CN114437228B - Double-function fusion protein composed of IL-2 and antibody subunit - Google Patents

Double-function fusion protein composed of IL-2 and antibody subunit Download PDF

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CN114437228B
CN114437228B CN202011189127.4A CN202011189127A CN114437228B CN 114437228 B CN114437228 B CN 114437228B CN 202011189127 A CN202011189127 A CN 202011189127A CN 114437228 B CN114437228 B CN 114437228B
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CN114437228A (en
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彭华
曹帅帅
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Institute of Biophysics of CAS
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Abstract

The invention relates to a bifunctional fusion protein composed of IL-2 and an antibody subunit, which comprises the following components: a heterodimer consisting of a first monomer and a second monomer as follows: (1) A first monomer formed by connecting interleukin 2 (IL-2) with an immunoglobulin Fc single chain; (2) The Fab/ScFv of the antibody of the TNF family costimulatory or cosuppressive molecule or CTLA4 molecule is linked to the Fc single chain. The bifunctional fusion protein further comprises a homodimer, and the homodimer monomers comprise: (1) an IL-2 functional block; and (2) a monomer of an antibody or a monomer formed by connecting Fab/ScFv of the antibody with Fc single chain, wherein the antibody is an antibody of a TNF family costimulatory or cosuppression molecule or CTLA4 molecule. The invention also relates to application of the bifunctional fusion protein in preparing antitumor drugs.

Description

Double-function fusion protein composed of IL-2 and antibody subunit
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a bifunctional fusion protein composed of IL-2 and an antibody subunit.
Background
Interleukin 2 (IL 2), also known as T cell growth factor, was found in 1976 to stimulate T cell clonal expansion in an in vitro experiment [1]. IL2 is a glycoprotein of four alpha helices, with a molecular weight of 15.5Kd 2, secreted primarily by activated CD 4T cells, and other cells such as activated CD8 cells, NK cells, NKT cells, and ILCs cells can also produce small amounts of IL2[3,4]. IL2 functions by binding to receptor cells via the autocrine and paracrine pathways, and has an important regulatory role on T, NK cells.
The receptor for IL2 consists of three subunits, IL2R alpha (CD 25), IL2R beta (CD 122) and IL2R gamma c (CD 132), and there is a difference in affinity between IL2 and the three subunits [5,6]. IL2 binds with low affinity (Kd-10-8M) to the alpha subunit, with medium affinity (Kd-10-9M) to the beta gamma dimer and with high affinity (Kd-10-11M) to the alpha beta gamma trimer [7,8].
The expression levels of different affinity receptors on different cells are different: beta gamma receptors are mainly expressed on T cells, CD 8T memory cells and NK cells in resting state; the alpha receptor is up-regulated after cell activation and the activated T cells express a high affinity alpha beta gamma receptor. Treg cells continuously express CD25 molecules in high degree, and have high affinity to IL2 molecules [9 ]]. Because of the variability in receptor expression, low doses of IL2 preferentially expand Treg cells expressing high affinity receptors, and high doses of IL2 are effective in activating CD8 + T cells and NK cells [10 ]]。
IL2 has the highest affinity for the trimeric receptor, which is predominantly expressed on Treg cells continuously and only transiently up-regulated on activated effector cells; clinically, low doses of IL2 will preferentially expand Treg cells, whereas high doses of IL2 will activate effector T cells. High doses of IL2 can lead to greater toxic side effects that limit the use of IL 2.
To overcome this problem, attempts have been made to engineer IL2 molecules by different means to reduce Treg binding and to increase effector T cell and NK cell binding to enhance their effect in tumor therapy while avoiding the toxicity resulting from high dose use. At present, the IL2 is modified by the following methods:
(1) PEGylated IL2
IL2 has a molecular weight of 15.5KD and a half-life in serum of 10-85min in vivo, and requires multiple repeated administrations to achieve therapeutic effects. PEGylated IL2 increases the overall molecular weight of IL2 and increases the half-life of IL2, while IL2 may be biased to bind IL2-Rα or IL2-Rβ [11] by differences in the PEGylated modification sites.
(2) IL2/aIL2 antibody complexes
Binding of IL2 to the aIL2 antibody complex can extend the half-life of IL2 on the one hand and the IL2/aIL antibody complex can exhibit a different propensity to bind to the receptor CD25 or CD122 on the other hand, as the aIL2 antibody can bind to different sites of the IL2 molecule.
(3) IL2 mutant engineering
The affinity of IL2 for different receptor subunits can also be altered by engineering point mutations at different receptor binding sites on IL 2. By point mutation of the five bases L80F, R81D, L85V, I V and I92F of the IL2 molecule, conformational changes of IL2 occur, increasing binding to CD122 while rendering downstream signaling independent of the CD25 molecule. The mutated IL2 preferentially amplifies CD8+ T cells and NK cells, has lower toxic side effects and shows better tumor treatment effect in tumor models of mice [12].
In our pre-laboratory engineering, we performed mutations on IL2 that reduced CD25 subunit binding (F42A) and increased CD122 subunit binding simultaneously. Such double mutated IL2 molecules have better therapeutic effects than either the reduced affinity mutation of the CD25 subunit alone or the increased affinity mutation of the CD122 subunit alone [13]. The double-mutation IL2 not only has good treatment effect in MC38 tumor model; in the TUBO and other cold tumor models, the combination treatment with TKI or 7.16.4 antibody also has good treatment effect. It was demonstrated that further decreasing the affinity of the CD25 subunit while increasing the affinity of the CD122 subunit may even further enhance the therapeutic effect of IL 2.
The above-described modifications to IL2 have been made by either decreasing IL2 binding to the CD25 molecule or increasing binding to the CD122 subunit. Although this engineered strategy has improved therapeutic efficacy compared to wild-type IL2 molecules, it is still not possible to completely eliminate the inhibitory effect of intratumoral Treg cells on therapeutic efficacy.How to further compare and analyze intratumoral Treg cells and CD25 and CD122 receptor surface on effector T cellsThe variability achieved to further enhance the therapeutic effect of IL2 remains a key Problem(s)。
The co-stimulatory or co-inhibitory molecules of the TNF family are also highly expressed on Treg cells and are target molecules for regulating the functions of the Treg cells, and common molecules include ICOS, GITR, OX, 4-1BB and the like.
Some co-stimulatory molecules such as GITR and OX40 molecules are highly expressed on intratumoral tregs and also up-regulate expression on activated CD4 and CD 8T cells. OX40 molecules are expressed on T cells within the tumor and are highly expressed on tregs within the tumor; systemic administration of the activating aOX antibody can lead to a decrease in the number and proportion of intratumoral tregs to exert an antitumor effect [14]. No significant decrease in Treg number but an increase in the number of effector T cells was observed in the clinical trial of the aOX antibody [15].
OX40 molecules are co-expressed on effector T cells and Treg cells, and currently used OX40 antibodies exert antitumor effects, both activating effector cells and deleting Treg cells. OX40 exerted antitumor effects depended on the expansion of intratumoral CD 4T and CD 8T cells, and tumors did not recur after mice had immune memory re-challenge after treatment [16]. On the other hand, OX40 antibody OX86 used in the mouse experiments may also function to delete tregs, which deletion depends on activated fcγr and not on inhibitory fcγriib. Moreover, after changing the rIgG1Fc of OX86 to mouse IgG2a and IgG2An297A (not binding to FcR), respectively, the Fc of IgG2a was found to have better tumor control, demonstrating that the function of OX40 antibody on this tumor model was mainly dependent on the function of Treg deletion; moreover, the deletion occurs mainly in Treg cells, but not in CD 4T cells and CD 8T cells of Treg, and the number and absolute number are not reduced [17]. When using OX40 targets for treatment, we can exploit the difference in abundance of OX40 expression on tregs and effector T cells to exert different effects on different T cell populations. In addition to OX40 antibodies alone, OX40 molecules can be used in combination with chemotherapy, radiation therapy, and other cytokines, all with synergistic effects to exert better antitumor effects [18].
Therefore, IL2/aOX40 bifunctional molecules, as well as fusion protein bifunctional antibodies composed of antibodies to CTLA4 and other costimulatory or cosuppressive molecules of the TNF family ICOS, GITR, 4-1BB, etc. and IL-2 cytokines, are clinically novel tumor treatment protocols with great development potential.
Disclosure of Invention
The invention firstly relates to a bifunctional fusion protein which is a heterodimer and is characterized in that,
the heterodimer comprises:
(1) A heterodimeric first monomer formed by connecting interleukin 2 (IL-2) and an immunoglobulin Fc single chain;
(2) A heterodimeric second monomer formed by linking Fab or ScFv of an antibody against a CTLA4 molecule or a costimulatory or cosuppressive molecule of the TNF family with an immunoglobulin Fc single chain;
the first monomer and the second monomer are connected through dimerization of Fc single chains to form the heterodimer;
the CTLA4 or TNF family co-stimulatory or co-inhibitory molecules 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 with ADCC effect knocked out by gene mutation;
preferably, 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; more preferably, the immunoglobulin Fc single chain is that of human IgG.
The Co-stimulatory or co-inhibitory molecules of CTLA4 or TNF family are OX40, 4-1BB, and the antibodies against CTLA4 (aCTLA 4) or anti-TNF family are anti-OX 40 (aOX 40), anti-4-1 BB (a 4-1 BB);
in the second monomer, the monomer is a monomer,
the Fab of the antibody is the Fab of the humanized antibody or the Fab of the fully humanized antibody;
the ScFv of the antibody is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody;
more preferably, the second monomer is:
monomers of an antibody against a co-stimulatory or co-inhibitory molecule of CTLA4 or anti-TNF family, the monomers of the antibody 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) The first monomer, first monomer from N end contains in proper order:
1) A wild type IL-2 protein with a sequence shown as SEQ ID NO.1 or a 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) The necessary linkage (G4S linkage) is preferably as shown in SEQ ID NO. 6;
3) An Fc single chain of IgG shown in SEQ ID NO.2, or an Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or a knob mutant Fc shown in SEQ ID NO.4, or a hole mutant Fc shown in SEQ ID NO. 5;
(2) A second monomer comprising:
1) An anti-OX 40 antibody Fab region consisting of an anti-OX 40 antibody light chain VL-KCL with a sequence shown as SEQ ID NO.7 and an anti-OX 40 antibody heavy chain VH & CH1 with a sequence shown as SEQ ID NO. 8;
or: 2) An anti-OX 40 single chain antibody (ScFv) having the sequence shown in SEQ ID NO. 9:
and: 3) An Fc single chain of IgG shown in SEQ ID NO.2, or an Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or a knob mutant Fc shown in SEQ ID NO.4, or a hole mutant Fc shown in SEQ ID NO. 5;
more preferably, the heterodimer comprises:
a first monomer: the polypeptide is a polypeptide (IL 2-Fc) with a sequence shown as SEQ ID NO. 10;
and a second monomer: it is:
(1) A second monomer consisting of an anti-OX 40 antibody VH-CH1-Fc (knob) having the sequence shown in SEQ ID NO.11 and an anti-OX 40 antibody light chain VL-KCL having the sequence shown in SEQ ID NO. 7;
or (2) a polypeptide (aOX ScFv-Fc (knob)) having a sequence shown in SEQ ID NO. 12.
The invention also relates to a double-function fusion protein, which is a homodimer and is characterized in that,
The monomer of the homodimer is as follows:
a monomer comprising a molecule of interleukin 2 (IL-2) and a molecule of anti-OX 40 antibody Fab linked by any means, or,
a molecule of interleukin 2 (IL-2) and a molecule of anti-OX 40 single chain antibody (ScFv) are connected by any means.
Preferably, the monomers of the homodimer sequentially comprise, from the N-terminal:
(1) A wild-type IL-2 protein as shown in SEQ ID No.1, or a mutant of said wild-type IL-2 protein, said mutant comprising a mutation site of any one or any combination of the following; f42A, L80F, R81D, L85V, I86V and I92F;
(2) The necessary linkage (G4S linkage) is preferably as shown in SEQ ID NO. 6;
(3) Fab or ScFv of anti-OX 40 antibodies; the Fab is the Fab of the humanized antibody or the Fab of the fully humanized antibody, and the ScFv is the ScFv of the humanized antibody or the ScFv of the fully humanized antibody;
(4) An Fc of an antibody; the antibody Fc is a fully human wild-type Fc or a 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) SEQ ID NO.14 (IL 2-aOX40: IL2-VL-VH (ScFv) -Fc (wt)).
The invention also relates to another bifunctional fusion protein, wherein the bifunctional fusion protein is formed by combining an anti-CTLA 4 antibody and IL2, and the fusion protein is a heterodimer;
The heterodimer comprises:
(1) The first monomer, first monomer from N end contains in proper order:
1) A wild-type IL-2 protein as shown in SEQ ID No.1, or a mutant of said wild-type IL-2 protein, said mutant comprising a mutation site of any one or any combination of the following; f42A, L80F, R81D, L85V, I86V and I92F;
2) The necessary linkage (G4S linkage) is preferably as shown in SEQ ID NO. 6;
3) An Fc single chain of IgG shown as SEQ ID NO.2, or an Fc of No-ADCC mutant IgG shown as SEQ ID NO.3, or a knob mutant Fc shown as SEQ ID NO.4, or a hole mutant Fc shown as SEQ ID NO. 5;
(2) A second monomer comprising:
1) An antibody Fab region composed of an anti-CTLA 4 antibody light chain VL-KCL with a sequence shown as SEQ ID NO.21 and an anti-CTLA 4 antibody heavy chain VH & CH1 with a sequence shown as SEQ ID NO. 22;
or: 2) An anti-CTLA 4 single chain antibody (ScFv) with a sequence shown in SEQ ID NO. 23:
and: 3) An Fc single chain of IgG shown in SEQ ID NO.2, or an Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or a knob mutant Fc shown in SEQ ID NO.4, or a hole mutant Fc shown in SEQ ID NO. 5;
More preferably, the heterodimer comprises:
a first monomer: the polypeptide is a polypeptide (IL 2-Fc) with a sequence shown as SEQ ID NO. 10;
and a second monomer: it is:
(1) A second monomer composed of a polypeptide (aCTLA 4 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 (b)
(2) A polypeptide (a CTLA4 ScFv-Fc (knob)) shown in SEQ ID NO. 25.
The invention also relates to a double-function fusion protein, which is a homodimer and is characterized in that,
the monomer of the homodimer is as follows:
a monomer formed by connecting a molecule of interleukin 2 (IL-2) and a molecule of anti-CTLA 4 antibody Fab through any way, or,
a monomer formed by connecting a molecule of interleukin 2 (IL-2) and a molecule of anti-CTLA 4 single chain antibody (ScFv) in any mode.
Preferably, the monomers of the homodimer sequentially comprise, from the N-terminal:
(1) A wild-type IL-2 protein as shown in SEQ ID No.1, or a mutant of said wild-type IL-2 protein, said mutant comprising a mutation site of any one or any combination of the following; f42A, L80F, R81D, L85V, I86V and I92F;
(2) The necessary linkage (G4S linkage) is preferably as shown in SEQ ID NO. 6;
(3) Fab or ScFv of anti-CTLA 4 antibodies; the Fab is the Fab of the humanized antibody or the Fab of the fully humanized antibody, and the ScFv is the ScFv of the humanized antibody or the ScFv of the fully humanized antibody;
(4) An Fc of an antibody; the antibody Fc is a fully human wild-type Fc or a 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 (IL 2-aCTLA 4: IL2-VL-VH (ScFv) -Fc (wt)).
The invention also relates to another bifunctional fusion protein, wherein the bifunctional fusion protein is formed by combining an anti-4-1 BB antibody and IL2, and the fusion protein is a hetero-or homodimer;
the heterodimer comprises:
(1) The first monomer, first monomer from N end contains in proper order:
1) A wild-type IL-2 protein as shown in SEQ ID No.1, or a mutant of said wild-type IL-2 protein, said mutant comprising a mutation site of any one or any combination of the following; f42A, L80F, R81D, L85V, I86V and I92F;
2) The necessary linkage (G4S linkage) is preferably as shown in SEQ ID NO. 6;
3) An Fc single chain of IgG shown as SEQ ID NO.2, or an Fc of No-ADCC mutant IgG shown as SEQ ID NO.3, or a knob mutant Fc shown as SEQ ID NO.4, or a hole mutant Fc shown as SEQ ID NO. 5;
(2) A second monomer comprising:
1) An antibody Fab region composed of an anti-4-1 BB antibody light chain with the sequences shown as SEQ ID NO.28 and 29 and an anti-4-1 BB antibody heavy chain VH & CH1 with the sequences shown as SEQ ID NO.30 and 31;
or: 2) Anti-4-1 BB single-chain antibody (ScFv) with the sequences shown in SEQ ID NO.32 and 33:
and: 3) An Fc single chain of IgG shown in SEQ ID NO.2, or an Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or a knob mutant Fc shown in SEQ ID NO.4, or a hole mutant Fc shown in SEQ ID NO. 5;
more preferably, the heterodimer comprises:
a first monomer: the polypeptide is a polypeptide (IL 2-Fc) with a sequence shown as SEQ ID NO. 10;
and a second monomer: it is:
(1) A second monomer composed of a polypeptide (a 4-1BB VH-CH1-Fc (knob)) with the sequences shown in SEQ ID No.34 and 35 and an anti-4-1 BB antibody light chain with the sequences shown in SEQ ID No.28 and 29; or (b)
(2) The polypeptide (a 4-1BB ScFv-Fc (knob)) shown in SEQ ID NO.36 and 37.
The fusion protein is a homodimer and is characterized in that,
the monomer of the homodimer is as follows:
a monomer formed by connecting a molecule of interleukin 2 (IL-2) and a molecule of anti-4-1 BB antibody Fab through any means, or,
A molecule of interleukin 2 (IL-2) and a molecule of anti-4-1 BB single-chain antibody (ScFv) are connected by any mode.
The monomer of the homodimer sequentially comprises the following components from the N end:
(1) A wild-type IL-2 protein as shown in SEQ ID No.1, or a mutant of said wild-type IL-2 protein, said mutant comprising a mutation site of any one or any combination of the following; f42A, L80F, R81D, L85V, I86V and I92F;
(2) The necessary linkage (G4S linkage) is preferably as shown in SEQ ID NO. 6;
(3) Fab or ScFv of an anti-4-1 BB antibody; the Fab is the Fab of the humanized antibody or the Fab of the fully humanized antibody, and the ScFv is the ScFv of the humanized antibody or the ScFv of the fully humanized antibody;
(4) An Fc of an antibody; the antibody Fc is a fully human wild-type Fc or a 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 (IL 2-a4-1BB (3H 3): IL2-VL-VH (ScFv) -Fc (wt)).
It should also be clear to a person skilled in the art that:
the following were used:
functional structure of anti-human OX40 antibody encoded by the amino acid sequences shown in SEQ ID nos. 52-55
Functional structure of anti-human CTLA4 antibody encoded by amino acid sequences shown in SEQ ID No.56-57
Functional structure of anti-human 4-1BB antibody encoded by amino acid sequence shown in SEQ ID No.58-61
The technical effects described in the present invention can be achieved as well by replacing the similar functional blocks of the respective bifunctional fusion proteins described above.
The invention also relates to nucleotide sequences encoding said heterodimers and homodimers.
Preferably, the method comprises the steps of,
the nucleotide sequence of the first monomer of the encoding hetero-dimer is shown as SEQ ID NO.15 (IL 2-Fc (hole));
the nucleotide sequence encoding the heterodimeric second monomer 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))、
nucleotide sequence shown in SEQ ID NO.47 (a 4-1BB (3H 3): scFv-Fc (knob));
the nucleotide sequences encoding the homodimers are:
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 as SEQ ID NO.51 (IL 2-a4-1BB (3H 3): IL2-VL-VH (ScFv) -Fc (wt)).
The invention also relates to the following application of the bifunctional fusion protein:
(1) Preparing an antitumor drug;
(2) Preparing an anti-tumor drug for use in combination with an immune checkpoint inhibitor;
(3) Preparing an anti-tumor drug which overcomes tolerance of an immune checkpoint inhibitor;
(4) Preparing an anti-tumor drug for use in combination with a TKI antagonist;
(5) Preparing the anti-tumor medicine for overcoming the tolerance of the TKI antagonist.
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 the IL2 are provided, the IL2/aOX40-hIgG1 antibody deletes the intratumoral Treg cells while keeping the activation of the IL2 to the effector cells, overcomes the adverse factors which can cause the expansion of the Treg in the use process of the IL2, and provides a new idea for the clinical application of the IL 2;
(2) Has important significance in overcoming tolerance of immune checkpoint blocking therapy and resistance generated after the use of TKI chemotherapeutics.
Drawings
FIG. 1, IL2-Fc fusion protein can partially control tumor growth, the molecular structure of 1A, IL-Fc fusion protein, the lower dimer is the Fc region dimer of human IgG, and one molecule of wild type IL-2 molecule is coupled to the N end of each Fc monomer; inhibition of tumor growth by 1B, IL2-Fc fusion protein.
Fig. 2, aOX antibody stimulates T cell activation and exerts antitumor function, 2A, aOX antibody is able to effectively activate CD 3T cells; the 2B, aOX antibody can effectively control tumor growth.
FIG. 3, IL2-Fc and aOX40 combination therapy did not show significant synergy
FIG. 4, construction of a schematic diagram of the structure of an IL2/aOX40-Fc bispecific antibody, 4A, IL2/aOX40-Fc bispecific antibody; 4B, SDS-PAGE analysis constructed IL 2/aOX-Fc bispecific antibody; 4C, IL 2/aOX-Fc heterodimer form, aOX40 being Fab or scFv form, the antibody comprising 1 IL2 molecule; 4D, IL 2/aOX-Fc, aOX40 in the form of scFv, 1 or 2 IL2 molecules linked at the N-terminus of the V region or at the C-terminus of the Fc region; 4E, IL 2/aOX-Fc homodimer form, aOX40 being in Fab form, 2 IL2 molecules linked at the N-terminus or C-terminus of the light chain, at the N-terminus or C-terminus of the heavy chain; 4F, IL 2/aOX-Fc heterodimeric form, aOX40 is in the form of a Fab, with 1 IL2 molecule attached at the N-terminus or C-terminus of the light chain, at the N-terminus or C-terminus of the heavy chain, and at the Fc.
FIG. 5, IL2/aOX40-Fc antibody has synergistic therapeutic effect, 5A, in vitro CTLL2 proliferation assay; tumor suppression effect of 5B, IL2/aOX wt Fc fusion protein (MC 38 tumor model); 5C, re-challenge test results (MC 38 tumor model) in IL2/aOX40-Fc treatment group; 5D, IL2/aOX wt. Fc fusion protein (B16F 10 tumor model).
FIG. 6, IL2/aOX40-Fc antibody therapeutic effect is NK cell independent; one day after 6A, NK cell-deleted antibody injection, the proportion of NK cells in the peripheral blood of mice was flow analyzed; 6B, growth curves of MC38 tumors of different treatment groups.
FIG. 7, IL2/aOX40wt Fc function was dependent on CD 8T cells; 7A, 200ug of the deletion antibody is injected for one day, and the deletion efficiency of the CD 4T cells and the CD 8T cells in the peripheral blood is detected in a flow mode; 7B, tumor growth curves for different treatment groups.
FIG. 8, intratumoral T cells play a key role in the treatment of IL2/aOX 40-Fc; 20ug FTY720 was injected intraperitoneally into 8A, MC tumor-bearing mice, and peripheral blood CD 3T cells were detected after 2 days; 8B, tumor growth curves of different treatment groups.
FIG. 9, in situ tumor local therapy induced immune response can control distal tumor growth; 9A, tumor treatment effect on the right side (administration side) of different treatment groups; tumor treatment effect on the left side (non-treatment side) of the different treatment groups 9B.
Fig. 10, CD25 and OX40 were highly expressed on intratumoral Treg cells; 10A, flow analysis of CD4, CD8 and CD25, OX40 expression on Treg cells in tumors, comparison of CD25, OX40 expression on intratumoral, draining lymph nodes and spleen Treg cells; 10B, MFI statistical analysis of different cellular expression levels.
FIG. 11, IL2/aOX40-Fc treatment of intratumoral Treg increase CD 8T cell to Treg ratio; 11A, different treatment groups of intratumoral Treg cell flow charts; 11B, different treatment groups intratumoral Treg/CD 4T and CD 8T/Treg ratios; 11C, draining lymph nodes and spleen Treg/CD 4T ratio.
Fig. 12, the function of antibodies, in part, depends on Fc binding to fcγr.
FIG. 13 shows that aPDL1 antibody can synergistically improve the anti-tumor effect of IL2/aOX 40-Fc.
FIG. 14, IL2/aOX40-Fc was able to overcome resistance to Afatinib treatment, with more T cells infiltrating into the tumor during Afatinib treatment, but no change in the proportion of tregs; combination therapy with 14C, afatinib and IL2/aOX40-Fc antibody is effective in controlling tumor growth and recurrence.
FIG. 15, IL2/aCTLA4 wt Fc fusion protein enhances tumor suppression (MC 38 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
And (3) strain: top10 E.coli, DH 5. Alpha. E.coli competent cells (Beijing full gold biotechnology Co., ltd.)
Plasmid:
pEE12.4-IgG kappa-hIgG 1 comprising a signal peptide of mouse IgG kappa and an Fc sequence of human IgG1 for expression of the antibody.
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 expressing the antibody portion of the heterodimeric protein; pEE12.4-Wt IL-2-Fc-hole, IL-2 portion for expression of heterodimeric proteins.
2. Experimental animal
Wild type C57BL/6, BALB/C mice and BALB/C-nude mice were purchased from Beijing veteh Liwa laboratory animal hearts, china. All experiments used female mice of 8-10 weeks of age, except for the specific instructions. Mice were kept in a barrier environment free of specific pathogenic microorganisms (specific pathogen-free, SPF). Animal feeding and experimental operations are in compliance with the relevant regulations of the animal management committee of the national academy of sciences of biophysics research.
3. Cell lines
MC38 is a C57 background mouse colorectal cancer cell line,
MC38-EGFR5 is a monoclonal cell line obtained by respectively infecting a slow virus expressing human-mouse chimeric Epidermal Growth Factor (EGFR) with MC38 and screening,
TUBO is a BALB/C background mouse breast cancer cell line and B16F10 is a C57 background mouse melanoma cell line.
These 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-210 (TM) hybridoma cell line (ATCC) for expression of a deleted antibody of CD8+ T cells (clone: 2.43).
TIB-207 (TM) hybridoma cell line (ATCC) for expression of deleted antibodies to CD4+ T cells (clone: GK 1.5).
The PK136 hybridoma cell line was used to produce deleted antibodies to NK cells.
HB-197TM hybridoma cell line (ATCC) for the expression of antibodies blocking mouse FcgammaRII/III (clone: 2.4G2).
The freeform 293F cell line (Invitrogen) is a suspension cell derived from HEK293 cell line, cultured in SMM293-TII or CD OptiCHOTM medium, and is mainly used for transient transfection expression of fusion proteins.
CTLL-2 cell lines are mouse T cell lines for detecting the biological activity of IL-2
The above cell lines were cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2mmol/L L-glutamine, 0.1mmol/L nonessential amino acids, 100U penicillin and 100. Mu.g/ml streptomycin, 100IU/ml recombinant IL 2).
Design and synthesis of genes and primers
The wild type and mutant IL-2 gene sequence of human is shown in SEQ ID NO. 1. Primers used in the experiments were all designed by DNAMAN software and synthesized by Invitrogen company.
Mouse tumor inoculation and treatment
(1) Tumor inoculation and measurement:
the establishment of a tumor model is carried out,
5-7.5×10 5 the MC38, MC38-EGFR5 single cells were suspended in 100 μl PBS and inoculated subcutaneously on the backs of C57BL/6 mice;
5-7.5×10 5 each TUBO single cell was suspended in 100. Mu.l PBS and inoculated subcutaneously into the back of BALB/c mice.
When the re-challenge experiment of the same tumor cells is carried out on a tumor-resolved mouse, the inoculation quantity of the tumor cells is 5 times that of the initial tumor modeling, and the inoculation part is subcutaneous on the other side of the back of the mouse. Tumor size was monitored twice a week and tumor long diameter (a), short diameter (b) and high (c) were measured using vernier calipers, with mouse tumor volume = a x b x c/2.
(2) Treatment:
the antibodies or antibody fusion proteins are administered intraperitoneally, and in some experiments, intratumoral administration is also used, and the specific dosages will be described in the specific experiments.
Monoclonal antibody preparation (mouse ascites method)
CD4+ T cell deletion antibody GK1.5, CD8+ T cell deletion antibody TIB210, NK cell deletion antibody PK136 and FcRII/III blocking antibody used in the experiment are all from corresponding hybridoma cells, and are produced and purified by the laboratory.
Cell deletion in mice
(1) Deletion of cd4+ T cells, cd8+ T cells:
200 μg GK1.5 or TIB210 antibody was intraperitoneally injected to delete CD4+ T cells, CD8+ T cells, respectively, the day before treatment with IL-2 or IL-2 fusion protein, and then injected once every 3 days, and the number of injections was adjusted according to the treatment cycle. Deletion efficiency is detected by streaming.
(2) Deletion of NK cells and neutrophils:
the NK cells or neutrophils were deleted by intraperitoneal injection of 400. Mu.g of PK136 or 1A8 antibody, respectively, the day prior to treatment with IL-2 or IL-2 fusion protein. And detecting deletion efficiency by streaming.
T cell migration block
FTY720 (available from Sigma) is an immunosuppressant that reduces T cell migration from lymphoid organs to peripheral blood circulation. In the present invention, FTY720 blocking was performed at different times of tumor inoculation in mice to alter tumor microenvironment. Blocking was performed during tumor treatment in mice: 20 mug of FTY720 is injected intraperitoneally 1 day before tumor treatment, 10 mug of FTY720 is injected intraperitoneally every other day, and the blocking time is dependent on the treatment period, so that no T cells which are newly migrated into tumor tissues exist in the tumor treatment process. The importance of infiltrating lymphocytes in tumor tissue can be studied by means of FTY720 blocking protocol.
The molecular structure of the IL2/aOX40-Fc bispecific antibodies in examples 1-5 below are, unless otherwise specified, the molecular structure shown in FIG. 4A, i.e.a heterodimer consisting of one molecule of IL2-Fc and one molecule of aOX (Fab) -Fc.
Example 1, IL2/aOX40-Fc bispecific antibody significantly improved over treatment alone
1. Treatment with IL2-Fc fusion proteins can partially control tumors
Free IL2 alone has a short half-life and requires multiple doses, and in order to extend the half-life of IL2 we designed IL2-Fc fusion proteins (for structural schematic see FIG. 1A) and validated the function of the fusion proteins in MC38 tumor models. IL2-Fc was able to effectively control tumor growth compared to untreated control, but IL2-Fc treatment alone failed to completely clear the tumor when the tumor was large (FIG. 1B). Subcutaneous inoculation of C57BL/6 mice 5X 10 5 MC38 tumor cells, tumor-bearing mice began group treatment on day D12 (n=5/group): intraperitoneal injection (ip) of 9ug IL2-Fc fusion protein was treated every three days for a total of three treatments.
The results show that IL2-Fc fusion protein therapy alone does not continuously inhibit tumor growth.
2. anti-OX40 antibodies promote T cell activation and exert antitumor function
Treatment with the IL2-Fc fusion protein alone only partially controls tumor growth, and we adopted a combination strategy to further enhance the therapeutic effect. OX40 molecules are expressed only on Treg cells and activated T cellsThe antibody targeting OX40 has higher specificity because the antibody is not expressed on T cells; furthermore, the expression abundance of OX40 on Treg cells and effector cells is different and the same The target provides a theoretical basis for different effects on different cells. To verify whether the OX40 signaling pathway activates T cells and exerts anti-tumor effects, we performed spleen +.>T cell in vitro stimulation experiments. The specific method comprises the following steps: spleen cells of mice were isolated at 3X 10 5 Cells/well were plated in 96-well plates and assayed for CD69 expression levels on CD 3T cells in a flow-through assay after 72h stimulation with either aCD3 and aCD28 or aCD3 and aOX antibodies, respectively, with aCD3 and aCD28 antibody concentrations of 1ug/ml and aOX antibody concentrations of 2ug/ml.
The results showed that the aOX antibody and aCD3 combined used gave similar stimulation to aCD28, compared to the co-stimulatory effect of aCD3 and aCD28 on T cells, and was able to effectively activate CD 3T cells (fig. 2A).
We further validated the function of anti-OX40 in anti-tumor experiments in mice, the specific method was: subcutaneous inoculation of wild-type C57BL/6 mice with 5X 10 5 MC38 tumor cells, tumor growth to 100-150mm 3 Start grouping treatment (n=5/group): the 16ug of aOX40 antibody was injected intraperitoneally (ip) and treated three times per day.
The results showed that intraperitoneal injection (ip) 16ug of ax40 antibody was effective in controlling tumor growth compared to untreated groups (fig. 2B).
3. IL2-Fc fusion protein and aOX40 combined therapy does not obviously improve the treatment effect of tumors
IL2-Fc alone expands tregs, while aOX antibody can delete intratumoral Treg cells, in order to verify whether the combined use of IL2-Fc and aOX antibody can delete expanded tregs simultaneously with activating effector T cells, exerting the effect of synergistic treatment. We simultaneously administered two protein therapies on MC38 tumor-bearing mice by: subcutaneous inoculation of C57BL6 mice with 5X 10 5 MC38 tumor cells, tumor growth to D14 and treatment are started, 6ug of IL2-Fc or 9ug of aOX40 antibody are respectively injected into the abdominal cavity of a single treatment group, and two antibodies are simultaneously injected into the abdominal cavity of a combined treatment group; three times of treatment are carried out once every three days.
The results showed that the combination therapy did not further increase the therapeutic effect of IL2-Fc compared to aOX antibody or IL2-Fc alone (figure 3).
4. IL2/aOX40-Fc bispecific antibodies with synergistic therapeutic effects
4.1 construction of bispecific antibodies
The analysis of the experimental results shows that the simple mixed combination treatment does not have obvious synergistic treatment effect or even additive effect, which suggests that the two antibodies are singly combined and cannot simultaneously exert the effect of activating effector cells and deleting tregs. Accordingly, to reduce IL2 binding, expand Treg cells, we constructed two structurally identical bispecific antibodies (IL 2/aOX WT Fc and IL2/aOX no ADCC Fc), each containing only one IL2 molecule (fusion protein structure see fig. 4A), in which the Fc dimer is that of human IgG or that of a deletion of ADCC effector function, one Fc monomer was linked to the WT IL2 protein and the other Fc monomer was linked to the Fab fragment of the aOX antibody. The constructed fusion protein plasmid was transfected into 293F cells, and the cell supernatant was harvested seven days later. Protein A column is used for purifying protein after supernatant, and the purified antibody is identified by protein gel. Analysis of protein molecular composition by SDS-PAGE demonstrated that bispecific molecules were able to express the light chain, heavy chain and IL2 molecules of the aOX antibody intact simultaneously (fig. 4B).
We designed and constructed antibodies comprising different forms of IL2 and aOX:
the structure illustrated in fig. 4C is a heterodimer, the antibody comprising one IL2 molecule, aOX (anti-OX 40 antibody) in Fab or scFv form, the Fc dimer being a heterodimer formed by the Fc monomer of human IgG and the Fc monomer of human IgG with the ADCC effector function deleted.
In the structure illustrated in fig. 4D, aOX (anti-OX 40 antibody) is in the form of scFv, with one or two IL2 molecules attached at the N-terminus of the V region or at the C-terminus of the Fc, the Fc dimer being the Fc homodimer of wild-type human IgG, or a heterodimer of the Fc monomer of human IgG and the Fc monomer of human IgG with the ADCC effector function deleted;
in the structure illustrated in fig. 4E-F, aOX (anti-OX 40 antibody) is in Fab form, with one or two IL2 molecules attached at the N-or C-terminus of the antibody light chain, at the N-or Fc-terminus of the antibody heavy chain, and the Fc dimer is the Fc homodimer of wild-type human IgG, or a heterodimer of the Fc monomer of human IgG and the Fc monomer of human IgG with the ADCC effector function deleted.
4.2, verifying the function of bispecific antibodies,
(1) In vitro CTLL2 proliferation assay:
IL2-Fc, IL2/aOX wt Fc and IL2/aOX no ADCC Fc protein (structure shown in FIG. 4A) were added to CTLL2 cell culture medium at different concentrations, and the proliferation level of CTLL2 cells at different protein concentrations was detected by CCK8 kit after 72 hours, which showed that IL2/aOX40-Fc antibody was able to effectively amplify CTLL2 cells as IL2-Fc molecule, indicating that the constructed antibody molecule retained IL2 activity (FIG. 5A).
(2) In vivo mouse tumor-bearing experiment:
subcutaneous inoculation of 5X 10 in C57BL6 mice 5 MC38 tumor cells, D14 treatment was initiated after tumor inoculation; 9ug IL2-Fc, 16ug aOX40 antibody, IL2-Fc+ aOX40 antibody equimolar to aOX antibody or IL2/aOX wt Fc protein, respectively, were injected intraperitoneally. Tumor size was measured twice weekly three times a day of co-treatment on days D14, D17, D20; the results showed that the IL2/aOX40-wtFc bispecific antibody had better therapeutic effect compared to aOX antibody alone, IL2-Fc, and a combination of both treatments (FIG. 5B).
The same experiment was performed on a B16F10 tumor-bearing mouse model except for the MC38 model, and a C57BL6 mouse was inoculated subcutaneously with 5X 10 5 B16F10 tumor cells, D8 treatment started after tumor inoculation; 15ug IL2/aOX wt Fc protein by intraperitoneal injection; three times co-treatments were performed on days D8, D11 and D14, and the results showed that IL2/aOX wt 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 removal. Mice treated for complete tumor elimination were subjected to the tumor re-challenge test two months after IL2/aOX wt Fc group, subcutaneously vaccinated with five times the dose (2.5X10) 6 Individual) MC38 tumor cells, re-challenge were inoculated with tumor cells only, and no treatment was given to each group after inoculation.
The results showed that the re-inoculation of five doses of MC38 cells did not cause tumor growth compared to the control group, indicating that bispecific antibody treatment induced the mice to produce memory immune cells (fig. 5C).
Example 2 IL2/aOX40-Fc was able to activate CD 8T cells
1. IL2/aOX40-Fc bispecific antibody therapeutic efficacy is independent of NK cells
Since CD25 (IL-2 receptor α) and OX40 are predominantly expressed on activated effector T cells and NK cells, we performed deletion experiments of different cell populations, respectively, in order to determine which group of immune cells the treatment of IL2/aOX40-Fc antibody is predominantly dependent on.
Subcutaneous inoculation of C57BL6 mice with 5X 10 5 MC38 tumor cells were intraperitoneally injected with 25ug IL2/aOX wt Fc fusion protein on days 12, 15, 18. 400ug of NK cell deleted antibody PK136 (prepared in this laboratory) was intraperitoneally injected 3 times every 4 days before the start of the treatment.
FIG. 6A shows the change in peripheral blood NK cells after 1 day of NK cell deletion. In the mouse tumor experiments, the IL2/aOX40-Fc antibody still had therapeutic effects after NK cells were deleted, indicating that NK cells were not the primary effector cells for the therapeutic effects of the antibody (FIG. 6B).
2. IL2/aOX40-Fc antibody therapeutic effect depends on CD 8T cells
We further validated the role of T cells in antibody therapy.
Subcutaneous inoculation of the back of C57 mice 5X 10 5 Treatment was started on day 12 after MC38 tumor cells, and 25ug IL2/aOX wt Fc protein was intraperitoneally injected (administered on days 12, 15, 18, respectively, after tumor inoculation). 200ug of CD4T cell deleted antibody (clone number: GK1.5, prepared in this laboratory), 200ug of CD8T cell deleted antibody (clone number: TIB210, prepared in this laboratory) or two deleted antibodies were injected intraperitoneally at the same time during the treatment, and the injections were three times in total.
The deletion experiments of the CD 4T and the CD 8T cells show that the antibody treatment effect is not obviously reduced after the CD 4T cells are deleted, but the treatment effect is obviously reduced after the CD 8T cells are deleted; after simultaneous deletion of CD 4T and CD 8T cells, the therapeutic effect of the antibodies was also completely abolished. It was demonstrated that the therapeutic effect of IL2/aOX40-Fc antibodies was T cell dependent and was predominantly CD 8T cells (FIG. 7B).
3. IL2/aOX40-Fc bispecific antibody function dependent on intratumoral T cells
To further examine whether intratumoral infiltrating T cells play a major role or peripheral migrating T cells play a major role during treatment, we performed FTY720 blocking experiments. FTY720 is a drug that can clinically inhibit immune rejection during organ transplantation and inhibit T cell migration from lymphoid organs to peripheral blood [19].
Treatment with IL2/aOX40wt Fc antibody alone can clear tumors, and ftY720 blocking was performed simultaneously with treatment, as follows:
back subcutaneous inoculation of C57BL6 mice 4X 10 5 MC38 tumor cells, 15ug IL2/aOX-Fc antibody was injected intraperitoneally on days 7, 10, and 13 after tumor inoculation. In the FTY720 treatment group, 25ug of FTY720 was injected intraperitoneally at day 6 after tumor inoculation, and 10ug was continued to be injected on days 8, 10 and 12, respectively.
The results showed that inhibition of peripheral lymphocyte migration to the tumor did not affect the therapeutic effect of the bispecific antibody (fig. 8B), suggesting that IL2/aOX40-Fc antibody play an anti-tumor role primarily dependent on effector T cells infiltrating into the tumor.
4. Protection of distal metastatic tumors by treatment with IL2/aOX40-Fc
To verify whether the immune protection generated by in situ tumor therapy has an inhibitory effect on tumors at other metastatic sites, we performed a mouse dual tumor model experiment. At the same time, the mouse is inoculated with tumor cells on the left and right sides of the skin respectively and only one side of the mouse is treated in situ, so as to observe whether the tumor at the far end can be controlled. The experimental protocol was as follows:
c57BL6 mice were inoculated with 2X 10 mice on the left and right sides of the back, respectively 5 MC38 and 4X 10 5 MC38 tumor cells (seeding 4X 10 on the right side of the back) 5 MC38 tumor cells, 2X 10 on the left side 5 MC38 tumor cells). Tumors on the right side grew to 5 on day seven0mm 3 Left tumor at 30mm 3 Treatment was started left and right:
treatment group 1, intratumoral injection of 7.5ug IL2/aOX wt Fc antibody (therapeutic antibody three times total treatment on days 7, 10, 13) on right tumor, left tumor was untreated;
treatment group 2, intratumoral injection of 7.5ug IL2/aOX wt Fc antibody in right tumor, while intratumoral injection of 20ug tib210 antibody in right tumor, deleted intratumoral CD8T cells. Treatment and deletion antibodies were co-treated three times on days 7, 10, 13, left tumor was not treated.
The results are shown in figure 9 of the drawings,
(1) In situ treatment by intratumoral injection of only right tumor can not only clear in situ tumor but also control the untreated tumor on the far side.
(2) Also to verify if control of distant tumors is dependent on CD8T cells of the in situ tumor on the treatment side, we deleted CD8T cells of the in situ tumor at the same time as treatment. After deletion of CD8T cells, both in situ and remote tumor therapeutic effects disappeared, indicating that CD8T cells within in situ tumors play an important role in controlling remote tumors.
Example 3 IL2/aOX40-Fc was able to delete intratumoral Treg and increase CD8/Treg cell ratio
1. Intratumoral Treg cells highly express CD25 and OX40 molecules
To further investigate the anti-tumor mechanism of the IL2/aOX40-Fc antibody, we analyzed the expression levels of CD25 and OX40 molecules on tumor-bearing mouse T cells to analyze to which population of cells the IL2/aOX40-Fc antibody was primarily binding to function. On day 12 after inoculation of MC38 tumors we analyzed expression of IL2 receptors CD25 and OX40 on mouse intratumoral, draining lymph node and spleen T cells, respectively: the specific scheme is as follows: mice were vaccinated 5X 10 5 MC38 tumor cells tumor tissue, spleen and draining lymph node (dLN) of tumor-bearing mice were taken on day 12, respectively, and CD25 and OX40 expression on different cells was flow-tested.
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 external Zhou Yinliu lymph node and spleen tregs. The expression levels of CD 4T and CD8T were lower for non-tregs than for Treg cells, with minimal CD25 and OX40 expression levels for CD8T cells within the tumor (fig. 10A, B).
2. IL2/aOX40-Fc antibody deletes intratumoral Treg cells and improves CD8/Treg cell proportion
Since CD25 and OX40 are highly expressed on intratumoral Treg cells, we speculate that IL2/aOX40-Fc antibody may bind more to Treg after intraperitoneal injection. Treatment was started on day 12 after tumor inoculation of MC38 tumor-bearing mice, and 25ug of IL2/aOX-Fc antibody was intraperitoneally injected, tumor tissue, draining lymph nodes and spleen were harvested on day 15, and the proportion of intratumoral Treg cells, CD8T/Treg, was flow analyzed. Through flow-through experimental analysis, we found that IL2/aOX40-Fc was able to delete intratumoral Treg cells more effectively and increase CD8T/Treg ratio with the best therapeutic effect compared to IL2-Fc and aOX40 alone (fig. 11a, b). IL2/aOX40-Fc did not alter the Treg/CD 4T ratio in the peripheral tumor draining lymph nodes and spleen compared to the deletion of intratumoral tregs, demonstrating that IL2/aOX40-Fc treatment did not break peripheral immune tolerance with better safety (FIG. 11C).
3. IL2/aOX40-Fc antibody therapeutic efficacy depends on Fc binding to FcgammaR
Through analysis of MC38 tumor-bearing mouse intratumoral lymphocytes, we find that the IL2/aOX40-Fc antibody has the effect of deleting tregs, and in order to verify whether Treg deletion is a main mechanism of anti-tumor effect of the IL 2/aOX-Fc antibody, we construct an IL2/aOX40-no ADCC Fc antibody through point mutation of an Fc region, and the mutated Fc does not bind with an Fc gamma R receptor and loses ADCC and ADCP mediated deletion functions. Using the MC38 tumor model, C57BL6 mice were vaccinated with MC38 tumor cells and treatment was started on day 13 post-vaccination. The 15ug wt Fc or no ADCC Fc antibody was intraperitoneally injected, once every three days for a total of three treatments.
The results showed that comparing the therapeutic effects of wt Fc with that of no ADCC Fc after tumor inoculation, the mutant Fc was found to only partially reduce the therapeutic effects of IL2/aOX antibody (fig. 12). In the IL2/aOX40-no ADCC Fc antibody treatment group, the antibodies still retain partial anti-tumor effect, which indicates that the IL2/aOX40-Fc antibody functions not only by deleting Treg cells, but also by combining with effector T cells.
EXAMPLE 4 IL2/aOX40-Fc combination PDL1 antibody treatment
1. aPDL1 can cooperate with IL2/aOX40-Fc antibody to enhance anti-tumor effect
Tumor of MC38 tumor-bearing mice was 200mm 3 In the following, IL2/aOX40-Fc antibody therapy alone had good clearance. However, when tumors are larger, antibody alone therapy can only control tumor growth but cannot completely clear; to better enhance the therapeutic effect, we observed whether the combination of bispecific antibody and immune checkpoint inhibitory antibody could enhance the therapeutic effect.
Specific test scheme:
scheme one: c57BL6 mice were inoculated subcutaneously with MC38 cells and treatment was started on day 16,
(1) IL2/aOX40-Fc treated groups alone were intraperitoneally injected with 25ug IL2/aOX40-Fc on days 16, 19 and 22;
(2) aOX40 and aCTLA4 combination group was given by intraperitoneal injection of 250ug antibody two days later (only once, three times per 25ug IL2/aOX 40-Fc) by intraperitoneal injection of 25ug IL2/aOX-Fc (aOX and aCTLA4 antibody were given by only one time per 25ug IL2/aOX-Fc);
(3) The aPDL1 combination treatment group was injected with 250ug aPDL1 antibody (16, 19, 22 days) at the same time as 25ug IL2/aOX 40-Fc.
Scheme II: MC38 tumor bearing mice, 200ug aPDL1 antibody was injected intraperitoneally on days 12, 15, and 18, 15ug wt Fc or IL2/aOX40 antibody no ADCC Fc was injected intraperitoneally on days 14, 17, and 20.
The results show that:
(1) During the course of combination therapy of IL2/aOX40-Fc with aCTLA4 or aOX40, the combined use of these two classes of antibodies was found not to further enhance the therapeutic effect of IL2/aOX40-Fc (FIG. 13A).
(2) The combined use of the two was found to have a synergistic effect during the combination treatment with aPDL1 (FIG. 13A).
(3) To further analyze the mechanism of action of aPDL1 with IL2/aOX40-Fc antibodies, we used either wt Fc or the IL2/aOX40-Fc antibody of no ADCC Fc, respectively, in combination with aPDL1, and found that better combination therapeutic effect was dependent on wt Fc (FIG. 13B).
EXAMPLE 5 tumor recurrence following IL2/aOX40-Fc bifunctional antibody antagonistic TKI treatment
Therapeutic regimens for patients with EGFR mutations have been mainly the use of antibodies targeting the receptor or kinase inhibitors against downstream signaling pathways. TKI has good therapeutic effect on tumors related to ErBB gene family, and Afatinib (Afatinib) is a second-generation kinase inhibitor which can irreversibly inhibit the tyrosine kinase activity of the receptor in a covalent binding manner. The mechanism of action of afatinib was analyzed on the TUBO model and an increase in the proportion of CD 3T/CD 45 lymphocytes in the tumor after treatment was found, indicating more T cells infiltrating into the tumor during treatment with the chemotherapeutic agent (fig. 14A). Afatinib alone, while increasing T cell infiltration into tumors, was not effective in reducing the proportion of intratumoral Treg cells (FIG. 14B). Clinical TKI alone treatment is difficult to completely clear tumors, and we speculate that intratumoral Treg cells inhibit killing of tumors by effector cells, thereby leading to tumor recurrence after treatment.
To verify whether IL2/aOX40-Fc was able to antagonize recurrence following chemotherapy, we used IL2/aOX40-Fc antibodies in combination with chemotherapy in tumor bearing mice.
The specific experimental scheme is as follows:
BALB/c mice were inoculated subcutaneously 5X 10 5 TUBO tumor cells,
treatment regimen 1: 1mg of Afatinib was administered for intragastric treatment on day 9, and tumor tissue was taken for flow analysis after 6 days.
Treatment regimen 2: tumor-bearing mice began treatment on day 14 post tumor inoculation,
(1) The Afatinib treatment group was gavaged with 1mg Afatinib on day 14,
(2) IL2/aOX40-Fc treated groups were given by intraperitoneal injection of 25ug of antibody on days 14, 17 and 20,
(3) The combination treatment group was perfused once with Afatinib, 25ug IL2/aOX40-Fc peritoneal antibody, as per the protocol described above, three times.
The results show that the afatinib alone treatment group in treatment regimen 1, tumors recurred soon after partial control; in treatment regimen 2, the combination therapy with the IL2/aOX40-Fc antibody was effective in controlling tumor growth and the tumor was completely cleared in some mice (FIG. 14C).
Example 6 IL2/aCTLA4-Fc bispecific antibodies significantly improve tumor therapeutic efficacy
In vivo mouse tumor-bearing experiment: subcutaneous inoculation of 5X 10 in C57BL6 mice 5 MC38 tumor cells, D11 treatment after tumor inoculation; 15ug IL2/aCTLA4 wt Fc protein was injected intraperitoneally, respectively (the specific structure of the IL2/aCTLA4 wt Fc protein is shown in FIG. 15). Tumor size was measured twice weekly three times a day of co-treatment on days D11, D14, D17;
the results showed that the IL2/aCTLA4-Fc bispecific antibody was effective in controlling tumors (FIG. 15).
Example 7 IL2/a4-1BB-Fc bispecific antibodies significantly improve tumor therapeutic efficacy
Subcutaneous inoculation of C57BL/6 mice with 5X 10 5 MC38 tumor cells, D10 treatment after tumor inoculation; 10ug3H3-IL2 and 10ug LOB12.3-IL2 wt Fc fusion protein (the schematic structure of IL2/a4-1BB-Fc fusion protein is shown in FIG. 16) were injected intraperitoneally, respectively. Tumor size was measured twice weekly for three treatments on days 10, 16 and 19 after tumor inoculation; the results show that the tumor volumes of the treatment groups of 2 fusion proteins are obviously reduced and the differences are very obvious compared with the control group.
Experiments prove that the IL2/a4-1BB-Fc fusion protein has good tumor treatment effect (figure 16).
Finally, it should be noted that the above embodiments are merely used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of the present invention.
[ reference ]
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SEQUENCE LISTING
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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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
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Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
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Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
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Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
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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
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Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
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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
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Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
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Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
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Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
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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 (7)

1. A bifunctional fusion protein, which is a heterodimer, characterized in that,
the heterodimer consists of the following domains:
(1) A first monomer formed by connecting interleukin 2 (IL-2) with an immunoglobulin Fc single chain;
the first monomer comprises the following components in sequence from the N end:
1) Wild type IL-2 protein with a sequence shown as SEQ ID NO. 1;
2) A linker sequence as shown in SEQ ID NO. 6;
3) An immunoglobulin Fc single chain;
(2) A second monomer formed by linking Fab of an antibody of the anti-TNF family costimulatory molecule with an immunoglobulin Fc single chain; the co-stimulatory molecules of the TNF family are: OX40;
the second monomer comprises:
1) An anti-OX 40 antibody Fab region consisting of an anti-OX 40 antibody light chain VL-KCL with a sequence shown as SEQ ID NO.7 and an anti-OX 40 antibody heavy chain VH & CH1 with a sequence shown as SEQ ID NO. 8;
and: 2) An immunoglobulin Fc single chain;
the first monomer and the second monomer are connected through dimerization of Fc single chains to form the heterodimer;
The immunoglobulin Fc single chain is the Fc single chain of human IgG or the immunoglobulin Fc single chain with ADCC effect knocked out by gene mutation.
2. The bifunctional fusion protein of claim 1, wherein the bifunctional fusion protein comprises,
the Fc single-chain sequence of the IgG is shown as SEQ ID NO. 2; the sequence of the immunoglobulin Fc single chain for knocking out ADCC effect through gene mutation is shown in SEQ ID NO. 3.
3. A bifunctional fusion protein as claimed in claim 1 or 2, wherein,
the heterodimer comprises:
a first monomer: the polypeptide is shown as SEQ ID NO. 10;
and a second monomer: it is: the anti-OX 40 antibody VH-CH1-Fc shown in SEQ ID NO.11 and the anti-OX 40 antibody light chain VL-KCL shown in SEQ ID NO. 7.
4. A nucleic acid encoding the bifunctional fusion protein of any one of claims 1-3.
5. The nucleic acid of claim 4, wherein the nucleic acid,
the sequence of the nucleic acid encoding the heterodimer first monomer is shown in SEQ ID NO. 15;
the sequences of the nucleic acids encoding the second monomer of the heterodimer are shown in SEQ ID NO.16 and SEQ ID NO. 17.
6. Use of a bifunctional fusion protein of any one of claims 1-3, for:
(1) Preparing an anti-colorectal cancer or melanoma medicine;
(2) Preparing an anti-colorectal or breast cancer drug in combination with an immune checkpoint inhibitor;
(3) Preparing an anti-colorectal or breast cancer drug in combination with a TKI antagonist;
the immune checkpoint inhibitor is an antagonist for resisting PD-L1, and the antagonist for resisting PD-L1 is an antibody for resisting PD-L1;
the TKI antagonist is a small molecule TKI antagonist; the small molecule TKI antagonist is afatinib or a structural analogue thereof.
7. A medicament or pharmaceutical composition comprising the bifunctional fusion protein of any one of claims 1-3.
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