells; the alpha receptor is up-regulated after cell activation, and the activated T cell expresses high-affinity alpha beta gamma receptor. Treg cells continuously and highly express CD25 molecules, and have high affinity to IL2 molecules [ 9]]. Due to the difference of receptor expression, the low dose of IL2 preferentially expanded Treg cells expressing high affinity receptor, and the high dose of IL2 could effectively activate CD8+T cells and NK cells [10]。
IL2 has the highest affinity for the trimeric receptor, which is predominantly expressed persistently on Treg cells and only transiently upregulated on activated effector cells; clinically, low doses of IL2 preferentially expanded Treg cells, whereas high doses of IL2 activated effector T cells. High doses of IL2 resulted in greater toxic side effects and limited the use of IL 2.
To overcome this problem, IL2 molecules were engineered by different means to decrease binding to tregs and increase binding to effector T cells and NK cells to enhance their efficacy in tumor therapy, while avoiding toxicity due to high dose use. There are several current approaches to the modification of IL 2:
(1) PEGylated IL2
IL2 has a molecular weight of 15.5KD and a half-life in serum of 10-85min, and needs to be administered repeatedly for several times to achieve therapeutic effect. PEGylated IL2 increased the overall molecular weight of IL2 and extended the half-life of IL2, while IL2 could be biased toward binding to IL 2-Ra or IL 2-Rbeta by differences in the PEGylation modification sites [11 ].
(2) IL2/aIL2 antibody complex
Binding of IL2 to the aIL2 antibody complex can, on the one hand, prolong the half-life of IL2 and, on the other hand, lead to IL2/aIL2 antibody complexes exhibiting a different propensity to bind to the receptor CD25 or CD122, since the aIL2 antibody can bind to different sites of the IL2 molecule.
(3) IL2 mutation engineering
The affinity of IL2 for different receptor subunits may also be altered by point mutation engineering of different receptor binding sites on IL 2. IL2 was conformationally altered by point mutations of five bases L80F, R81D, L85V, I86V and I92F of IL2 molecules to increase binding to CD122 while rendering downstream signal transduction independent of CD25 molecules. Mutant IL2 preferentially expanded CD8+ T cells and NK cells, had lower toxic side effects and showed better tumor therapeutic effect in mouse tumor models [12 ].
In our pre-laboratory adaptation, we simultaneously mutate IL2 to reduce CD25 subunit binding (F42A) and increase CD122 subunit binding. This double mutant IL2 molecule had better therapeutic efficacy than either the CD25 subunit alone, or the CD122 subunit alone, with the increased affinity mutation [13 ]. The double-mutation IL2 has good treatment effect in an MC38 tumor model; in the cold tumor model such as TUBO and the like, the combined treatment with TKI or the 7.16.4 antibody also has good treatment effect. It is demonstrated that increasing the affinity of the CD122 subunit and further decreasing the affinity of the CD25 subunit can further improve the therapeutic effect of IL 2.
The modifications described above for IL2 were either reduced by reducing IL2 binding to the CD25 molecule or increased binding to the CD122 subunit. Although this is the caseOne strategy for modification has been to improve the therapeutic efficacy compared to the wild-type IL2 molecule, but still does not completely eliminate the inhibitory effect of intratumoral Treg cells on the therapeutic efficacy.How to further compare and analyze intratumoral Treg cells and it is still crucial that the difference in CD25 and CD122 receptor expression on effector T cells further enhances the therapeutic effect of IL2 Problem(s)。
The co-stimulatory or co-inhibitory molecules of the TNF family are also highly expressed on Treg cells, are target molecules for regulating the functions of the Treg cells, and are common molecules such as ICOS, GITR, OX40, 4-1BB and the like.
Some costimulatory molecules such as GITR and OX40 molecules are highly expressed on intratumoral tregs and are also up-regulated on activated CD4 and CD8T cells. OX40 molecule is expressed on T cells within the tumor and is highly expressed on tregs within the tumor; systemic administration of the activating aOX40 antibody can result in a decrease in the number and proportion of intratumoral tregs and thus exert an anti-tumor effect [14 ]. No significant decrease in Treg numbers was observed in the clinical trial of the aOX40 antibody, but an increase in the number of effector T cells was observed [15 ].
OX40 molecules are co-expressed on effector T cells and Treg cells, and the OX40 antibody used at present can play an anti-tumor role and can play a role in activating effector cells and deleting Treg cells. OX40 was dependent on the expansion of intratumoral CD 4T and CD8T cells, and tumors did not recur after re-challenge with immunological memory in mice after treatment [16 ]. On the other hand, OX40 antibody OX86 used in mouse experiments may also function to delete tregs that are dependent on activating Fc γ R and not on inhibiting Fc γ RIIB. Furthermore, after replacing rIgG1Fc of OX86 with IgG2a and IgG2An297A of mice (which do not bind to FcR), respectively, Fc of IgG2a is found to have better tumor control capability, which indicates that the function of OX40 antibody in the tumor model is mainly dependent on the function of deleting Treg; moreover, deletion occurred mainly on Treg cells, but not on the number and absolute number of CD 4T cells and CD8T cells of tregs [17 ]. In using OX40 targets for treatment, we can exploit the differences in abundance of OX40 expression on tregs and effector T cells to exert different effects on different T cell populations. In addition to the use of OX40 antibody alone, OX40 molecules have also been shown to exert synergistic antitumor effects in combination with chemotherapy, radiation therapy and other cytokines [18 ].
Therefore, IL2/aOX40 bifunctional molecules, and fusion protein bifunctional antibodies composed of antibodies of CTLA4 and co-stimulatory or co-inhibitory molecules of other TNF families, such as ICOS, GITR, 4-1BB and the like, and IL-2 cytokines are new tumor treatment schemes with great development potential in clinic.
Disclosure of Invention
The invention firstly relates to a bifunctional fusion protein, which is a heterodimer and is characterized in that,
the heterodimer includes:
(1) a heterodimer first monomer formed by connecting interleukin 2(IL-2) and an immunoglobulin Fc single chain;
(2) a heterodimer second monomer in which a Fab or ScFv of an antibody against CTLA4 molecule or a co-stimulatory or co-inhibitory molecule of the TNF family is linked to an immunoglobulin Fc single chain;
the first monomer and the second monomer are connected through dimerization of Fc single chains to form the heterodimer;
co-stimulatory or co-inhibitory molecules of the CTLA4 or TNF family include, but are not limited to: OX40, 4-1BB, ICOS, GITR.
The immunoglobulin Fc single chain is a natural immunoglobulin Fc single chain or an immunoglobulin Fc single chain for knocking ADCC effect out through gene mutation;
preferably, the immunoglobulin Fc single chain is a natural immunoglobulin Fc single chain or an immunoglobulin Fc single chain for knocking out ADCC effect through gene mutation; more preferably, the immunoglobulin Fc single chain is the Fc single chain of human IgG.
The co-stimulatory or co-inhibitory molecule of CTLA4 or TNF family is OX40, 4-1BB, and the antibody against CTLA4 (aCTLA4) or the antibody against the co-stimulatory or co-inhibitory molecule of TNF family is an antibody against OX40 (aOX40) or an antibody against 4-1BB (a4-1 BB);
in the second monomer, the monomer is a monomer,
the Fab of the antibody is the Fab of a humanized antibody or the Fab of a fully humanized antibody;
the ScFv of the antibody is a humanized antibody ScFv or a fully humanized antibody ScFv;
more preferably, the second monomer is:
a monomer of an antibody against CTLA4 or a co-stimulatory or co-inhibitory molecule of the TNF family, said antibody monomer comprising one light chain and one heavy chain; preferably, the antibody is a humanized antibody or a fully human antibody.
More preferably, the heterodimer comprises:
(1) a first monomer, said first monomer comprising in order from the N-terminus:
1) the wild type IL-2 protein with the sequence shown in SEQ ID NO.1 or the mutant of the wild type IL-2 protein, wherein the mutant comprises the mutation site of any one or any combination of the following parts; F42A, L80F, R81D, L85V, I86V and I92F;
2) (ii) an essential linker structure (G4S linker sequence), preferably, said linker sequence is as shown in SEQ ID No. 6;
3) fc single chain of IgG with the sequence shown as SEQ ID NO.2, or Fc of No-ADCC mutant IgG with the sequence shown as SEQ ID NO.3, or knob mutant Fc with the sequence shown as SEQ ID NO.4, or hole mutant Fc with the sequence shown as SEQ ID NO. 5;
(2) a second monomer comprising:
1) an anti-OX40 antibody Fab region consisting of an anti-OX40 antibody light chain VL-KCL with the sequence shown as SEQ ID No.7 and an anti-OX40 antibody heavy chain VH & CH1 with the sequence shown as SEQ ID No. 8;
or: 2) an anti-OX40 single-chain antibody (ScFv) with the sequence shown as SEQ ID NO. 9:
and: 3) fc single chain of IgG with the sequence shown as SEQ ID NO.2, or Fc of No-ADCC mutant IgG with the sequence shown as SEQ ID NO.3, or knob mutant Fc with the sequence shown as SEQ ID NO.4, or hole mutant Fc with the sequence shown as SEQ ID NO. 5;
more preferably, the heterodimer comprises:
a first monomer: it is polypeptide (IL2-Fc) with the sequence shown in SEQ ID NO. 10;
a second monomer: it comprises the following steps:
(1) a second monomer consisting of an anti-OX40 antibody VH-CH1-Fc (knob) having a sequence shown in SEQ ID NO.11 and an anti-OX40 antibody light chain VL-KCL having a sequence shown in SEQ ID NO. 7;
or (2) a polypeptide (aOX40 ScFv-Fc (knob)) with the sequence shown as SEQ ID NO. 12.
The invention also relates to a bifunctional fusion protein, which is a homodimer and is characterized in that,
the monomers of the homodimer are as follows:
a monomer consisting of a molecule of interleukin 2(IL-2) linked by any means to a molecule of anti-OX40 antibody Fab, or,
a monomer formed by connecting a molecule of interleukin 2(IL-2) and a molecule of anti-OX40 single-chain antibody (ScFv) in any mode.
Preferably, the monomers of the homodimer comprise, in order from the N-terminus:
(1) the wild type IL-2 protein shown in SEQ ID NO.1 or the mutant of the wild type IL-2 protein, wherein the mutant comprises a mutation site of any one or any combination of the following parts; F42A, L80F, R81D, L85V, I86V and I92F;
(2) (ii) an essential linker structure (G4S linker sequence), preferably, said linker sequence is as shown in SEQ ID No. 6;
(3) fab or ScFv of anti-OX40 antibody; the Fab is Fab of a humanized antibody or Fab of a fully humanized antibody, and the ScFv is ScFv of the humanized antibody or ScFv of the fully humanized antibody;
(4) fc of an antibody; the antibody Fc is fully human wild-type Fc or No-ADCC mutant Fc.
More preferably, the monomers of the homodimer are:
(1)SEQ ID NO.13(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
(2) the sequence shown in SEQ ID NO.14(IL2-aOX 40: IL2-VL-VH (ScFv) -Fc (wt)).
The invention also relates to another bifunctional fusion protein, which is a bifunctional fusion protein formed by combining an anti-CTLA 4 antibody and IL2, and is a heterodimer;
the heterodimer includes:
(1) a first monomer, said first monomer comprising in order from the N-terminus:
1) the wild type IL-2 protein shown in SEQ ID NO.1 or the mutant of the wild type IL-2 protein, wherein the mutant comprises a mutation site of any one or any combination of the following parts; F42A, L80F, R81D, L85V, I86V and I92F;
2) (ii) an essential linker structure (G4S linker sequence), preferably, said linker sequence is as shown in SEQ ID No. 6;
3) an Fc single chain of IgG shown as SEQ ID NO.2, or Fc of No-ADCC mutant IgG shown as SEQ ID NO.3, or knob mutant Fc shown as SEQ ID NO.4, or hole mutant Fc shown as SEQ ID NO. 5;
(2) a second monomer comprising:
1) an antibody Fab region consisting of an anti-CTLA 4 antibody light chain VL-KCL with the sequence shown as SEQ ID No.21 and an anti-CTLA 4 antibody heavy chain VH & CH1 with the sequence shown as SEQ ID No. 22;
or: 2) an anti-CTLA 4 single-chain antibody (ScFv) with a sequence shown as SEQ ID NO. 23:
and: 3) fc single chain of IgG with the sequence shown as SEQ ID NO.2, or Fc of No-ADCC mutant IgG with the sequence shown as SEQ ID NO.3, or knob mutant Fc with the sequence shown as SEQ ID NO.4, or hole mutant Fc with the sequence shown as SEQ ID NO. 5;
more preferably, the heterodimer comprises:
a first monomer: it is polypeptide (IL2-Fc) with the sequence shown in SEQ ID NO. 10;
a second monomer: it comprises the following steps:
(1) a second monomer consisting of a polypeptide (aCTLA4 VH-CH1-Fc (knob)) with a sequence shown as SEQ ID NO.24 and an anti-CTLA 4 antibody light chain with a sequence shown as SEQ ID NO. 21; or
(2) The polypeptide (a CTLA4 ScFv-Fc (knob)) with the sequence shown as SEQ ID NO. 25.
The invention also relates to a bifunctional fusion protein, which is a homodimer and is characterized in that,
the monomers of the homodimer are as follows:
a monomer formed by connecting a molecule of interleukin 2(IL-2) and a molecule of anti-CTLA 4 antibody Fab in any mode, or,
one molecule of interleukin 2(IL-2) and one molecule of anti-CTLA 4 single-chain antibody (ScFv) are connected by any mode to form a monomer.
Preferably, the monomers of the homodimer comprise, in order from the N-terminus:
(1) the wild type IL-2 protein shown in SEQ ID NO.1 or the mutant of the wild type IL-2 protein, wherein the mutant comprises a mutation site of any one or any combination of the following parts; F42A, L80F, R81D, L85V, I86V, and I92F;
(2) (ii) an essential linker structure (G4S linker sequence), preferably said linker sequence is as shown in SEQ ID No. 6;
(3) fab or ScFv of anti-CTLA 4 antibodies; the Fab is Fab of a humanized antibody or Fab of a fully humanized antibody, and the ScFv is ScFv of the humanized antibody or ScFv of the fully humanized antibody;
(4) fc of an antibody; the antibody Fc is fully human wild-type Fc or No-ADCC mutant Fc.
More preferably, the monomers of the homodimer are:
(1)SEQ ID NO.26(aCTLA4-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
(2) SEQ ID NO.27(IL2-a CTLA 4: IL2-VL-VH (ScFv) -Fc (wt)).
The invention also relates to another bifunctional fusion protein, which is a bifunctional fusion protein formed by combining an anti-4-1 BB antibody and IL2, and is a heterologous or homodimer;
the heterodimer includes:
(1) a first monomer, said first monomer comprising in order from the N-terminus:
1) the wild type IL-2 protein shown in SEQ ID NO.1 or the mutant of the wild type IL-2 protein, wherein the mutant comprises a mutation site of any one or any combination of the following parts; F42A, L80F, R81D, L85V, I86V and I92F;
2) (ii) an essential linker structure (G4S linker sequence), preferably, said linker sequence is as shown in SEQ ID No. 6;
3) an Fc single chain of IgG shown as SEQ ID NO.2, or Fc of No-ADCC mutant IgG shown as SEQ ID NO.3, or knob mutant Fc shown as SEQ ID NO.4, or hole mutant Fc shown as SEQ ID NO. 5;
(2) a second monomer comprising:
1) an antibody Fab region consisting of an anti-4-1 BB antibody light chain with sequences shown as SEQ ID NO.28 and 29 and an anti-4-1 BB antibody heavy chain VH & CH1 with sequences shown as SEQ ID NO.30 and 31;
or: 2) an anti-4-1 BB single-chain antibody (ScFv) with the sequence shown in SEQ ID NO.32 and 33:
and: 3) fc single chain of IgG with the sequence shown as SEQ ID NO.2, or Fc of No-ADCC mutant IgG with the sequence shown as SEQ ID NO.3, or knob mutant Fc with the sequence shown as SEQ ID NO.4, or hole mutant Fc with the sequence shown as SEQ ID NO. 5;
more preferably, the heterodimer comprises:
a first monomer: it is polypeptide (IL2-Fc) with the sequence shown in SEQ ID NO. 10;
a second monomer: it comprises the following steps:
(1) a second monomer consisting of a polypeptide (a4-1BB VH-CH1-Fc (knob)) with sequences shown as SEQ ID NO.34 and 35 and an anti-4-1 BB antibody light chain with sequences shown as SEQ ID NO.28 and 29; or
(2) The polypeptide (a4-1BB ScFv-Fc (knob)) with the sequence shown in SEQ ID NO.36 and 37.
The fusion protein is a homodimer and is characterized in that,
the monomers of the homodimer are as follows:
a monomer comprising a molecule of interleukin-2 (IL-2) linked to a molecule of anti-4-1 BB antibody Fab by any means, or,
one molecule of interleukin-2 (IL-2) and one molecule of anti-4-1 BB single-chain antibody (ScFv) are connected in any mode to form a monomer.
The monomer of the homodimer sequentially comprises from the N end:
(1) the wild type IL-2 protein shown in SEQ ID NO.1 or the mutant of the wild type IL-2 protein, wherein the mutant comprises a mutation site of any one or any combination of the following parts; F42A, L80F, R81D, L85V, I86V and I92F;
(2) (ii) an essential linker structure (G4S linker sequence), preferably, said linker sequence is as shown in SEQ ID No. 6;
(3) fab or ScFv of anti-4-1 BB antibody; the Fab is Fab of a humanized antibody or Fab of a fully humanized antibody, and the ScFv is ScFv of the humanized antibody or ScFv of the fully humanized antibody;
(4) fc of an antibody; the antibody Fc is fully human wild-type Fc or No-ADCC mutant Fc.
More preferably, the monomers of the homodimer are: such as
SEQ ID NO.38(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.39(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.40(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、
SEQ ID NO.41(IL2-a4-1BB (3H 3): IL2-VL-VH (ScFv) -Fc (wt)).
The person skilled in the art should likewise make clear:
the method comprises the following steps:
functional structure of anti-human OX40 antibody encoded by amino acid sequence shown in SEQ ID NO.52-55
Functional structure of anti-human CTLA4 antibody coded by amino acid sequence shown in SEQ ID NO.56-57
Functional structure of antihuman 4-1BB antibody coded by amino acid sequence shown in SEQ ID NO.58-61
The technical effects of the invention can also be achieved by replacing similar functional blocks of the above bifunctional fusion proteins.
The invention also relates to nucleotide sequences encoding the heterodimers and homodimers.
Preferably, the first and second liquid crystal materials are,
the nucleotide sequence of the first monomer of the heterodimer is the nucleotide sequence shown as SEQ ID NO.15(IL2-Fc (hole));
the nucleotide sequence encoding the second monomer of the heterodimer is:
SEQ ID NO.16(aOX40：VL-KCL)、
SEQ ID NO.17(aOX40：VH-CH1-Fc(knob))、
SEQ ID NO.18(aOX40 ScFv-Fc(knob))、
SEQ ID NO.42(a4-1BB(LOB12.3)：VL-KCL)、
SEQ ID NO.43(a4-1BB(3H3)：VL-KCL)、
SEQ ID NO.44(a4-1BB(LOB12.3)：VH-CH1-Fc(knob))、
SEQ ID NO.45(a4-1BB(3H3)：VH-CH1-Fc(knob))、
SEQ ID NO.46(a4-1BB(LOB12.3)：ScFv-Fc(knob))、
a nucleotide sequence shown in SEQ ID NO.47(a4-1BB (3H 3): ScFv-Fc (knob));
the nucleotide sequence encoding the homodimer is:
SEQ ID NO.19(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.20(IL2-aOX40：IL2-VL-VH(ScFv)-Fc(wt))、
SEQ ID NO.48(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.49(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.50(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、
the nucleotide sequence shown in SEQ ID NO.51(IL2-a4-1BB (3H 3): IL2-VL-VH (ScFv) -Fc (wt)).
The invention also relates to the following applications of the bifunctional fusion protein:
(1) preparing an anti-tumor medicament;
(2) preparing an anti-tumor drug in combination with an immune checkpoint inhibitor;
(3) preparing an anti-tumor drug for overcoming the tolerance of an immune checkpoint inhibitor;
(4) preparing an anti-tumor medicament combined with the TKI antagonist;
(5) preparing the antitumor drug for overcoming the TKI antagonist tolerance.
Preferably, the immune checkpoint inhibitor is a PD-L1 antibody;
preferably, the TKI antagonist is a small molecule TKI antagonist; more preferably, the small molecule TKI antagonist is afatinib or a structural analogue thereof.
The invention has the beneficial effects that:
(1) the bottleneck and the solution of the clinical application of IL2 are provided, the IL2/aOX40-hIgG1 antibody deletes the Treg cells in the tumor while keeping the activation of IL2 on effector cells, the adverse factor that the Treg is expanded in the use process of IL2 is overcome, and the results provide a new idea for the clinical application of IL 2;
(2) has important significance for overcoming the tolerance of the immune checkpoint blockade therapy and the resistance generated after the TKI chemotherapeutic drug is used.
Drawings
FIG. 1, IL2-Fc fusion protein is capable of partially controlling tumor growth, and the molecular structure of 1A, IL2-Fc fusion protein, the lower dimer is Fc region dimer of human IgG, and N-terminal of each Fc monomer is coupled with one molecule of wild type IL-2 molecule; 1B, IL2-Fc fusion protein has tumor growth inhibiting effect.
Fig. 2, aOX40 antibody stimulated T cell activation and exerted anti-tumor function, 2A, aOX40 antibody was able to effectively activate CD 3T cells; the 2B, aOX40 antibody is effective in controlling tumor growth.
FIG. 3, IL2-Fc and aOX40 in combination did not show any significant synergistic effect
FIG. 4 is a diagram of the structural pattern of the construction of IL2/aOX40-Fc bispecific antibody, 4A, IL2/aOX40-Fc bispecific antibody; 4B, SDS-PAGE analysis of the constructed IL2/aOX40-Fc bispecific antibody; 4C, IL2/aOX40-Fc heterodimer form, aOX40 in Fab or scFv form, the antibody comprising 1 IL2 molecule; 4D, IL2/aOX40-Fc, aOX40 is in scFv form, and 1 or 2 IL2 molecules are connected at the N end of a V region or the C end of Fc; 4E, IL2/aOX40-Fc homodimer form, aOX40 in Fab form, linked 2 molecules of IL2 at the N-or C-terminus of the light chain, at the N-or C-terminus of the heavy chain; 4F, IL2/aOX40-Fc heterodimer form, aOX40 in Fab form, with 1 molecule of IL2 attached to the N-or C-terminus of the light chain, the N-terminus of the heavy chain, or the C-terminus of the Fc chain.
FIG. 5, IL2/aOX40-Fc antibody has synergistic therapeutic effect, 5A, in vitro CTLL2 proliferation assay; tumor suppression effect of 5B, IL2/aOX40wt Fc fusion protein (MC38 tumor model); 5C, re-challenge test results in IL2/aOX40-Fc treatment group (MC38 tumor model); tumor-inhibiting effect of 5D, IL2/aOX40wt Fc fusion protein (B16F10 tumor model).
FIG. 6, IL2/aOX40-Fc antibody therapeutic effect is independent of NK cells; 6A, NK one day after injection of the cell-deleted antibody, the proportion of NK cells in peripheral blood of the mice was analyzed by flow analysis; 6B, growth curve of MC38 tumor in different treatment groups.
FIG. 7, IL2/aOX40wt dependent on CD8T cell function for Fc function; 7A, after injecting 200ug of the deletion antibody for one day, detecting the deletion efficiency of CD 4T cells and CD8T cells in peripheral blood in a flow mode; 7B, tumor growth curves of different treatment groups.
FIG. 8, intratumoral T cells play a key role in the treatment of IL2/aOX 40-Fc; 8A, MC38 tumor-bearing mice were injected intraperitoneally with 20ug FTY720, and 2 days later, peripheral blood CD 3T cells were detected; and 8B, tumor growth curves of different treatment groups.
FIG. 9, local tumor treatment in situ induced immune response can control distal tumor growth; 9A, tumor treatment effect of the right side (administration side) of different treatment groups; 9B, tumor treatment effect on the left side (non-treatment side) of different treatment groups.
Figure 10, CD25 and OX40 were highly expressed on intratumoral Treg cells; 10A, flow analysis of CD4, CD8 and CD25 and OX40 expression on Treg cells in tumors, and comparison of CD25 and OX40 expression on Treg cells in tumors, draining lymph nodes and spleen; 10B, MFI statistical analysis of different cell expression levels.
Figure 11, IL2/aOX40-Fc treatment deleting intratumoral tregs increases the ratio of CD8T cells to tregs; 11A, flow charts of intratumoral Treg cells of different treatment groups; 11B, intratumoral Treg/CD 4T and CD8T/Treg ratios in different treatment groups; 11C, draining lymph nodes and spleen Treg/CD 4T ratio.
Figure 12, the performance of antibody function depends in part on Fc binding to Fc γ R.
FIG. 13, aPDL1 antibody was able to increase antitumor effect in cooperation with IL2/aOX 40-Fc.
FIG. 14, IL2/aOX40-Fc was able to overcome the resistance of Afatinib treatment, with more T cells infiltrating into the tumor during 14A, 14B, Afatinib treatment, but the proportion of Tregs was not altered; the 14C, afatinib and IL2/aOX40-Fc antibody combined treatment can effectively control the growth and recurrence of tumors.
FIG. 15, IL2/aCTLA4 wt Fc fusion protein enhanced tumor suppression effect (MC38 tumor model).
FIG. 16, IL2/a4-1BB-wt Fc bispecific antibody significantly improved tumor treatment effect.
Detailed Description
Experimental materials
1. Bacterial species and plasmids
Strain: top10 E.coli, DH 5. alpha. E.coli competent cell (Beijing Quanyujin Biotechnology Co., Ltd.)
Plasmid:
pEE12.4-IgG kappa-hIgG 1, containing a signal peptide of mouse IgG kappa and an Fc sequence of human IgG1, was used to express antibodies.
pEE12.4-IgG kappa-hIgG 1-Fc-hole and pEE12.4-IgG kappa-hIgG 1-Fc-knob were used to express heterodimeric proteins.
001-OX40 VH-CH1-Fc-knob, 002-OX40 VL-CL for expression of the antibody portion of the heterodimeric protein; pEE12.4-Wt IL-2-Fc-hole, the IL-2 moiety used to express heterodimeric proteins.
2. Laboratory animal
Wild type C57BL/6, BALB/C mice and BALB/C-nude mice were purchased from laboratory animals of Beijing Wintolite, China. Except for special instructions, 8-10 week old female mice were used for all experiments. Mice were all raised in a barrier environment free of specific pathogenic microorganisms (SPF). Animal feeding and experimental procedures were in compliance with the regulations of the animal care committee of the institute of biophysics, academy of sciences, china.
3. Cell lines
MC38 is C57 background mouse colorectal cancer cell line,
MC38-EGFR5 is a monoclonal cell line obtained by infecting lentivirus expressing human and mouse chimeric Epidermal Growth Factor (EGFR) with MC38, respectively, and screening,
TUBO is a BALB/C background mouse breast cancer cell line, B16F10 is a C57 background mouse melanoma cell line.
These cell lines were all cultured in DMEM complete medium (containing 10% inactivated fetal calf serum, 2mmol/l L-glutamine, 0.1mmol/l non-essential amino acids, 100U penicillin and 100. mu.g/ml streptomycin).
TIB-210TM hybridoma cell line (ATCC) for expressing the deletion antibody for CD8+ T cells (clone: 2.43).
TIB-207TM hybridoma cell line (ATCC) for expressing the deletion antibody for CD4+ T cells (clone: GK 1.5).
The PK136 hybridoma cell line was used to produce deletion antibodies for NK cells.
HB-197TM hybridoma cell line (ATCC), for the expression of antibodies blocking Fc γ RII/III in mice (clone:2.4G 2).
FreeStyleTM 293F cell line (Invitrogen) is a suspension cell derived from the HEK293 cell line cultured in SMM293-TII or CD OptiCHOTM medium, primarily for transient transfection expression of fusion proteins.
CTLL-2 cell line is mouse T cell line and is used for detecting IL-2 bioactivity
The above cell lines were cultured in RPMI1640 complete medium (containing 10% inactivated fetal calf serum, 2mmol/L L-glutamine, 0.1mmol/L non-essential amino acids, 100U penicillin and 100. mu.g/ml streptomycin, 100IU/ml recombinant IL 2).
Design and Synthesis of Gene and primer
The human wild type and mutant IL-2 gene sequences are shown in SEQ ID NO. 1. The primers used in the experiments were all designed by DNAMAN software and synthesized by Invitrogen.
Tumor inoculation and treatment in mice
(1) Tumor inoculation and measurement:
the establishment of a tumor model is carried out,
5-7.5×105individual MC38, MC38-EGFR5 single cells were suspended in 100 μ l PBS and inoculated subcutaneously on the back of C57BL/6 mice;
5-7.5×105a single TUBO cell was suspended in 100. mu.l PBS and inoculated subcutaneously in the dorsal side of BALB/c mice.
When the re-challenge experiment of the same tumor cell is carried out on mice with tumor regression, the inoculation quantity of the tumor cell is 5 times of that of the initial tumor model, and the inoculation part is subcutaneous on the other side of the back of the mice. Tumor size was monitored twice weekly and tumor length (a), length (b) and height (c) were measured using a vernier caliper, mouse tumor volume ═ a × b × c/2.
(2) Treatment:
the antibody or antibody fusion protein is injected intraperitoneally, and the intratumoral administration is also adopted in part of experiments, and the specific administration dosage is described in the specific experiments.
Monoclonal antibody preparation (mouse ascites method)
The CD4+ T cell deletion antibody GK1.5, the CD8+ T cell deletion antibody TIB210, the NK cell deletion antibody PK136 and the FcRII/III blocking antibody used in the experiment are all from corresponding hybridoma cells, and are produced and purified by the laboratory.
In vivo cell deletion in mice
(1) Deletion of CD4+ T cells, CD8+ T cells:
before IL-2 or IL-2 fusion protein treatment, 200 mu g of GK1.5 or TIB210 antibody is intraperitoneally injected one day respectively to delete CD4+ T cells and CD8+ T cells, and then the injection is injected once every 3 days, and the injection frequency is adjusted according to the treatment period. The deletion efficiency is detected by streaming.
(2) Deletion of NK cells and neutrophils:
NK cells or neutrophils were deleted by intraperitoneal injection of 400 μ g of PK136 or 1A8 antibody, respectively, one day prior to IL-2 or IL-2 fusion protein treatment. And detecting deletion efficiency by streaming.
Blocking of T cell emigration
FTY720 (available from Sigma) is an immunosuppressant that reduces T cell migration from lymphoid organs into peripheral blood circulation. In the present invention, FTY720 blockade was performed at different stages of tumor inoculation in mice to alter the tumor microenvironment. Blocking during tumor treatment in mice: 20 mu g of FTY720 is intraperitoneally injected 1 day before the tumor treatment, 10 mu g of FTY is intraperitoneally injected every other day, and the blocking time is determined according to the treatment period, so that no T cells newly transplanted into the tumor tissue exist in the process of the tumor treatment. With the FTY720 blocking protocol, the importance of infiltrating lymphocytes in tumor tissue can be studied.
Unless otherwise specified, the molecular structures of IL2/aOX40-Fc bispecific antibodies in examples 1-5 below are all the molecular structures shown in FIG. 4A, i.e., a heterodimer consisting of one molecule of IL2-Fc and one molecule of aOX40(Fab) -Fc.
Example 1 IL2/aOX40-Fc bispecific antibody has a significantly improved effect over monotherapy
1. Treatment with IL2-Fc fusion proteins can in part control tumors
Free IL2 alone has a short half-life and requires multiple administrations, and to prolong the half-life of IL2 we designed IL2-Fc fusion proteins (see figure 1A for a schematic structural diagram) and validated the function of the fusion proteins in the MC38 tumor model. IL2-Fc was able to effectively control tumor growth compared to untreated controls, but treatment with IL2-Fc alone failed to completely eliminate tumors when they were larger (FIG. 1B). C57BL/6 mice were inoculated subcutaneously with 5X 105MC38 tumor cells, tumor-bearing mice started on day D12 with group treatment (n-5/group): intraperitoneal injection (ip) of 9ug of IL2-Fc fusion protein was performed once every three days for three treatments.
The results show that treatment with IL2-Fc fusion protein alone does not consistently inhibit tumor growth.
2. anti-OX40 antibody promotes T cell activation and exerts antitumor function
Treatment with IL2-Fc fusion protein alone can only partially control tumor growth, and to further enhance the efficacy of the treatment we adopted a combination therapy strategy. OX40 molecule is expressed only on Treg cells and activated T cells and on
Figure BDA0002752274300000101
(ii) is not expressed on T cells, such that antibodies targeting OX40 have higher specificity; and the expression abundance of OX40 on the Treg cells and the effector cells is different, thereby providing a theoretical basis for different effects of the same target point on different cells. To verify whether the OX40 signaling pathway could activate T cells and exert an anti-tumor effect, we performed the spleen
Figure BDA0002752274300000102
T cell in vitro stimulation experiments. The specific method comprises the following steps: spleen cells from mice were isolated and plated at 3X 105Cells/well were plated in 96-well plates and CD69 expression levels on CD 3T cells were flow analyzed after 72h stimulation with either aCD3 and aCD28 or aCD3 and aOX40 antibodies, respectively, at 1ug/ml for aCD3, aCD28 and 2ug/ml for aOX 40.
The results show that, in comparison to the co-stimulatory effects of aCD3 and aCD28 on T cells, the combined use of aOX40 antibody and aCD3 exerted a stimulatory effect similar to that of aCD28, enabling efficient activation of CD 3T cells (fig. 2A).
We further verified the function of anti-OX40 in the anti-tumor experiment of mice, and the specific method is as follows: wild type C57BL/6 mice were inoculated subcutaneously with 5X 105MC38 tumor cells, the tumor growth to 100-150mm3Start group therapy (n-5/group): intraperitoneal (ip)16ug of aOX40 antibody was administered as a three day treatment three times.
The results show that intraperitoneal injection of (ip)16ug aOX40 antibody was effective in controlling tumor growth compared to the untreated group (fig. 2B).
3. The combined treatment of IL2-Fc fusion protein and aOX40 does not obviously improve the treatment effect of tumors
While IL2-Fc alone expanded tregs, antibody aOX40 was able to delete intratumoral Treg cells, in order to verify whether the combined use of IL2-Fc and aOX40 antibodies could simultaneously delete expanded tregs while activating effector T cells, exerting a synergistic therapeutic effect. We administered two protein treatments simultaneously on MC38 tumor-bearing mice by: c57BL6 mice were inoculated subcutaneously with 5X 105MC38 tumor cell, tumor growth to D14 startTreatment, i.e. the single treatment group was intraperitoneally injected with (ip)6ug IL2-Fc or 9ug aOX40 antibody, respectively, and the combined treatment group was intraperitoneally injected with both antibodies at the same time; three treatments were given every three days.
The results show that the combination treatment did not further improve the therapeutic effect of IL2-Fc compared to either the aOX40 antibody alone or IL2-Fc treatment (figure 3).
4. IL2/aOX40-Fc bispecific antibodies have synergistic therapeutic effects
4.1 construction of bispecific antibodies
The analysis of the experimental results shows that the simple mixed combination therapy does not have obvious synergistic treatment effect or even has no additive effect, and the suggestion that the two antibodies can not simultaneously exert the effects of activating effector cells and deleting tregs by singly combining. Accordingly, in order to reduce IL2 binding and expand Treg cells, we constructed two structurally identical bispecific antibodies (IL2/aOX40WT Fc and IL2/aOX40 no ADCC Fc) each containing only one IL2 molecule (see fig. 4A for the fusion protein structure), in which the Fc dimer is that of human IgG or one that eliminates ADCC effector function, one Fc monomer is linked to WT-type IL2 protein and the other Fc monomer is linked to the Fab fragment of aOX40 antibody. The constructed fusion protein plasmid is transfected into 293F cells, and cell supernatant is collected after seven days. After centrifugation of the supernatant, the protein was purified using protein A column and the purified antibody was characterized by protein gel. Analysis of the protein molecular composition by SDS-PAGE demonstrated that the bispecific molecule was able to express well the light, heavy and IL2 molecules of aOX40 antibody simultaneously (FIG. 4B).
We have simultaneously designed and constructed antibodies comprising different forms of IL2 and aOX 40:
FIG. 4C schematically shows a heterodimer, an antibody comprising one IL2 molecule, aOX40 (anti-OX 40 antibody) in Fab or scFv form, and an Fc dimer formed from an Fc monomer of a human IgG and an Fc monomer of a human IgG that eliminates the ADCC effector function.
FIG. 4D schematically shows a structure wherein aOX40 (anti-OX 40 antibody) is in scFv form, one or two IL2 molecules are attached to the N-terminus of the V-region or C-terminus of the Fc region, and the Fc dimer is a Fc homodimer of a wild-type human IgG or a heterodimer formed by an Fc monomer of a human IgG and an Fc monomer of a human IgG that eliminates the ADCC effector function;
FIGS. 4E-F schematically show structures wherein aOX40 (anti-OX 40 antibody) is in the Fab form, with one or two IL2 molecules attached to the N-or C-terminus of the antibody light chain, to the N-or C-terminus of the antibody heavy chain, and with the Fc dimer being a Fc homodimer of a wild-type human IgG, or a heterodimer of a Fc monomer of a human IgG and a Fc monomer of a human IgG that eliminates the ADCC effector function.
4.2, verifying the function of the bispecific antibody,
(1) in vitro CTLL2 proliferation assay:
by adding different concentrations of IL2-Fc, IL2/aOX40wt Fc and IL2/aOX40 no ADCC Fc protein (the structure shown in FIG. 4A) into the CTLL2 cell culture medium, the proliferation level of the CTLL2 cells at different protein concentrations is detected by a CCK8 kit after 72h, and the result shows that an IL2/aOX40-Fc antibody can effectively amplify CTLL2 cells as well as an IL2-Fc molecule, which indicates that the constructed antibody molecule retains the IL2 activity (FIG. 5A).
(2) In vivo mouse tumor-bearing experiment:
c57BL6 mice were inoculated subcutaneously with 5X 105MC38 tumor cells, treatment started at D14 after tumor inoculation; 9ug IL2-Fc, 16ug aOX40 antibody, an equal molar number of IL2-Fc + aOX40 antibody to aOX40 antibody or IL2/aOX40wt Fc protein were injected intraperitoneally, respectively. Tumor size was measured twice weekly for three total treatments on days D14, D17, D20; the results show that the IL2/aOX40-wtFc bispecific antibody has a better therapeutic effect than the aOX40 antibody alone, IL2-Fc and the combination therapy of both (FIG. 5B).
In addition to the MC38 model, the same experiment was performed on a B16F10 tumor-bearing mouse model, and C57BL6 mice were inoculated subcutaneously with 5X 10 cells5B16F10 tumor cells, treatment started at D8 after tumor inoculation; by intraperitoneal injection of 15ug IL2/aOX40wt Fc protein; three total treatments at days D8, D11 and D14 showed that IL2/aOX40wt Fc also exerted significant tumor control (fig. 5D).
Further, in the IL2/aOX40-Fc treated group, tumor re-challenge experiments were performed two months after tumor clearance. In the IL2/aOX40wt Fc group, treatmentTwo months after treatment of mice with completely eliminated tumors, tumor re-challenge experiments were performed, and five times the dose (2.5X 10)6One) MC38 tumor cells, re-challenge only inoculated tumor cells, and no treatment was given to each group after inoculation.
The results showed that re-inoculation of five-fold doses of MC38 cells did not induce tumor growth compared to the control group, indicating that bispecific antibody treatment induced memory immune cell production in mice (fig. 5C).
Example 2 IL2/aOX40-Fc was able to activate CD8T cells
1. IL2/aOX40-Fc bispecific antibody therapeutic effect is independent of NK cells
Since CD25(IL-2 receptor alpha) and OX40 are predominantly expressed on activated effector T cells and NK cells, we performed deletion experiments for different cell populations separately in order to determine which population of immune cells the treatment of IL2/aOX40-Fc antibodies was predominantly dependent on.
C57BL6 mice were inoculated subcutaneously with 5X 105MC38 tumor cells were injected intraperitoneally on days 12, 15, and 18 with 25ug IL2/aOX40wt Fc fusion protein. 400ug of NK cell-depleting antibody PK136 (prepared in the laboratory) was intraperitoneally injected one day before treatment, once every 4 days for a total of 3 injections.
FIG. 6A shows the change in peripheral blood NK cells 1 day after deletion of NK cells. In mouse tumor experiments, IL2/aOX40-Fc antibody still had therapeutic effect after deletion of NK cells, suggesting that NK cells are not the main effector cells for therapeutic effect of the antibody (FIG. 6B).
2. The therapeutic effect of IL2/aOX40-Fc antibodies was dependent on CD8T cells
We further validated the role played by T cells in the course of antibody therapy.
C57 mice were inoculated subcutaneously on the back at 5X 105Treatment was initiated on day 12 after MC38 tumor cells and 25ug of IL2/aOX40wt Fc protein was intraperitoneally injected (administered on days 12, 15, and 18, respectively, after tumor inoculation). 200ug of CD 4T cell-deleted antibody (clone No.: GK1.5, prepared in this laboratory), 200ug of CD8T cell-deleted antibody (clone No.: TIB210, prepared in this laboratory) or two kinds of antibodies were injected intraperitoneally at the same time of treatmentDeleted antibodies were injected three times in total.
Deletion experiments of CD 4T and CD8T cells show that the treatment effect of the antibody is not obviously reduced after the CD 4T cells are deleted, but the treatment effect is obviously reduced after the CD8T cells are deleted; the therapeutic effect of the antibody was also completely lost after the deletion of both CD 4T and CD8T cells. Indicating that the therapeutic effect of IL2/aOX40-Fc antibodies was T cell dependent and was predominantly CD8T cells (FIG. 7B).
3. The function of IL2/aOX40-Fc bispecific antibodies is dependent on intratumoral T cells
To further examine whether intratumoral infiltrating T cells play a major role or peripherally migrating T cells play a major role during treatment, we performed FTY720 blocking experiments. FTY720 is a drug that can clinically inhibit immune rejection in the process of organ transplantation, and inhibits migration of T cells from lymphoid organs to peripheral blood [19 ].
Treatment with IL2/aOX40wt Fc antibody alone can eliminate tumors, and FTY720 blockade is performed simultaneously with treatment, according to the following experimental protocol:
c57BL6 mice were inoculated subcutaneously on the back at 4X 105MC38 tumor cells were injected intraperitoneally with 15ug of IL2/aOX40-Fc antibody on days 7, 10, and 13 post tumor inoculation. In the FTY720 treatment group, 25ug of FTY720 was intraperitoneally injected on day 6 post tumor inoculation, and continued to be injected with 10ug on days 8, 10, and 12, respectively.
The results showed that inhibition of peripheral lymphocyte migration to the tumor did not affect the effect of bispecific antibody therapy (FIG. 8B), indicating that IL2/aOX40-Fc antibody exerts anti-tumor effects mainly depending on effector T cells infiltrating into the tumor.
4. Protective effects of IL2/aOX40-Fc treatment on distant metastatic tumors
To verify whether immunoprotection from in situ tumor therapy also inhibits tumors at other metastatic sites, we performed a mouse dual tumor model experiment. Tumor cells are respectively inoculated on the left side and the right side of the subcutaneous part of a mouse at the same time, and in-situ treatment is carried out on only one side of the mouse, so that whether the tumor at the far end can be controlled or not is observed. The experimental protocol was as follows:
C57Bl6 mice were inoculated with 2X 10 cells on the left and right sides of the back, respectively5MC38 and 4X 105MC38 tumor cells (4X 10 inoculation on the right side of the back)5MC38 tumor cells, inoculated 2X 10 cells on the left5MC38 tumor cells). Tumors on the right side of day seven grew to 50mm3Left and right, left tumors 30mm3Starting treatment from left to right:
treatment group 1, right tumors were intratumorally injected with 7.5ug IL2/aOX40wt Fc antibody (treatment antibody was treated three times on days 7, 10, 13) and left tumors were untreated;
treatment group 2, intratumorally injected with 7.5ug IL2/aOX40wt Fc antibody in the right tumor, while intratumorally injected with 20ug TIB210 antibody in the right tumor, deleted intratumoral CD8T cells. Treatment and deletion antibodies were treated three times on days 7, 10, and 13, with left tumors untreated.
The results are shown in figure 9 of the drawings,
(1) the in situ treatment of intratumoral injection of only the right tumor can not only eliminate the in situ tumor but also control the contralateral distal untreated tumor.
(2) Also to verify whether control of distal tumors was dependent on CD8T cells of the tumor in situ on the treated side, we deleted CD8T cells of the tumor in situ while treatment. After deletion of CD8T cells, both in situ and distant tumor treatment effects disappeared, suggesting that CD8T cells within the in situ tumor play an important role in controlling distant tumors.
Example 3 IL2/aOX40-Fc was able to delete intratumoral Tregs and increase the CD8/Treg cell ratio
1. Intratumoral Treg cells highly express CD25 and OX40 molecules
To further investigate the anti-tumor mechanism of IL2/aOX40-Fc antibodies, we analyzed the expression levels of CD25 and OX40 molecules on T cells of tumor-bearing mice to analyze to which population of cells IL2/aOX40-Fc antibodies were primarily functional. After inoculation of MC38 tumors, we analyzed the expression of IL2 receptors CD25 and OX40 in mice tumors, on draining lymph nodes and spleen T cells, respectively, at day 12: the specific scheme is as follows: mice were inoculated with 5X 105MC38 tumor cells, tumor tissue, spleen and drainage of tumor-bearing mice were taken and drained on day 12Lymph node (dLN), flow-assayed for CD25 and OX40 expression on different cells.
Both CD25 and OX40 were found to be highly expressed on Treg cells in tumor-bearing mice, and intratumoral tregs expressed higher levels of CD25 and OX40 than peripheral draining lymph node and spleen tregs. Non-tregs expressed CD 4T and CD8T at lower levels than Treg cells, and intratumoral CD8T cells had the lowest expression levels of CD25 and OX40 (fig. 10A, B).
2. IL2/aOX40-Fc antibody deletes intratumoral Treg cells and improves the proportion of CD8/Treg cells
Since CD25 and OX40 are highly expressed on intratumoral Treg cells, we speculate that IL2/aOX40-Fc antibodies may bind more to tregs following intraperitoneal injection. Treatment was initiated in MC38 tumor-bearing mice on day 12 after tumor inoculation, with intraperitoneal injection of 25ug IL2/aOX40-Fc antibody, tumor tissue, draining lymph nodes and spleen taken on day 15, and flow analysis of intratumoral Treg cell proportion, CD 8T/Treg. Through flow experimental analysis, we found that IL2/aOX40-Fc was able to delete intratumoral Treg cells more efficiently and increased the CD8T/Treg ratio with the best therapeutic effect compared to treatment with IL2-Fc and aOX40 alone (fig. 11A, B). Compared to the deletion of intratumoral tregs, IL2/aOX40-Fc did not alter the Treg/CD 4T ratio in peripheral tumor draining lymph nodes and spleen, suggesting that IL2/aOX40-Fc treatment did not break peripheral immune tolerance with greater safety (fig. 11C).
3. The therapeutic efficacy of IL2/aOX40-Fc antibodies is dependent on the binding of Fc to FcyR
Through the analysis of the lymphocytes in the MC38 tumor-bearing mice, the IL2/aOX40-Fc antibody has the effect of deleting Tregs, and in order to verify whether the deletion of Tregs is the main mechanism of the anti-tumor effect of the IL2/aOX40-Fc antibody, the IL2/aOX40-no ADCC Fc antibody is constructed through point mutation on the Fc region, and the mutated Fc does not bind with the Fc gamma R receptor and loses ADCC and ADCP mediated deletion functions. Using the MC38 tumor model, C57BL6 mice were inoculated with MC38 tumor cells and treatment was started on day 13 post-inoculation. Intraperitoneal injection of 15ug wt Fc or no ADCC Fc antibody was performed once every three days for three treatments.
The results show that, after tumor inoculation, comparing the therapeutic effect of wt Fc with no ADCC Fc, mutated Fc was found to only partially reduce the therapeutic effect of IL2/aOX40 antibody (figure 12). In the IL2/aOX40-no ADCC Fc antibody treatment group, the antibody still retains partial antitumor effect, and the result shows that the IL2/aOX40-Fc antibody can play a role not only in deleting Treg cells but also in playing a role in treatment through binding with effector T cells.
Example 4 IL2/aOX40-Fc in combination with PDL1 antibody treatment
1. aPDL1 capable of potentiating antitumor effect in cooperation with IL2/aOX40-Fc antibody
The tumor of the MC38 tumor-bearing mouse is 200mm3The IL2/aOX40-Fc antibody alone treatment had a good clearance effect as follows. However, when tumors are larger, antibody therapy alone can only control tumor growth and not completely eliminate it; to better enhance the therapeutic effect, we combined the bispecific antibody and the immune checkpoint inhibitory antibody to see if the therapeutic effect could be enhanced.
The specific test scheme is as follows:
the first scheme is as follows: c57BL6 mice were inoculated subcutaneously with MC38 cells and treatment was started on day 16,
(1) the IL2/aOX40-Fc treatment group alone was injected intraperitoneally on days 16, 19, and 22 with 25ug of IL2/aOX 40-Fc;
(2) aOX40 and aCTLA4 combination group on day 16, i.p. 250ug of antibody two days later, 25ug of IL2/aOX40-Fc (aOX40 and aCTLA4 antibodies were administered only once, 25ug of IL2/aOX40-Fc was administered three times i.p);
(3) the aPDL1 combination treatment group was injected with 25ug IL2/aOX40-Fc along with 250ug aPDL1 antibody (16, 19, 22 days).
Scheme II: MC38 tumor-bearing mice were intraperitoneally injected with 200ug aPDL1 antibody on days 12, 15 and 18, and IL2/aOX40 antibody with 15ug wt Fc or no ADCC Fc on days 14, 17 and 20.
The results show that:
(1) during the combination therapy of IL2/aOX40-Fc with aCTLA4 or aOX40, it was found that the combination of these two classes of antibodies did not further improve the therapeutic effect of IL2/aOX40-Fc (FIG. 13A).
(2) The combination of the two was found to have a synergistic effect during the combined treatment with aPDL1 (FIG. 13A).
(3) To further analyze the mechanism of action of aPDL1 and IL2/aOX40-Fc antibodies, we used a combination of IL2/aOX40-Fc antibody and aPDL1, with either wt Fc or no ADCC Fc, respectively, and found that better combination therapy was dependent on wt Fc (FIG. 13B).
Example 5 IL2/aOX40-Fc bifunctional antibody antagonizes tumor recurrence following TKI treatment
Clinical treatment regimens for patients with EGFR-mutated tumors have primarily used antibodies targeting the receptor or kinase inhibitors directed to downstream signaling pathways. The TKI has a good treatment effect on ErBB gene family-related tumors, and Afatinib (Afatinib) is a second-generation kinase inhibitor and irreversibly inhibits the tyrosine kinase activity of a receptor in a covalent binding manner. The mechanism of action of afatinib was analyzed on the tobo model and the increase in the proportion of intratumoral CD 3T/CD 45 lymphocytes was found after treatment, indicating that more T cells infiltrated the tumor during the course of treatment with the chemotherapeutic drugs (fig. 14A). Treatment with Afatinib alone, while increasing T cell infiltration into the tumor, was not effective in reducing the proportion of intratumoral Treg cells (fig. 14B). Clinically, the tumor is difficult to completely remove by independent TKI treatment, and the Treg cells in the tumor are supposed to inhibit the killing of effector cells to the tumor, so that the treated tumor recurs.
To verify whether IL2/aOX40-Fc could antagonize recurrence following chemotherapy treatment, we combined IL2/aOX40-Fc antibody with chemotherapy treatment in tumor-bearing mice.
The specific experimental scheme is as follows:
BALB/c mice were inoculated subcutaneously with 5X 105(ii) a TUBO tumor cell,
treatment protocol 1: 1mg of Afatinib was given for intragastric administration on day 9 for treatment, and tumor tissue was taken 6 days later for flow analysis.
Treatment protocol 2: tumor-bearing mice began treatment on day 14 post tumor inoculation,
(1) the Afatinib treatment group was gavaged with 1mg of Afatinib on day 14,
(2) IL2/aOX40-Fc treatment group was intraperitoneally injected with 25ug of antibody on days 14, 17 and 20,
(3) the combination treatment group was administered with Afatinib once and three treatments with 25ug IL2/aOX40-Fc intraperitoneal antibody according to the above protocol.
The results show that in the afatinib-treated group alone in treatment regimen 1, tumors recurred soon after being partially controlled; in the treatment scheme 2, the combination treatment group with IL2/aOX40-Fc antibody was effective in controlling tumor growth, and some mice were completely cleared of tumors (FIG. 14C).
Example 6 IL2/aCTLA4-Fc bispecific antibody significantly improved tumor treatment effect
In vivo mouse tumor-bearing experiment: c57BL6 mice were inoculated subcutaneously with 5X 105MC38 tumor cells, treatment was initiated D11 after tumor inoculation; 15ug IL2/aCTLA4 wt Fc protein was intraperitoneally injected (the detailed structure of IL2/aCTLA4 wt Fc protein is shown in FIG. 15). Tumor size was measured twice weekly for three total treatments on days D11, D14, D17;
the results showed that IL2/aCTLA4-Fc bispecific antibody was effective in controlling tumors (FIG. 15).
Example 7 IL2/a4-1BB-Fc bispecific antibody significantly improves tumor treatment effects
C57BL/6 mice were inoculated subcutaneously with 5X 105MC38 tumor cells, treatment started at D10 after tumor inoculation; 10ug3H3-IL2 and 10ug LOB12.3-IL2 wt Fc fusion proteins were injected intraperitoneally (the schematic structure of IL2/a4-1BB-Fc fusion protein is shown in FIG. 16). Three treatments on days 10, 16 and 19 after tumor inoculation, tumor size was measured twice weekly; the results show that compared with the control group, the tumor volume of the treatment group of 2 fusion proteins is obviously reduced, and the difference is extremely obvious.
The experiment proves that the IL2/a4-1BB-Fc fusion protein has good tumor treatment effect (FIG. 16).
Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.
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SEQUENCE LISTING
<110> institute of biophysics of Chinese academy of sciences
<120> bifunctional fusion protein consisting of IL-2 and antibody subunit
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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
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Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
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Ile Ser Thr Leu Thr
130
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Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
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Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
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Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
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His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
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Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
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Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
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Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
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Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
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Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
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His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
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Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
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Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
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Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
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Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
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Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
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His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
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Pro Gly Lys
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Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
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Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
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His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
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Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
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Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
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His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
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Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
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Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
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Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
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Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
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Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val
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Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
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His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
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Pro Gly Lys
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Asp Ile Val Met Thr Gln Ala Ala Leu Pro Asn Pro Val Pro Ser Gly
1 5 10 15
Glu Ser Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val Tyr Lys
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Asp Gly Gln Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser
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Pro Gln Leu Leu Thr Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Arg Ala Glu Asp Ala Gly Val Tyr Tyr Cys Gln Gln Val
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Arg Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
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Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
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Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205
Thr Lys Ser Phe Asn Arg Gly Glu Cys
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35 40 45
Gly Arg Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Thr
85 90 95
Arg Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
<210> 9
<211> 244
<212> PRT
<213> Artificial sequence
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Asp Ile Val Met Thr Gln Ala Ala Leu Pro Asn Pro Val Pro Ser Gly
1 5 10 15
Glu Ser Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val Tyr Lys
20 25 30
Asp Gly Gln Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Thr Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Arg Ala Glu Asp Ala Gly Val Tyr Tyr Cys Gln Gln Val
85 90 95
Arg Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Thr
130 135 140
Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Asn
145 150 155 160
Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met Gly
165 170 175
Arg Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser Val Leu Lys Ser
180 185 190
Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu Lys
195 200 205
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Thr Arg
210 215 220
Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Val Met Val
225 230 235 240
Thr Val Ser Ser
<210> 10
<211> 375
<212> PRT
<213> Artificial sequence
<400> 10
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
145 150 155 160
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
165 170 175
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
180 185 190
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
195 200 205
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
210 215 220
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
225 230 235 240
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
245 250 255
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
260 265 270
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
275 280 285
Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
290 295 300
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
305 310 315 320
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Val Ser
325 330 335
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
340 345 350
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
355 360 365
Leu Ser Leu Ser Pro Gly Lys
370 375
<210> 11
<211> 447
<212> PRT
<213> Artificial sequence
<400> 11
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Asn Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Thr
85 90 95
Arg Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Val Met
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 12
<211> 471
<212> PRT
<213> Artificial sequence
<400> 12
Asp Ile Val Met Thr Gln Ala Ala Leu Pro Asn Pro Val Pro Ser Gly
1 5 10 15
Glu Ser Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val Tyr Lys
20 25 30
Asp Gly Gln Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Thr Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Arg Ala Glu Asp Ala Gly Val Tyr Tyr Cys Gln Gln Val
85 90 95
Arg Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Thr
130 135 140
Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Asn
145 150 155 160
Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met Gly
165 170 175
Arg Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser Val Leu Lys Ser
180 185 190
Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu Lys
195 200 205
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Thr Arg
210 215 220
Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Val Met Val
225 230 235 240
Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
340 345 350
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys
370 375 380
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 13
<211> 619
<212> PRT
<213> Artificial sequence
<400> 13
Asp Ile Val Met Thr Gln Ala Ala Leu Pro Asn Pro Val Pro Ser Gly
1 5 10 15
Glu Ser Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val Tyr Lys
20 25 30
Asp Gly Gln Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Thr Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Arg Ala Glu Asp Ala Gly Val Tyr Tyr Cys Gln Gln Val
85 90 95
Arg Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Thr
130 135 140
Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Asn
145 150 155 160
Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met Gly
165 170 175
Arg Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser Val Leu Lys Ser
180 185 190
Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu Lys
195 200 205
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Thr Arg
210 215 220
Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Val Met Val
225 230 235 240
Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
340 345 350
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
465 470 475 480
Ser Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr
485 490 495
Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn
500 505 510
Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe
515 520 525
Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys
530 535 540
Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln
545 550 555 560
Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn
565 570 575
Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
580 585 590
Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile
595 600 605
Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr
610 615
<210> 14
<211> 619
<212> PRT
<213> Artificial sequence
<400> 14
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ala Ala Leu Pro Asn Pro
145 150 155 160
Val Pro Ser Gly Glu Ser Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser
165 170 175
Leu Val Tyr Lys Asp Gly Gln Thr Tyr Leu Asn Trp Phe Leu Gln Arg
180 185 190
Pro Gly Gln Ser Pro Gln Leu Leu Thr Tyr Trp Met Ser Thr Arg Ala
195 200 205
Ser Gly Val Ser Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe
210 215 220
Thr Leu Lys Ile Ser Arg Val Arg Ala Glu Asp Ala Gly Val Tyr Tyr
225 230 235 240
Cys Gln Gln Val Arg Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys
245 250 255
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
260 265 270
Gly Gly Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln
275 280 285
Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
290 295 300
Thr Gly Tyr Asn Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
305 310 315 320
Glu Trp Met Gly Arg Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser
325 330 335
Val Leu Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln
340 345 350
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
355 360 365
Tyr Cys Thr Arg Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln
370 375 380
Gly Val Met Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro
385 390 395 400
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
405 410 415
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
420 425 430
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
435 440 445
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
450 455 460
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
465 470 475 480
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
485 490 495
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
500 505 510
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
515 520 525
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
530 535 540
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
545 550 555 560
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
565 570 575
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
580 585 590
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
595 600 605
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615
<210> 15
<211> 1185
<212> DNA
<213> Artificial sequence
<400> 15
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60
gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 120
ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 180
acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 240
gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 300
agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 360
acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 420
tggattacct tttgtcaaag catcatctca acactgactg gcggcggcgg cagcggcggc 480
ggcggcagcg gcggcggcgg cagcgacaaa actcacacat gcccaccgtg cccagctccg 540
gaactcctgg gcggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 600
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 660
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 720
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 780
tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 840
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgtg taccctgccc 900
ccatcccggg atgagctgac caagaaccag gtcagcctga gttgcgcggt caaaggcttc 960
tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1020
accacgcctc ccgtgttgga ctccgacggc tccttcaagc tcgtcagcaa gctcaccgtg 1080
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1140
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa 1185
<210> 16
<211> 651
<212> DNA
<213> Artificial sequence
<400> 16
gatattgtga tgacgcaggc tgcactcccc aatcctgtcc cttctggaga gtcagcttcc 60
atcacctgca ggtctagtca gagtctggta tacaaagacg gccagacata cttgaattgg 120
tttctgcaga ggccaggaca gtctcctcag cttctgacct attggatgtc tacccgtgca 180
tcaggagtct cagacaggtt cagtggcagt gggtcaggaa catatttcac actgaaaatc 240
agtagagtga gggctgagga tgcgggtgtg tattactgtc agcaagttcg agagtatcct 300
ttcactttcg gctcagggac gaagttggaa ataaaagtgg ctgcaccatc tgtcttcatc 360
ttcccgccat ctgatgagca gttgaaatct ggtaccgcta gcgttgtgtg cctgctgaat 420
aacttttatc cacgggaggc taaggtgcag tggaaagtgg acaatgccct ccagagcgga 480
aatagccaag agtccgttac cgaacaggac tctaaagact ctacatactc cctgtcctcc 540
acactgaccc tctccaaggc cgactatgag aaacacaagg tttacgcatg cgaggtcaca 600
caccagggac tctcctctcc cgtgaccaag agcttcaacc ggggagaatg c 651
<210> 17
<211> 1341
<212> DNA
<213> Artificial sequence
<400> 17
caggtgcagc tgaaggagtc aggacctggt ctggtgcagc cctcacagac cctgtccctc 60
acctgcactg tctctgggtt ctcactaacc ggttacaatt tacactgggt tcgccagcct 120
ccaggaaagg gtctggagtg gatgggaaga atgaggtatg atggagacac atattataat 180
tcagttctca aatcccgact gagcatcagc agggacacct ccaagaacca agttttcttg 240
aaaatgaaca gtctgcaaac ggatgacaca gccatttact attgtaccag agacgggcgt 300
ggtgactcct ttgattactg gggccaagga gtcatggtca cagtctcctc cgccagcact 360
aaggggccct ctgtgtttcc actcgcccct tctagcaaaa gcacttccgg aggaactgcc 420
gctctgggct gtctggtgaa agattacttc cccgaaccag tcactgtgtc atggaactct 480
ggagcactga catctggagt tcacaccttt cctgctgtgc tgcagagttc tggactgtac 540
tccctgtcat ctgtggtcac cgtgccatct tcatctctgg ggacccagac ctacatctgt 600
aacgtgaacc acaaaccctc caacacaaaa gtcgacaaac gagtcgaacc aaaatcttgt 660
gacaaaactc acacatgccc accgtgccca gctccggaac tcctgggcgg accgtcagtc 720
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat gtcgggatga gctgaccaag 1080
aaccaggtca gcctgtggtg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gttggactcc 1200
gacggctcct tcttcctcta cagcgcgctc accgtggaca agagcaggtg gcagcagggg 1260
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320
ctctccctgt ctccgggtaa a 1341
<210> 18
<211> 1413
<212> DNA
<213> Artificial sequence
<400> 18
gatattgtga tgacgcaggc tgcactcccc aatcctgtcc cttctggaga gtcagcttcc 60
atcacctgca ggtctagtca gagtctggta tacaaagacg gccagacata cttgaattgg 120
tttctgcaga ggccaggaca gtctcctcag cttctgacct attggatgtc tacccgtgca 180
tcaggagtct cagacaggtt cagtggcagt gggtcaggaa catatttcac actgaaaatc 240
agtagagtga gggctgagga tgcgggtgtg tattactgtc agcaagttcg agagtatcct 300
ttcactttcg gctcagggac gaagttggaa ataaaaggcg gcggcggcag cggcggcggc 360
ggcagcggcg gcggcggcag ccaggtgcag ctgaaggagt caggacctgg tctggtgcag 420
ccctcacaga ccctgtccct cacctgcact gtctctgggt tctcactaac cggttacaat 480
ttacactggg ttcgccagcc tccaggaaag ggtctggagt ggatgggaag aatgaggtat 540
gatggagaca catattataa ttcagttctc aaatcccgac tgagcatcag cagggacacc 600
tccaagaacc aagttttctt gaaaatgaac agtctgcaaa cggatgacac agccatttac 660
tattgtacca gagacgggcg tggtgactcc tttgattact ggggccaagg agtcatggtc 720
acagtctcct ccgacaaaac tcacacatgc ccaccgtgcc cagctccgga actcctgggc 780
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020
aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atgtcgggat 1140
gagctgacca agaaccaggt cagcctgtgg tgcctggtca aaggcttcta tcccagcgac 1200
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260
gtgttggact ccgacggctc cttcttcctc tacagcgcgc tcaccgtgga caagagcagg 1320
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380
acgcagaaga gcctctccct gtctccgggt aaa 1413
<210> 19
<211> 1917
<212> DNA
<213> Artificial sequence
<400> 19
gatattgtga tgacgcaggc tgcactcccc aatcctgtcc cttctggaga gtcagcttcc 60
atcacctgca ggtctagtca gagtctggta tacaaagacg gccagacata cttgaattgg 120
tttctgcaga ggccaggaca gtctcctcag cttctgacct attggatgtc tacccgtgca 180
tcaggagtct cagacaggtt cagtggcagt gggtcaggaa catatttcac actgaaaatc 240
agtagagtga gggctgagga tgcgggtgtg tattactgtc agcaagttcg agagtatcct 300
ttcactttcg gctcagggac gaagttggaa ataaaaggcg gcggcggcag cggcggcggc 360
ggcagcggcg gcggcggcag ccaggtgcag ctgaaggagt caggacctgg tctggtgcag 420
ccctcacaga ccctgtccct cacctgcact gtctctgggt tctcactaac cggttacaat 480
ttacactggg ttcgccagcc tccaggaaag ggtctggagt ggatgggaag aatgaggtat 540
gatggagaca catattataa ttcagttctc aaatcccgac tgagcatcag cagggacacc 600
tccaagaacc aagttttctt gaaaatgaac agtctgcaaa cggatgacac agccatttac 660
tattgtacca gagacgggcg tggtgactcc tttgattact ggggccaagg agtcatggtc 720
acagtctcct ccgacaaaac ccacacatgc ccaccttgtc ccgcccctga actcctgggc 780
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020
aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260
gtgttggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380
acgcagaaga gcctctccct gtctccgggt aaaggcggcg gcggcagcgg cggcggcggc 1440
agcggcggcg gcggcagcat gtacaggatg caactcctgt cttgcattgc actaagtctt 1500
gcacttgtca caaacagtgc acctacttca agttctacaa agaaaacaca gctacaactg 1560
gagcatttac tgctggattt acagatgatt ttgaatggaa ttaataatta caagaatccc 1620
aaactcacca ggatgctcac atttaagttt tacatgccca agaaggccac agaactgaaa 1680
catcttcagt gtctagaaga agaactcaaa cctctggagg aagtgctaaa tttagctcaa 1740
agcaaaaact ttcacttaag acccagggac ttaatcagca atatcaacgt aatagttctg 1800
gaactaaagg gatctgaaac aacattcatg tgtgaatatg ctgatgagac agcaaccatt 1860
gtagaatttc tgaacagatg gattaccttt tgtcaaagca tcatctcaac actgact 1917
<210> 20
<211> 1917
<212> DNA
<213> Artificial sequence
<400> 20
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60
gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 120
ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 180
acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 240
gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 300
agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 360
acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 420
tggattacct tttgtcaaag catcatctca acactgactg gcggcggcgg cagcggcggc 480
ggcggcagcg gcggcggcgg cagcgatatt gtgatgacgc aggctgcact ccccaatcct 540
gtcccttctg gagagtcagc ttccatcacc tgcaggtcta gtcagagtct ggtatacaaa 600
gacggccaga catacttgaa ttggtttctg cagaggccag gacagtctcc tcagcttctg 660
acctattgga tgtctacccg tgcatcagga gtctcagaca ggttcagtgg cagtgggtca 720
ggaacatatt tcacactgaa aatcagtaga gtgagggctg aggatgcggg tgtgtattac 780
tgtcagcaag ttcgagagta tcctttcact ttcggctcag ggacgaagtt ggaaataaaa 840
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagccaggt gcagctgaag 900
gagtcaggac ctggtctggt gcagccctca cagaccctgt ccctcacctg cactgtctct 960
gggttctcac taaccggtta caatttacac tgggttcgcc agcctccagg aaagggtctg 1020
gagtggatgg gaagaatgag gtatgatgga gacacatatt ataattcagt tctcaaatcc 1080
cgactgagca tcagcaggga cacctccaag aaccaagttt tcttgaaaat gaacagtctg 1140
caaacggatg acacagccat ttactattgt accagagacg ggcgtggtga ctcctttgat 1200
tactggggcc aaggagtcat ggtcacagtc tcctccgaca aaacccacac atgcccacct 1260
tgtcccgccc ctgaactcct gggcggaccg tcagtcttcc tcttcccccc aaaacccaag 1320
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1380
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1440
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1500
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1560
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1620
tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1680
gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1740
aacaactaca agaccacgcc tcccgtgttg gactccgacg gctccttctt cctctacagc 1800
aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1860
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa 1917
<210> 21
<211> 218
<212> PRT
<213> Artificial sequence
<400> 21
Asp Val Met Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly
1 5 10 15
Glu Lys Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser
20 25 30
Asn Ala Lys Thr Asn Tyr Leu Asn Trp Tyr Met Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg His Thr Gly Val
50 55 60
Pro Asp Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Asp Glu Asp Gln Ala Phe Tyr Tyr Cys Gln Gln
85 90 95
Trp Tyr Asp Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys 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> 22
<211> 219
<212> PRT
<213> Artificial sequence
<400> 22
Gln Val Lys Leu Glu Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gly
1 5 10 15
Ser Leu Ser Leu Ser Cys Ser Val Thr Gly Thr Ser Ile Thr Ser Gly
20 25 30
Tyr Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Val Glu Trp
35 40 45
Met Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr Asn Pro Ser Ile
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Val Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Gln Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215
<210> 23
<211> 244
<212> PRT
<213> Artificial sequence
<400> 23
Asp Val Met Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly
1 5 10 15
Glu Lys Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser
20 25 30
Asn Ala Lys Thr Asn Tyr Leu Asn Trp Tyr Met Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg His Thr Gly Val
50 55 60
Pro Asp Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Asp Glu Asp Gln Ala Phe Tyr Tyr Cys Gln Gln
85 90 95
Trp Tyr Asp Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Lys Leu Glu Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gly
130 135 140
Ser Leu Ser Leu Ser Cys Ser Val Thr Gly Thr Ser Ile Thr Ser Gly
145 150 155 160
Tyr Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Val Glu Trp
165 170 175
Met Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr Asn Pro Ser Ile
180 185 190
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
195 200 205
Leu Gln Val Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
210 215 220
Ala Arg Gln Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val
225 230 235 240
Thr Val Ser Ser
<210> 24
<211> 446
<212> PRT
<213> Artificial sequence
<400> 24
Gln Val Lys Leu Glu Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gly
1 5 10 15
Ser Leu Ser Leu Ser Cys Ser Val Thr Gly Thr Ser Ile Thr Ser Gly
20 25 30
Tyr Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Val Glu Trp
35 40 45
Met Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr Asn Pro Ser Ile
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Val Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Gln Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 25
<211> 471
<212> PRT
<213> Artificial sequence
<400> 25
Asp Val Met Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly
1 5 10 15
Glu Lys Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser
20 25 30
Asn Ala Lys Thr Asn Tyr Leu Asn Trp Tyr Met Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg His Thr Gly Val
50 55 60
Pro Asp Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Asp Glu Asp Gln Ala Phe Tyr Tyr Cys Gln Gln
85 90 95
Trp Tyr Asp Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Lys Leu Glu Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gly
130 135 140
Ser Leu Ser Leu Ser Cys Ser Val Thr Gly Thr Ser Ile Thr Ser Gly
145 150 155 160
Tyr Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Val Glu Trp
165 170 175
Met Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr Asn Pro Ser Ile
180 185 190
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
195 200 205
Leu Gln Val Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
210 215 220
Ala Arg Gln Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val
225 230 235 240
Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
340 345 350
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys
370 375 380
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 26
<211> 619
<212> PRT
<213> Artificial sequence
<400> 26
Asp Val Met Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly
1 5 10 15
Glu Lys Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser
20 25 30
Asn Ala Lys Thr Asn Tyr Leu Asn Trp Tyr Met Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg His Thr Gly Val
50 55 60
Pro Asp Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Asp Glu Asp Gln Ala Phe Tyr Tyr Cys Gln Gln
85 90 95
Trp Tyr Asp Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Lys Leu Glu Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gly
130 135 140
Ser Leu Ser Leu Ser Cys Ser Val Thr Gly Thr Ser Ile Thr Ser Gly
145 150 155 160
Tyr Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Val Glu Trp
165 170 175
Met Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr Asn Pro Ser Ile
180 185 190
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
195 200 205
Leu Gln Val Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
210 215 220
Ala Arg Gln Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val
225 230 235 240
Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
340 345 350
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
465 470 475 480
Ser Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr
485 490 495
Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn
500 505 510
Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe
515 520 525
Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys
530 535 540
Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln
545 550 555 560
Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn
565 570 575
Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
580 585 590
Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile
595 600 605
Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr
610 615
<210> 27
<211> 619
<212> PRT
<213> Artificial sequence
<400> 27
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Asp Val Met Met Thr Gln Ser Pro Ser Ser Leu Ser
145 150 155 160
Val Ser Ala Gly Glu Lys Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser
165 170 175
Leu Phe Asn Ser Asn Ala Lys Thr Asn Tyr Leu Asn Trp Tyr Met Gln
180 185 190
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg
195 200 205
His Thr Gly Val Pro Asp Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp
210 215 220
Phe Thr Leu Thr Ile Ser Ser Val Gln Asp Glu Asp Gln Ala Phe Tyr
225 230 235 240
Tyr Cys Gln Gln Trp Tyr Asp Tyr Pro Tyr Thr Phe Gly Ala Gly Thr
245 250 255
Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
260 265 270
Gly Gly Gly Ser Gln Val Lys Leu Glu Glu Ser Gly Pro Gly Leu Val
275 280 285
Asn Pro Ser Gly Ser Leu Ser Leu Ser Cys Ser Val Thr Gly Thr Ser
290 295 300
Ile Thr Ser Gly Tyr Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln
305 310 315 320
Lys Val Glu Trp Met Gly Phe Ile Tyr Tyr Glu Gly Ser Thr Tyr Tyr
325 330 335
Asn Pro Ser Ile Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
340 345 350
Asn Gln Phe Phe Leu Gln Val Asn Ser Val Thr Thr Glu Asp Thr Ala
355 360 365
Thr Tyr Tyr Cys Ala Arg Gln Thr Gly Tyr Phe Asp Tyr Trp Gly Gln
370 375 380
Gly Thr Met Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro
385 390 395 400
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
405 410 415
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
420 425 430
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
435 440 445
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
450 455 460
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
465 470 475 480
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
485 490 495
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
500 505 510
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
515 520 525
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
530 535 540
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
545 550 555 560
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
565 570 575
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
580 585 590
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
595 600 605
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615
<210> 28
<211> 214
<212> PRT
<213> Artificial sequence
<400> 28
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ala Trp Tyr His Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Gly Ser Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Ser Ser Gly Ser Gln Tyr Ser Leu Lys Ile Ser Arg Leu Gln Val
65 70 75 80
Glu Asp Ile Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr Gly Ala Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 29
<211> 213
<212> PRT
<213> Artificial sequence
<400> 29
Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Leu Asn Cys Arg Thr Ser Gln Asn Val Tyr Lys Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Gln Leu Gly Glu Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Asn Thr
85 90 95
Phe Gly Ala Gly Thr Asn Leu Glu Leu Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 30
<211> 223
<212> PRT
<213> Artificial sequence
<400> 30
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Tyr Phe
20 25 30
Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Asp Gly Ser Ile Pro Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Glu Asn Ala Lys Ser Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Arg Ser Tyr Gly Gly Tyr Ser Glu Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Val Met 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 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 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
210 215 220
<210> 31
<211> 227
<212> PRT
<213> Artificial sequence
<400> 31
Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Gly Ser Gly Phe Thr Ile Ser Asp Tyr
20 25 30
Gly Val Ala Trp Val Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Tyr Ala Gly Gly Thr Thr Tyr Tyr Arg Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Ile Asp Gly Tyr Gly Gly Tyr Ser Gly Ser His Trp Tyr Phe Asp
100 105 110
Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys
225
<210> 32
<211> 242
<212> PRT
<213> Artificial sequence
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ala Trp Tyr His Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Gly Ser Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Ser Ser Gly Ser Gln Tyr Ser Leu Lys Ile Ser Arg Leu Gln Val
65 70 75 80
Glu Asp Ile Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr Gly Ala Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu
115 120 125
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Lys Leu Ser Cys
130 135 140
Ala Ala Ser Gly Phe Ile Phe Ser Tyr Phe Asp Met Ala Trp Val Arg
145 150 155 160
Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Ser Ile Ser Pro Asp
165 170 175
Gly Ser Ile Pro Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Val
180 185 190
Ser Arg Glu Asn Ala Lys Ser Ser Leu Tyr Leu Gln Met Asp Ser Leu
195 200 205
Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Arg Ser Tyr Gly
210 215 220
Gly Tyr Ser Glu Leu Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val
225 230 235 240
Ser Ser
<210> 33
<211> 245
<212> PRT
<213> Artificial sequence
<400> 33
Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Leu Asn Cys Arg Thr Ser Gln Asn Val Tyr Lys Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Gln Leu Gly Glu Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Asn Thr
85 90 95
Phe Gly Ala Gly Thr Asn Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Met Gln Leu Val Glu Ser
115 120 125
Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Met Lys Leu Ser Cys Ala
130 135 140
Gly Ser Gly Phe Thr Ile Ser Asp Tyr Gly Val Ala Trp Val Arg Gln
145 150 155 160
Ala Pro Lys Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Tyr Ala Gly
165 170 175
Gly Thr Thr Tyr Tyr Arg Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
180 185 190
Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg
195 200 205
Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Ile Asp Gly Tyr Gly Gly
210 215 220
Tyr Ser Gly Ser His Trp Tyr Phe Asp Phe Trp Gly Pro Gly Thr Met
225 230 235 240
Val Thr Val Ser Ser
245
<210> 34
<211> 450
<212> PRT
<213> Artificial sequence
<400> 34
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Tyr Phe
20 25 30
Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Asp Gly Ser Ile Pro Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Glu Asn Ala Lys Ser Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Arg Ser Tyr Gly Gly Tyr Ser Glu Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Val Met 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 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 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 Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 35
<211> 454
<212> PRT
<213> Artificial sequence
<400> 35
Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Gly Ser Gly Phe Thr Ile Ser Asp Tyr
20 25 30
Gly Val Ala Trp Val Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Tyr Ala Gly Gly Thr Thr Tyr Tyr Arg Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Ile Asp Gly Tyr Gly Gly Tyr Ser Gly Ser His Trp Tyr Phe Asp
100 105 110
Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
355 360 365
Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys
450
<210> 36
<211> 469
<212> PRT
<213> Artificial sequence
<400> 36
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ala Trp Tyr His Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Gly Ser Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Ser Ser Gly Ser Gln Tyr Ser Leu Lys Ile Ser Arg Leu Gln Val
65 70 75 80
Glu Asp Ile Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr Gly Ala Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu
115 120 125
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Lys Leu Ser Cys
130 135 140
Ala Ala Ser Gly Phe Ile Phe Ser Tyr Phe Asp Met Ala Trp Val Arg
145 150 155 160
Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Ser Ile Ser Pro Asp
165 170 175
Gly Ser Ile Pro Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Val
180 185 190
Ser Arg Glu Asn Ala Lys Ser Ser Leu Tyr Leu Gln Met Asp Ser Leu
195 200 205
Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Arg Ser Tyr Gly
210 215 220
Gly Tyr Ser Glu Leu Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val
225 230 235 240
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys
465
<210> 37
<211> 472
<212> PRT
<213> Artificial sequence
<400> 37
Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Leu Asn Cys Arg Thr Ser Gln Asn Val Tyr Lys Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Gln Leu Gly Glu Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Asn Thr
85 90 95
Phe Gly Ala Gly Thr Asn Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Met Gln Leu Val Glu Ser
115 120 125
Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Met Lys Leu Ser Cys Ala
130 135 140
Gly Ser Gly Phe Thr Ile Ser Asp Tyr Gly Val Ala Trp Val Arg Gln
145 150 155 160
Ala Pro Lys Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Tyr Ala Gly
165 170 175
Gly Thr Thr Tyr Tyr Arg Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
180 185 190
Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg
195 200 205
Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Ile Asp Gly Tyr Gly Gly
210 215 220
Tyr Ser Gly Ser His Trp Tyr Phe Asp Phe Trp Gly Pro Gly Thr Met
225 230 235 240
Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 38
<211> 617
<212> PRT
<213> Artificial sequence
<400> 38
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ala Trp Tyr His Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Gly Ser Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Ser Ser Gly Ser Gln Tyr Ser Leu Lys Ile Ser Arg Leu Gln Val
65 70 75 80
Glu Asp Ile Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr Gly Ala Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu
115 120 125
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Lys Leu Ser Cys
130 135 140
Ala Ala Ser Gly Phe Ile Phe Ser Tyr Phe Asp Met Ala Trp Val Arg
145 150 155 160
Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Ser Ile Ser Pro Asp
165 170 175
Gly Ser Ile Pro Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Val
180 185 190
Ser Arg Glu Asn Ala Lys Ser Ser Leu Tyr Leu Gln Met Asp Ser Leu
195 200 205
Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Arg Ser Tyr Gly
210 215 220
Gly Tyr Ser Glu Leu Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val
225 230 235 240
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
465 470 475 480
Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu
485 490 495
Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile
500 505 510
Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe
515 520 525
Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
530 535 540
Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys
545 550 555 560
Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
565 570 575
Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala
580 585 590
Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe
595 600 605
Cys Gln Ser Ile Ile Ser Thr Leu Thr
610 615
<210> 39
<211> 620
<212> PRT
<213> Artificial sequence
<400> 39
Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Leu Asn Cys Arg Thr Ser Gln Asn Val Tyr Lys Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Gln Leu Gly Glu Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Asn Thr
85 90 95
Phe Gly Ala Gly Thr Asn Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Met Gln Leu Val Glu Ser
115 120 125
Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Met Lys Leu Ser Cys Ala
130 135 140
Gly Ser Gly Phe Thr Ile Ser Asp Tyr Gly Val Ala Trp Val Arg Gln
145 150 155 160
Ala Pro Lys Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Tyr Ala Gly
165 170 175
Gly Thr Thr Tyr Tyr Arg Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
180 185 190
Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg
195 200 205
Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Ile Asp Gly Tyr Gly Gly
210 215 220
Tyr Ser Gly Ser His Trp Tyr Phe Asp Phe Trp Gly Pro Gly Thr Met
225 230 235 240
Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly
465 470 475 480
Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys
485 490 495
Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu
500 505 510
Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr
515 520 525
Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln
530 535 540
Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala
545 550 555 560
Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile
565 570 575
Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys
580 585 590
Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp
595 600 605
Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr
610 615 620
<210> 40
<211> 617
<212> PRT
<213> Artificial sequence
<400> 40
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser
145 150 155 160
Ala Ser Leu Glu Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp
165 170 175
Ile Gly Asn Trp Leu Ala Trp Tyr His Gln Lys Pro Gly Lys Ser Pro
180 185 190
Gln Leu Leu Ile Tyr Gly Ser Thr Ser Leu Ala Asp Gly Val Pro Ser
195 200 205
Arg Phe Ser Gly Ser Ser Ser Gly Ser Gln Tyr Ser Leu Lys Ile Ser
210 215 220
Arg Leu Gln Val Glu Asp Ile Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr
225 230 235 240
Gly Ala Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly
245 250 255
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val
260 265 270
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu
275 280 285
Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Tyr Phe Asp Met
290 295 300
Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Ser
305 310 315 320
Ile Ser Pro Asp Gly Ser Ile Pro Tyr Tyr Arg Asp Ser Val Lys Gly
325 330 335
Arg Phe Thr Val Ser Arg Glu Asn Ala Lys Ser Ser Leu Tyr Leu Gln
340 345 350
Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg
355 360 365
Arg Ser Tyr Gly Gly Tyr Ser Glu Leu Asp Tyr Trp Gly Gln Gly Val
370 375 380
Met Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
385 390 395 400
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
405 410 415
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
420 425 430
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
435 440 445
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
450 455 460
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
465 470 475 480
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
485 490 495
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
500 505 510
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
515 520 525
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
530 535 540
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
545 550 555 560
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
565 570 575
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
580 585 590
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
595 600 605
Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615
<210> 41
<211> 620
<212> PRT
<213> Artificial sequence
<400> 41
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser
145 150 155 160
Ala Ser Val Gly Asp Arg Val Thr Leu Asn Cys Arg Thr Ser Gln Asn
165 170 175
Val Tyr Lys Asn Leu Ala Trp Tyr Gln Gln Gln Leu Gly Glu Ala Pro
180 185 190
Lys Leu Leu Ile Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser
195 200 205
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
210 215 220
Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr
225 230 235 240
Ser Gly Asn Thr Phe Gly Ala Gly Thr Asn Leu Glu Leu Lys Gly Gly
245 250 255
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Met Gln
260 265 270
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Met Lys
275 280 285
Leu Ser Cys Ala Gly Ser Gly Phe Thr Ile Ser Asp Tyr Gly Val Ala
290 295 300
Trp Val Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Val Ala Tyr Ile
305 310 315 320
Ser Tyr Ala Gly Gly Thr Thr Tyr Tyr Arg Glu Ser Val Lys Gly Arg
325 330 335
Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met
340 345 350
Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Ile Asp
355 360 365
Gly Tyr Gly Gly Tyr Ser Gly Ser His Trp Tyr Phe Asp Phe Trp Gly
370 375 380
Pro Gly Thr Met Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro
385 390 395 400
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
405 410 415
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
420 425 430
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
435 440 445
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
450 455 460
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
465 470 475 480
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
485 490 495
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
500 505 510
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
515 520 525
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
530 535 540
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
545 550 555 560
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
565 570 575
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
580 585 590
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
595 600 605
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 42
<211> 642
<212> DNA
<213> Artificial sequence
<400> 42
gacatccaga tgacccagag ccccgcctct ctgagcgctt ctctggagga gatcgtgacc 60
atcacatgcc aagccagcca agacatcggc aactggctgg cttggtatca ccagaagccc 120
ggcaagagcc cccagctgct gatctacggc agcacatctc tggctgatgg cgtgccctcc 180
agatttagcg gcagcagcag cggaagccag tactctctga agatctctag actgcaagtg 240
gaggacattg gcatctacta ctgtctgcaa gcctatggcg ccccttggac attcggaggc 300
ggcacaaagc tggagctgaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agttgaaatc tggtaccgct agcgttgtgt gcctgctgaa taacttttat 420
ccacgggagg ctaaggtgca gtggaaagtg gacaatgccc tccagagcgg aaatagccaa 480
gagtccgtta ccgaacagga ctctaaagac tctacatact ccctgtcctc cacactgacc 540
ctctccaagg ccgactatga gaaacacaag gtttacgcat gcgaggtcac acaccaggga 600
ctctcctctc ccgtgaccaa gagcttcaac cggggagaat gc 642
<210> 43
<211> 639
<212> DNA
<213> Artificial sequence
<400> 43
gacatccaga tgacccagag cccttctctg ctgagcgctt ccgtgggaga cagagtgaca 60
ctgaactgta gaacctccca gaacgtgtac aagaatctgg cttggtatca gcagcagctg 120
ggcgaagccc ccaagctgct gatctacaac gccaactctc tgcaagccgg catccctagc 180
agattcagcg gctccggcag cggaaccgac tttacactga ccatcagctc tctgcagccc 240
gaggatgtgg ccacctactt ctgccagcag tactacagcg gcaacacctt cggcgccggc 300
accaatctgg agctgaagcg tacggtggct gcaccatctg tcttcatctt cccgccatct 360
gatgagcagt tgaaatctgg taccgctagc gttgtgtgcc tgctgaataa cttttatcca 420
cgggaggcta aggtgcagtg gaaagtggac aatgccctcc agagcggaaa tagccaagag 480
tccgttaccg aacaggactc taaagactct acatactccc tgtcctccac actgaccctc 540
tccaaggccg actatgagaa acacaaggtt tacgcatgcg aggtcacaca ccagggactc 600
tcctctcccg tgaccaagag cttcaaccgg ggagaatgc 639
<210> 44
<211> 1350
<212> DNA
<213> Artificial sequence
<400> 44
gacgtgcagc tggtggaatc cggcggagga ctggtgcagc ccggcagatc tctgaaactc 60
agctgcgccg ccagcggctt catcttcagc tacttcgaca tggcttgggt gagacaagcc 120
cctaccaaag gactggagtg ggtggccagc attagccccg acggcagcat tccctactat 180
agagacagcg tgaagggaag attcaccgtg agcagagaga acgccaagag ctctctgtat 240
ctgcagatgg actctctgag aagcgaggac accgccacct actactgcgc cagaagaagc 300
tacggcggct acagcgagct ggactactgg ggacaaggcg tgatggtgac agtgagcagc 360
gccagcacta aggggccctc tgtgtttcca ctcgcccctt ctagcaaaag cacttccgga 420
ggaactgccg ctctgggctg tctggtgaaa gattacttcc ccgaaccagt cactgtgtca 480
tggaactctg gagcactgac atctggagtt cacacctttc ctgctgtgct gcagagttct 540
ggactgtact ccctgtcatc tgtggtcacc gtgccatctt catctctggg gacccagacc 600
tacatctgta acgtgaacca caaaccctcc aacacaaaag tcgacaaacg agtcgaacca 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag ctccggaact cctgggcgga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg tcgggatgag 1080
ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ttggactccg acggctcctt cttcctctac agcgcgctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc tccgggtaaa 1350
<210> 45
<211> 1362
<212> DNA
<213> Artificial sequence
<400> 45
gagatgcagc tggtggagag cggaggagga ctggtgcagc ccggcagaag catgaagctg 60
agctgtgccg gcagcggctt caccatcagc gattacggcg tggcttgggt gagacaagcc 120
cctaagaagg gactggagtg ggtggcctac atcagctacg ccggcggcac cacctactac 180
agagagagcg tgaagggaag attcaccatt tctagagaca acgccaagag cacactgtat 240
ctgcagatgg actctctgag gagcgaggac accgccacct actactgcac catcgacggc 300
tatggcggct acagcggcag ccactggtac ttcgacttct ggggacccgg caccatggtg 360
accgtgagca gcgccagcac taaggggccc tctgtgtttc cactcgcccc ttctagcaaa 420
agcacttccg gaggaactgc cgctctgggc tgtctggtga aagattactt ccccgaacca 480
gtcactgtgt catggaactc tggagcactg acatctggag ttcacacctt tcctgctgtg 540
ctgcagagtt ctggactgta ctccctgtca tctgtggtca ccgtgccatc ttcatctctg 600
gggacccaga cctacatctg taacgtgaac cacaaaccct ccaacacaaa agtcgacaaa 660
cgagtcgaac caaaatcttg tgacaaaact cacacatgcc caccgtgccc agctccggaa 720
ctcctgggcg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 780
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 840
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 900
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 960
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1020
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1080
tgtcgggatg agctgaccaa gaaccaggtc agcctgtggt gcctggtcaa aggcttctat 1140
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1200
acgcctcccg tgttggactc cgacggctcc ttcttcctct acagcgcgct caccgtggac 1260
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1320
aaccactaca cgcagaagag cctctccctg tctccgggta aa 1362
<210> 46
<211> 1407
<212> DNA
<213> Artificial sequence
<400> 46
gacatccaga tgacccagag ccccgcctct ctgagcgctt ctctggagga gatcgtgacc 60
atcacatgcc aagccagcca agacatcggc aactggctgg cttggtatca ccagaagccc 120
ggcaagagcc cccagctgct gatctacggc agcacatctc tggctgatgg cgtgccctcc 180
agatttagcg gcagcagcag cggaagccag tactctctga agatctctag actgcaagtg 240
gaggacattg gcatctacta ctgtctgcaa gcctatggcg ccccttggac attcggaggc 300
ggcacaaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 360
ggcagcgacg tgcagctggt ggaatccggc ggaggactgg tgcagcccgg cagatctctg 420
aaactcagct gcgccgccag cggcttcatc ttcagctact tcgacatggc ttgggtgaga 480
caagccccta ccaaaggact ggagtgggtg gccagcatta gccccgacgg cagcattccc 540
tactatagag acagcgtgaa gggaagattc accgtgagca gagagaacgc caagagctct 600
ctgtatctgc agatggactc tctgagaagc gaggacaccg ccacctacta ctgcgccaga 660
agaagctacg gcggctacag cgagctggac tactggggac aaggcgtgat ggtgacagtg 720
agcagcgaca aaactcacac atgcccaccg tgcccagctc cggaactcct gggcggaccg 780
tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020
tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080
gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatgtcg ggatgagctg 1140
accaagaacc aggtcagcct gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1200
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgttg 1260
gactccgacg gctccttctt cctctacagc gcgctcaccg tggacaagag caggtggcag 1320
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380
aagagcctct ccctgtctcc gggtaaa 1407
<210> 47
<211> 1416
<212> DNA
<213> Artificial sequence
<400> 47
gacatccaga tgacccagag cccttctctg ctgagcgctt ccgtgggaga cagagtgaca 60
ctgaactgta gaacctccca gaacgtgtac aagaatctgg cttggtatca gcagcagctg 120
ggcgaagccc ccaagctgct gatctacaac gccaactctc tgcaagccgg catccctagc 180
agattcagcg gctccggcag cggaaccgac tttacactga ccatcagctc tctgcagccc 240
gaggatgtgg ccacctactt ctgccagcag tactacagcg gcaacacctt cggcgccggc 300
accaatctgg agctgaaggg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 360
agcgagatgc agctggtgga gagcggagga ggactggtgc agcccggcag aagcatgaag 420
ctgagctgtg ccggcagcgg cttcaccatc agcgattacg gcgtggcttg ggtgagacaa 480
gcccctaaga agggactgga gtgggtggcc tacatcagct acgccggcgg caccacctac 540
tacagagaga gcgtgaaggg aagattcacc atttctagag acaacgccaa gagcacactg 600
tatctgcaga tggactctct gaggagcgag gacaccgcca cctactactg caccatcgac 660
ggctatggcg gctacagcgg cagccactgg tacttcgact tctggggacc cggcaccatg 720
gtgaccgtga gcagcgacaa aactcacaca tgcccaccgt gcccagctcc ggaactcctg 780
ggcggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatgtcgg 1140
gatgagctga ccaagaacca ggtcagcctg tggtgcctgg tcaaaggctt ctatcccagc 1200
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260
cccgtgttgg actccgacgg ctccttcttc ctctacagcg cgctcaccgt ggacaagagc 1320
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380
tacacgcaga agagcctctc cctgtctccg ggtaaa 1416
<210> 48
<211> 1911
<212> DNA
<213> Artificial sequence
<400> 48
gacatccaga tgacccagag ccccgcctct ctgagcgctt ctctggagga gatcgtgacc 60
atcacatgcc aagccagcca agacatcggc aactggctgg cttggtatca ccagaagccc 120
ggcaagagcc cccagctgct gatctacggc agcacatctc tggctgatgg cgtgccctcc 180
agatttagcg gcagcagcag cggaagccag tactctctga agatctctag actgcaagtg 240
gaggacattg gcatctacta ctgtctgcaa gcctatggcg ccccttggac attcggaggc 300
ggcacaaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 360
ggcagcgacg tgcagctggt ggaatccggc ggaggactgg tgcagcccgg cagatctctg 420
aaactcagct gcgccgccag cggcttcatc ttcagctact tcgacatggc ttgggtgaga 480
caagccccta ccaaaggact ggagtgggtg gccagcatta gccccgacgg cagcattccc 540
tactatagag acagcgtgaa gggaagattc accgtgagca gagagaacgc caagagctct 600
ctgtatctgc agatggactc tctgagaagc gaggacaccg ccacctacta ctgcgccaga 660
agaagctacg gcggctacag cgagctggac tactggggac aaggcgtgat ggtgacagtg 720
agcagcgaca aaacccacac atgcccacct tgtcccgccc ctgaactcct gggcggaccg 780
tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020
tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080
gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1140
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgttg 1260
gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1320
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380
aagagcctct ccctgtctcc gggtaaaggc ggcggcggca gcggcggcgg cggcagcggc 1440
ggcggcggca gcatgtacag gatgcaactc ctgtcttgca ttgcactaag tcttgcactt 1500
gtcacaaaca gtgcacctac ttcaagttct acaaagaaaa cacagctaca actggagcat 1560
ttactgctgg atttacagat gattttgaat ggaattaata attacaagaa tcccaaactc 1620
accaggatgc tcacatttaa gttttacatg cccaagaagg ccacagaact gaaacatctt 1680
cagtgtctag aagaagaact caaacctctg gaggaagtgc taaatttagc tcaaagcaaa 1740
aactttcact taagacccag ggacttaatc agcaatatca acgtaatagt tctggaacta 1800
aagggatctg aaacaacatt catgtgtgaa tatgctgatg agacagcaac cattgtagaa 1860
tttctgaaca gatggattac cttttgtcaa agcatcatct caacactgac t 1911
<210> 49
<211> 1920
<212> DNA
<213> Artificial sequence
<400> 49
gacatccaga tgacccagag cccttctctg ctgagcgctt ccgtgggaga cagagtgaca 60
ctgaactgta gaacctccca gaacgtgtac aagaatctgg cttggtatca gcagcagctg 120
ggcgaagccc ccaagctgct gatctacaac gccaactctc tgcaagccgg catccctagc 180
agattcagcg gctccggcag cggaaccgac tttacactga ccatcagctc tctgcagccc 240
gaggatgtgg ccacctactt ctgccagcag tactacagcg gcaacacctt cggcgccggc 300
accaatctgg agctgaaggg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 360
agcgagatgc agctggtgga gagcggagga ggactggtgc agcccggcag aagcatgaag 420
ctgagctgtg ccggcagcgg cttcaccatc agcgattacg gcgtggcttg ggtgagacaa 480
gcccctaaga agggactgga gtgggtggcc tacatcagct acgccggcgg caccacctac 540
tacagagaga gcgtgaaggg aagattcacc atttctagag acaacgccaa gagcacactg 600
tatctgcaga tggactctct gaggagcgag gacaccgcca cctactactg caccatcgac 660
ggctatggcg gctacagcgg cagccactgg tacttcgact tctggggacc cggcaccatg 720
gtgaccgtga gcagcgacaa aacccacaca tgcccacctt gtcccgcccc tgaactcctg 780
ggcggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1140
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260
cccgtgttgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1320
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380
tacacgcaga agagcctctc cctgtctccg ggtaaaggcg gcggcggcag cggcggcggc 1440
ggcagcggcg gcggcggcag catgtacagg atgcaactcc tgtcttgcat tgcactaagt 1500
cttgcacttg tcacaaacag tgcacctact tcaagttcta caaagaaaac acagctacaa 1560
ctggagcatt tactgctgga tttacagatg attttgaatg gaattaataa ttacaagaat 1620
cccaaactca ccaggatgct cacatttaag ttttacatgc ccaagaaggc cacagaactg 1680
aaacatcttc agtgtctaga agaagaactc aaacctctgg aggaagtgct aaatttagct 1740
caaagcaaaa actttcactt aagacccagg gacttaatca gcaatatcaa cgtaatagtt 1800
ctggaactaa agggatctga aacaacattc atgtgtgaat atgctgatga gacagcaacc 1860
attgtagaat ttctgaacag atggattacc ttttgtcaaa gcatcatctc aacactgact 1920
<210> 50
<211> 1911
<212> DNA
<213> Artificial sequence
<400> 50
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60
gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 120
ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 180
acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 240
gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 300
agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 360
acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 420
tggattacct tttgtcaaag catcatctca acactgactg gcggcggcgg cagcggcggc 480
ggcggcagcg gcggcggcgg cagcgacatc cagatgaccc agagccccgc ctctctgagc 540
gcttctctgg aggagatcgt gaccatcaca tgccaagcca gccaagacat cggcaactgg 600
ctggcttggt atcaccagaa gcccggcaag agcccccagc tgctgatcta cggcagcaca 660
tctctggctg atggcgtgcc ctccagattt agcggcagca gcagcggaag ccagtactct 720
ctgaagatct ctagactgca agtggaggac attggcatct actactgtct gcaagcctat 780
ggcgcccctt ggacattcgg aggcggcaca aagctggagc tgaagggcgg cggcggcagc 840
ggcggcggcg gcagcggcgg cggcggcagc gacgtgcagc tggtggaatc cggcggagga 900
ctggtgcagc ccggcagatc tctgaaactc agctgcgccg ccagcggctt catcttcagc 960
tacttcgaca tggcttgggt gagacaagcc cctaccaaag gactggagtg ggtggccagc 1020
attagccccg acggcagcat tccctactat agagacagcg tgaagggaag attcaccgtg 1080
agcagagaga acgccaagag ctctctgtat ctgcagatgg actctctgag aagcgaggac 1140
accgccacct actactgcgc cagaagaagc tacggcggct acagcgagct ggactactgg 1200
ggacaaggcg tgatggtgac agtgagcagc gacaaaaccc acacatgccc accttgtccc 1260
gcccctgaac tcctgggcgg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 1320
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 1380
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 1440
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1500
caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1560
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1620
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1680
ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1740
tacaagacca cgcctcccgt gttggactcc gacggctcct tcttcctcta cagcaagctc 1800
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1860
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a 1911
<210> 51
<211> 1920
<212> DNA
<213> Artificial sequence
<400> 51
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60
gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 120
ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 180
acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 240
gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 300
agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 360
acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 420
tggattacct tttgtcaaag catcatctca acactgactg gcggcggcgg cagcggcggc 480
ggcggcagcg gcggcggcgg cagcgacatc cagatgaccc agagcccttc tctgctgagc 540
gcttccgtgg gagacagagt gacactgaac tgtagaacct cccagaacgt gtacaagaat 600
ctggcttggt atcagcagca gctgggcgaa gcccccaagc tgctgatcta caacgccaac 660
tctctgcaag ccggcatccc tagcagattc agcggctccg gcagcggaac cgactttaca 720
ctgaccatca gctctctgca gcccgaggat gtggccacct acttctgcca gcagtactac 780
agcggcaaca ccttcggcgc cggcaccaat ctggagctga agggcggcgg cggcagcggc 840
ggcggcggca gcggcggcgg cggcagcgag atgcagctgg tggagagcgg aggaggactg 900
gtgcagcccg gcagaagcat gaagctgagc tgtgccggca gcggcttcac catcagcgat 960
tacggcgtgg cttgggtgag acaagcccct aagaagggac tggagtgggt ggcctacatc 1020
agctacgccg gcggcaccac ctactacaga gagagcgtga agggaagatt caccatttct 1080
agagacaacg ccaagagcac actgtatctg cagatggact ctctgaggag cgaggacacc 1140
gccacctact actgcaccat cgacggctat ggcggctaca gcggcagcca ctggtacttc 1200
gacttctggg gacccggcac catggtgacc gtgagcagcg acaaaaccca cacatgccca 1260
ccttgtcccg cccctgaact cctgggcgga ccgtcagtct tcctcttccc cccaaaaccc 1320
aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 1380
cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1440
aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1500
gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1560
ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1620
gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1680
ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1740
gagaacaact acaagaccac gcctcccgtg ttggactccg acggctcctt cttcctctac 1800
agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1860
atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 1920
<210> 52
<211> 108
<212> PRT
<213> Artificial sequence
<400> 52
Glu Ile Glu 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 Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 53
<211> 120
<212> PRT
<213> Artificial sequence
<400> 53
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Met
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Tyr Asp Asn Val Met Gly Leu Tyr Trp Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 54
<211> 107
<212> PRT
<213> Artificial sequence
<400> 54
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Arg Thr Asp Tyr Ser Leu Thr Ile Thr Asp Leu Asp Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Ala Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 55
<211> 121
<212> PRT
<213> Artificial sequence
<400> 55
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Phe Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn His Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala
85 90 95
Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 56
<211> 128
<212> PRT
<213> Artificial sequence
<400> 56
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Leu Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Thr
35 40 45
Ser Ser Ile Thr Tyr Met Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala
50 55 60
Pro Lys Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
115 120 125
<210> 57
<211> 142
<212> PRT
<213> Artificial sequence
<400> 57
Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Thr Ser Tyr Gly Val Tyr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Thr Thr Asn Tyr Asn Ser
65 70 75 80
Ala Leu Met Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
85 90 95
Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Arg Gly Pro Pro His Ala Met Met Lys Arg Gly Tyr Ala
115 120 125
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
130 135 140
<210> 58
<211> 109
<212> PRT
<213> Artificial sequence
<400> 58
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 Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 59
<211> 120
<212> PRT
<213> Artificial sequence
<400> 59
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Thr
1 5 10 15
Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr
20 25 30
Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly
35 40 45
Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser
50 55 60
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys
65 70 75 80
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95
Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 60
<211> 108
<212> PRT
<213> Artificial sequence
<400> 60
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu
85 90 95
Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 61
<211> 116
<212> PRT
<213> Artificial sequence
<400> 61
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr
20 25 30
Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115

Claims (18)

1.一种双功能融合蛋白，所述的融合蛋白为异源二聚体，其特征在于，1. a bifunctional fusion protein, described fusion protein is a heterodimer, it is characterised in that, 所述的异源二聚体包括：Described heterodimer includes: (1)白介素2(IL-2)与免疫球蛋白Fc单链连接而成的第一单体；(1) The first monomer formed by linking interleukin 2 (IL-2) and immunoglobulin Fc single chain; (2)抗CTLA4或TNF家族的共刺激或共抑制性分子的抗体的Fab或ScFv与免疫球蛋白Fc单链连接而成的第二单体；(2) a second monomer formed by linking the Fab or ScFv of an antibody against a co-stimulatory or co-inhibitory molecule of the CTLA4 or TNF family to an immunoglobulin Fc single chain; 所述的第一单体与第二单体通过Fc单链的二聚化连接，构成所述的异源二聚体；The first monomer and the second monomer are connected by dimerization of the Fc single chain to form the heterodimer; 所述的CTLA4或TNF家族的共刺激或共抑制性分子包括但不限于：OX40、ICOS、GITR、4-1BB。Said co-stimulatory or co-inhibitory molecules of CTLA4 or TNF family include but are not limited to: OX40, ICOS, GITR, 4-1BB. 2.根据权利要求1所述的双功能融合蛋白，其特征在于，所述的免疫球蛋白Fc单链为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链；优选的，所述的免疫球蛋白Fc单链为人IgG的Fc单链。2. The bifunctional fusion protein according to claim 1, wherein the immunoglobulin Fc single chain is a natural immunoglobulin Fc single chain or an immunoglobulin Fc single chain with ADCC effect knocked out by gene mutation ; Preferably, the immunoglobulin Fc single chain is the Fc single chain of human IgG. 3.根据权利要求1任一所述的双功能融合蛋白，其特征在于，3. bifunctional fusion protein according to any one of claim 1, is characterized in that, 所述的第二单体中，In the second monomer, 所述的抗体的Fab为人源化抗体的Fab或全人化抗体的Fab；The Fab of the antibody is the Fab of a humanized antibody or the Fab of a fully humanized antibody; 所述的抗体的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；The ScFv of the antibody is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody; 优选的，所述的第二单体为：Preferably, the second monomer is: 抗CTLA4或TNF家族的共刺激或共抑制性分子的抗体的单体，所述抗体的单体包含一条轻链和一条重链；A monomer of an antibody against a co-stimulatory or co-inhibitory molecule of the CTLA4 or TNF family, the monomer of which comprises one light chain and one heavy chain; 更优选的，所述的第二单体为：More preferably, the second monomer is: 人源化抗CTLA4或抗TNF家族的共刺激或共抑制性分子的抗体的单体或全人化抗CTLA4或抗TNF家族的共刺激或共抑制性分子的抗体抗体的单体，所述抗体的单体包含一条轻链和一条重链。Monomer of a humanized anti-CTLA4 or antibody against a costimulatory or costinhibitory molecule of the TNF family or a monomer of a fully humanized antibody against a costimulatory or costinhibitory molecule of the TNF family, said antibody The monomer contains one light chain and one heavy chain. 4.根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，4. according to the arbitrary described bifunctional fusion protein of claim 1-3, it is characterised in that, 所述的CTLA4或TNF家族的共刺激或共抑制性分子为OX40、4-1BB，The co-stimulatory or co-inhibitory molecules of the CTLA4 or TNF family are OX40, 4-1BB, 所述的抗CTLA4的抗体(aCTLA4)或抗TNF家族的共刺激或共抑制性分子的抗体为抗OX40的抗体(aOX40)、或抗4-1BB的抗体(a4-1BB)。The anti-CTLA4 antibody (aCTLA4) or the anti-TNF family co-stimulatory or co-inhibitory molecule antibody is an anti-OX40 antibody (aOX40) or an anti-4-1BB antibody (a4-1BB). 5.根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，5. according to the arbitrary described bifunctional fusion protein of claim 1-3, it is characterised in that, 所述的异源二聚体包括：Described heterodimer includes: (1)第一单体，所述的第一单体自N端开始依次包含：(1) the first monomer, the first monomer comprises sequentially from the N-terminus: 1)序列如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；1) A wild-type IL-2 protein whose sequence is shown in SEQ ID NO.1, or a mutant of the wild-type IL-2 protein, wherein the mutant contains any one or any combination of the following mutation sites ;F42A, L80F, R81D, L85V, I86V and I92F; 2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；2) Necessary connection structure (G4S connection sequence), preferably, the connection sequence is shown in SEQ ID NO.6; 3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；3) The Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the knob mutation shown in SEQ ID NO.4 type Fc, or a hole mutant Fc as shown in SEQ ID NO.5; (2)第二单体，所述的第二单体包含：(2) a second monomer, the second monomer comprises: 1)序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL和序列如SEQ ID NO.8所示的抗OX40抗体重链VH&CH1组成的抗OX40抗体Fab区；1) the anti-OX40 antibody Fab region composed of the anti-OX40 antibody light chain VL-KCL whose sequence is shown in SEQ ID NO.7 and the anti-OX40 antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.8; 或：2)序列如SEQ ID NO.9所示的抗OX40单链抗体(ScFv)：Or: 2) Anti-OX40 single chain antibody (ScFv) whose sequence is shown in SEQ ID NO.9: 和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。and: 3) the Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the Fc shown in SEQ ID NO.4 A knob mutant Fc, or a hole mutant Fc as shown in SEQ ID NO.5. 6.根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，6. according to the arbitrary described bifunctional fusion protein of claim 1-3, it is characterised in that, 所述的异源二聚体包括：Described heterodimer includes: 第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；The first monomer: it is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO. 10; 第二单体：其为：Second monomer: which is: (1)序列如SEQ ID NO.11所示的抗OX40抗体VH-CH1-Fc(knob)和序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL组成；(1) The anti-OX40 antibody VH-CH1-Fc (knob) whose sequence is shown in SEQ ID NO.11 and the anti-OX40 antibody light chain VL-KCL whose sequence is shown in SEQ ID NO.7; 或(2)序列如SEQ ID NO.12所示的多肽(aOX40 ScFv-Fc(knob))。。Or (2) a polypeptide whose sequence is shown in SEQ ID NO. 12 (aOX40 ScFv-Fc(knob)). . 7.根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，7. according to the arbitrary described bifunctional fusion protein of claim 1-3, it is characterised in that, 所述的异源二聚体包括：Described heterodimer includes: (1)第一单体，所述的第一单体自N端开始依次包含：(1) the first monomer, the first monomer comprises sequentially from the N-terminus: 1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；1) The wild-type IL-2 protein shown in SEQ ID NO.1, or the mutant of the wild-type IL-2 protein, wherein the mutant comprises any one or any combination of the following mutation sites; F42A, L80F, R81D, L85V, I86V and I92F; 2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；2) Necessary connection structure (G4S connection sequence), preferably, the connection sequence is shown in SEQ ID NO.6; 3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；3) Fc single chain of IgG as shown in SEQ ID NO.2, or Fc of IgG of No-ADCC mutant as shown in SEQ ID NO.3, or knob mutant as shown in SEQ ID NO.4 Fc, or a hole mutant Fc as shown in SEQ ID NO.5; (2)第二单体，所述的第二单体包含：(2) a second monomer, the second monomer comprises: 1)序列如SEQ ID NO.21所示的抗CTLA4抗体轻链与序列如SEQ ID NO.22所示的抗CTLA4抗体重链VH&CH1组成的抗体Fab区；1) the antibody Fab region composed of the anti-CTLA4 antibody light chain whose sequence is shown in SEQ ID NO.21 and the anti-CTLA4 antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.22; 或：2)序列如SEQ ID NO.23所示的抗CTLA4单链抗体(ScFv)：Or: 2) an anti-CTLA4 single-chain antibody (ScFv) whose sequence is shown in SEQ ID NO. 23: 和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。and: 3) the Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the Fc shown in SEQ ID NO.4 A knob mutant Fc, or a hole mutant Fc as shown in SEQ ID NO.5. 8.根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，8. according to the arbitrary described bifunctional fusion protein of claim 1-3, it is characterised in that, 所述的异源二聚体包括：Described heterodimer includes: 第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；The first monomer: it is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO. 10; 第二单体：其为：Second monomer: which is: (1)序列如SEQ ID NO.24所示的多肽(aCTLA4 VH-CH1-Fc(knob))和序列如SEQ IDNO.21所示的抗CTLA4抗体轻链可变区组成；或(1) The polypeptide (aCTLA4 VH-CH1-Fc(knob)) whose sequence is shown in SEQ ID NO.24 and the light chain variable region of an anti-CTLA4 antibody whose sequence is shown in SEQ ID NO.21; or (2)序列如SEQ ID NO.25所示的多肽(a CTLA4 ScFv-Fc(knob))。(2) The polypeptide (a CTLA4 ScFv-Fc(knob)) whose sequence is shown in SEQ ID NO. 25. 9.根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，9. according to the arbitrary described bifunctional fusion protein of claim 1-3, it is characterised in that, 所述的异源二聚体包括：Described heterodimer includes: (1)第一单体，所述的第一单体自N端开始依次包含：(1) the first monomer, the first monomer comprises sequentially from the N-terminus: 1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；1) The wild-type IL-2 protein shown in SEQ ID NO.1, or the mutant of the wild-type IL-2 protein, wherein the mutant comprises any one or any combination of the following mutation sites; F42A, L80F, R81D, L85V, I86V and I92F; 2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；2) Necessary connection structure (G4S connection sequence), preferably, the connection sequence is shown in SEQ ID NO.6; 3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；3) Fc single chain of IgG as shown in SEQ ID NO.2, or Fc of IgG of No-ADCC mutant as shown in SEQ ID NO.3, or knob mutant as shown in SEQ ID NO.4 Fc, or a hole mutant Fc as shown in SEQ ID NO.5; (2)第二单体，所述的第二单体包含：(2) a second monomer, the second monomer comprises: 1)序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链与序列如SEQ ID NO.30、31所示的抗4-1BB抗体重链VH&CH1组成的抗体Fab区；1) the antibody Fab region composed of the anti-4-1BB antibody light chain whose sequence is shown in SEQ ID NO.28 and 29 and the anti-4-1BB antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.30 and 31; 或：2)序列如SEQ ID NO.32、33所示的抗4-1BB单链抗体(ScFv)：Or: 2) Anti-4-1BB single-chain antibody (ScFv) whose sequence is shown in SEQ ID NO.32 and 33: 和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。and: 3) the Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the Fc shown in SEQ ID NO.4 A knob mutant Fc, or a hole mutant Fc as shown in SEQ ID NO.5. 10.根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，10. The bifunctional fusion protein according to any one of claims 1-3, characterized in that, 所述的异源二聚体包括：Described heterodimer includes: 第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；The first monomer: it is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO. 10; 第二单体：其为：Second monomer: which is: (1)序列如SEQ ID NO.34、35所示的多肽(a4-1BB VH-CH1-Fc(knob))和序列如SEQ IDNO.28、29所示的抗4-1BB抗体轻链组成；或(1) The polypeptide (a4-1BB VH-CH1-Fc(knob)) whose sequence is shown in SEQ ID NO.34 and 35 and the light chain of anti-4-1BB antibody whose sequence is shown in SEQ ID NO.28 and 29; or (2)序列如SEQ ID NO.36、37所示的多肽(a4-1BB ScFv-Fc(knob))。(2) Polypeptides (a4-1BB ScFv-Fc(knob)) whose sequences are shown in SEQ ID NOs. 36 and 37. 11.一种双功能融合蛋白，所述的融合蛋白为同源二聚体，其特征在于，11. A bifunctional fusion protein, the fusion protein is a homodimer, characterized in that, 所述的同源二聚体的单体为：The monomer of the homodimer is: 一分子白介素2(IL-2)与一分子抗OX40的抗体(aOX40)、抗CTLA4的抗体(aCTLA4)或抗4-1BB的抗体(a4-1BB)的Fab通过任意方式连接构成的单体，或，A molecule of interleukin 2 (IL-2) and a molecule of anti-OX40 antibody (aOX40), anti-CTLA4 antibody (aCTLA4) or anti-4-1BB antibody (a4-1BB) Fab linked in any way to form a monomer, or, 一分子白介素2(IL-2)与一分子抗OX40的抗体(aOX40)、抗CTLA4的抗体(aCTLA4)或抗4-1BB的抗体(a4-1BB)的(ScFv)通过任意方式连接构成的单体。A single molecule of interleukin 2 (IL-2) linked to a molecule of anti-OX40 antibody (aOX40), anti-CTLA4 antibody (aCTLA4) or anti-4-1BB antibody (a4-1BB) (ScFv) in any manner body. 12.根据权利要求11所述的双功能融合蛋白，其特征在于，12. bifunctional fusion protein according to claim 11, is characterized in that, 所述的同源二聚体的单体自N端开始依次包含：The monomers of the homodimer, starting from the N-terminus, sequentially include: (1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；(1) The wild-type IL-2 protein shown in SEQ ID NO.1, or the mutant of the wild-type IL-2 protein, wherein the mutant contains any one or any combination of the following mutation sites ;F42A, L80F, R81D, L85V, I86V and I92F; (2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.4、SEQ IDNO.5所示；(2) Necessary connection structure (G4S connection sequence), preferably, the connection sequence is shown in SEQ ID NO.4 and SEQ ID NO.5; (3)抗OX40的抗体(aOX40)的Fab/ScFv、抗CTLA4的抗体(aCTLA4)的Fab/ScFv或抗4-1BB的抗体(a4-1BB)的Fab/ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；(3) Fab/ScFv of anti-OX40 antibody (aOX40), Fab/ScFv of anti-CTLA4 antibody (aCTLA4) or Fab/ScFv of anti-4-1BB antibody (a4-1BB); the Fab is humanized The Fab of an antibody or the Fab of a fully humanized antibody, the ScFv is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody; (4)抗体的Fc；所述的抗体Fc为全人野生型Fc或No-ADCC突变型Fc。(4) Fc of the antibody; the Fc of the antibody is a fully human wild-type Fc or a No-ADCC mutant Fc. 13.编码所述权利要求1-12任一所述的双功能融合蛋白的核苷酸序列。13. A nucleotide sequence encoding the bifunctional fusion protein of any one of claims 1-12. 14.根据权利要求13所述的核苷酸序列，其特征在于，14. The nucleotide sequence of claim 13, wherein 编码所述异源二聚体第一单体的核苷酸序列为SEQ ID NO.11所示的核苷酸序列；The nucleotide sequence encoding the first monomer of the heterodimer is the nucleotide sequence shown in SEQ ID NO.11; 编码所述异源二聚体第二单体的核苷酸序列为：The nucleotide sequence encoding the second monomer of the heterodimer is: SEQ ID NO.16(aOX40：VL-KCL)、SEQ ID NO. 16 (aOX40:VL-KCL), SEQ ID NO.17(aOX40：VH-CH1-Fc(knob))、SEQ ID NO. 17 (aOX40: VH-CH1-Fc(knob)), SEQ ID NO.18(aOX40 ScFv-Fc(knob))、SEQ ID NO. 18 (aOX40 ScFv-Fc(knob)), SEQ ID NO.42(a4-1BB(LOB12.3)：VL-KCL)、SEQ ID NO.42 (a4-1BB(LOB12.3):VL-KCL), SEQ ID NO.43(a4-1BB(3H3)：VL-KCL)、SEQ ID NO. 43 (a4-1BB(3H3):VL-KCL), SEQ ID NO.44(a4-1BB(LOB12.3)：VH-CH1-Fc(knob))、SEQ ID NO. 44 (a4-1BB(LOB12.3): VH-CH1-Fc(knob)), SEQ ID NO.45(a4-1BB(3H3)：VH-CH1-Fc(knob))、SEQ ID NO. 45 (a4-1BB(3H3): VH-CH1-Fc(knob)), SEQ ID NO.46(a4-1BB(LOB12.3)：ScFv-Fc(knob))、SEQ ID NO. 46 (a4-1BB(LOB12.3): ScFv-Fc(knob)), SEQ ID NO.47(a4-1BB(3H3)：ScFv-Fc(knob))所示的核苷酸序列；The nucleotide sequence shown in SEQ ID NO.47 (a4-1BB(3H3): ScFv-Fc(knob)); 编码所述同源二聚体的核苷酸序列为：The nucleotide sequence encoding the homodimer is: SEQ ID NO.19(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 19 (aOX40-IL2:VL-VH(ScFv)-Fc(wt)-IL2), SEQ ID NO.20(IL2-aOX40：IL2-VL-VH(ScFv)-Fc(wt))、SEQ ID NO. 20 (IL2-aOX40:IL2-VL-VH(ScFv)-Fc(wt)), SEQ ID NO.48(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 48 (a4-1BB(LOB12.3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2), SEQ ID NO.49(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 49 (a4-1BB(3H3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2), SEQ ID NO.50(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、SEQ ID NO. 50 (IL2-a4-1BB(LOB12.3): IL2-VL-VH(ScFv)-Fc(wt)), SEQ ID NO.51(IL2-a4-1BB(3H3)：IL2-VL-VH(ScFv)-Fc(wt))所示的核苷酸序列。The nucleotide sequence shown in SEQ ID NO. 51 (IL2-a4-1BB(3H3):IL2-VL-VH(ScFv)-Fc(wt)). 15.权利要求1-12任一所述的双功能融合蛋白的如下应用：15. the following application of the arbitrary described bifunctional fusion protein of claim 1-12: (1)制备抗肿瘤药物；(1) Preparation of antitumor drugs; (2)制备与免疫检查点抑制剂联用的抗肿瘤药物；(2) Preparation of anti-tumor drugs combined with immune checkpoint inhibitors; (3)制备克服免疫检查点抑制剂耐受的抗肿瘤药物；(3) Preparation of anti-tumor drugs that overcome immune checkpoint inhibitor tolerance; (4)制备与TKI拮抗剂联用的抗肿瘤药物；(4) preparation of antitumor drugs combined with TKI antagonists; (5)制备克服TKI拮抗剂耐受的抗肿瘤药物。(5) Preparation of antitumor drugs that overcome TKI antagonist tolerance. 16.根据权利要求15所述的应用，其特征在于，所述的免疫检查点抑制剂为抗PD-L1的拮抗剂，优选的，所述的抗PD-L1的拮抗剂为抗PD-L1的抗体。16. The use according to claim 15, wherein the immune checkpoint inhibitor is an anti-PD-L1 antagonist, preferably, the anti-PD-L1 antagonist is an anti-PD-L1 of antibodies. 17.根据权利要求15所述的应用，其特征在于，所述的TKI拮抗剂为小分子TKI拮抗剂；优选的，所述的小分子TKI拮抗剂为阿法替尼或其结构类似物。17. The use according to claim 15, wherein the TKI antagonist is a small molecule TKI antagonist; preferably, the small molecule TKI antagonist is afatinib or a structural analog thereof. 18.包含权利要求1-12任一所述的双功能融合蛋白的药物或药物组合物。18. A medicament or pharmaceutical composition comprising the bifunctional fusion protein of any one of claims 1-12.
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