Chimeric antigen receptor targeting CD123 and uses thereof

文档序号：795812 发布日期：2021-04-13 浏览：4次中文

阅读说明：本技术 靶向cd123的嵌合抗原受体及其用途 (Chimeric antigen receptor targeting CD123 and uses thereof ) 是由何霆齐菲菲鲁薪安丁艳萍梁梦梦于 2020-12-16 设计创作，主要内容包括：本发明提供了特异性结合CD123的单链抗体scFv,其包含选自SEQ ID NO：56、57、58、59、60和61的氨基酸序列。还提供了靶向CD123的嵌合抗原受体,其包含选自SEQ ID NO：65、66、67、68、69和70的氨基酸序列。还提供了编码所述scFv或嵌合抗原受体的核酸分子,包含所述核酸分子的载体和细胞,以及所述抗体、嵌合抗原受体、核酸分子、载体或细胞用于制备治疗表达CD123的肿瘤的药物的用途。(The present invention provides a single chain antibody scFv that specifically binds CD123 comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 56. 57, 58, 59, 60 and 61. Also provided is a CD123 targeting chimeric antigen receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 65. 66, 67, 68, 69 and 70. Also provided are nucleic acid molecules encoding the scFv or chimeric antigen receptor, vectors and cells comprising the nucleic acid molecules, and use of the antibody, chimeric antigen receptor, nucleic acid molecule, vector or cell for the manufacture of a medicament for the treatment of a tumor expressing CD 123.)

1. An antibody that specifically binds to CD123 comprising a heavy chain variable domain comprising SEQ ID NO: 1 (GYX)₁X₂X₃X₄(D)YX₅X₆X₇) The VH-CDR1 amino acid sequence of SEQ ID NO: 2 (X)₈X₉X₁₀(X₁₁)X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈NX₁₉X₂₀X₂₁KX₂₂X₂₃X₂₄) And the VH-CDR2 amino acid sequence of SEQ ID NO: 3 (AX)₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁(YD)X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀) The VH-CDR3 amino acid sequence of (a); wherein X₁Selected from S, T; x₂Selected from F, I; x₃Selected from T, S, M; x₄Selected from S, D, T, G; x₅Selected from W, Y, V, A, N; x₆Selected from M, V, I, W; x₇Selected from N, H; x₈Selected from Y, R; x₉Selected from I, C; x₁₀Selected from D, N, Y, S; x₁₁Selected from P, C; x₁₂Selected from Y, G; x₁₃Selected from D, N, S; x₁₄Selected from S, N, D, G; x₁₅Selected from E, A, G, S; x₁₆Selected from T, I, S; x₁₇Selected from H, S, N; x₁₈Selected from Y, S; x₁₉Selected from Q, E, P; x₂₀Selected from K, N, S; x₂₁Selected from F, L; x₂₂Selected from D, G, S; x₂₃Selected from K, R; x₂₄Selected from A, I; x₂₅Selected from R, G; x₂₆Selected from G, S, E; x₂₇Selected from E, P, R; x₂₈Selected from G, S; x₂₉Selected from N, Y, L, W; x₃₀Selected from W, Y, L; x₃₁Selected from G, D; x₃₂Selected from R, E; x₃₃Selected from Y, S, T; x₃₄Selected from D, Y, G; x₃₅Selected from G, Y; x₃₆Selected from Y, L; x₃₇Selected from A, G, P; x₃₈Selected from M, L; x₃₉Selected from D, A; x₄₀Selected from Y, C; the light chain variable domain comprises SEQ ID NO; 4 (X)₄₁SX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉(QK)X₅₀X₅₁X₅₂X₅₃W), the VL-CDR1 amino acid sequence of SEQ ID NO: 5 (X)₅₄SX₅₅X₅₆X₅₇X₅₈X₅₉) And the amino acid sequence of VL-CDR2 of SEQ ID NO: 6 (X)₆₀X₆₁X₆₂X₆₃X₆₄(P)X₆₅TF) VL-CDR3 amino acid sequence; wherein X₄₁Selected from A, S; x₄₂Selected from Q, K, S; x₄₃Selected from S, D; x₄₄Selected from I, V, L; x₄₅Selected from S, N, D, L; x₄₆Selected from K, Y, S, D, N; x₄₇Selected from D, S; x₄₈Selected from G, S; x₄₉Selected from D, N; x₅₀Selected from S, N; x₅₁Selected from D, Y; x₅₂Selected from L, I, M; x₅₃Selected from A, N, H; x₅₄Selected from G, T, A; x₅₅Selected from T, N, Q; x₅₆Selected from L, S,R；X₅₇Selected from Q, E, A, I, D; x₅₈Selected from S, P; x₅₉Selected from G, E; x₆₀Selected from Q, N; x₆₁Selected from H, Y, S, F, G; x₆₂Selected from N, D, H, Y; x₆₃Selected from K, L, E, R, S; x₆₄Selected from Y, L, D, S, F, T; x₆₅Selected from Y, R, W;

preferably, the VH-CDR1 is selected from the group consisting of SEQ ID NO: 8. 9, 10, 11, 12; the VH-CDR2 is selected from the group consisting of SEQ ID NO: 14. 15, 16, 17, 18; the VH-CDR3 is selected from the group consisting of SEQ ID NO: 20. 21, 22, 23, 24; the VL-CDR1 is selected from the group consisting of SEQ ID NO: 26. 27, 28, 29, 30; the VL-CDR2 is selected from the group consisting of SEQ ID NO: 32. 33, 34, 35, 36; the VL-CDR3 is selected from the group consisting of SEQ ID NO: 38. 39, 40, 41, 42;

more preferably, the antibody comprises:

1) comprises the amino acid sequence shown as SEQ ID NO: 8, VH-CDR1 as shown in SEQ ID NO: 14 and VH-CDR2 as shown in SEQ ID NO: 20 a heavy chain variable domain of VH-CDR 3; and comprising the amino acid sequence as set forth in SEQ ID NO: 26, VL-CDR1 as shown in SEQ ID NO: 32 and VL-CDR2 as shown in SEQ ID NO: 38 of VL-CDR 3; and/or

2) Comprises the amino acid sequence shown as SEQ ID NO: 9, VH-CDR1 as shown in SEQ ID NO: 15 and VH-CDR2 as shown in SEQ ID NO: 21 the heavy chain variable domain of VH-CDR 3; and comprising the amino acid sequence as set forth in SEQ ID NO: 27, VL-CDR1 as set forth in SEQ ID NO: 33 and VL-CDR2 as shown in SEQ ID NO: 39 of VL-CDR 3; and/or

3) Comprises the amino acid sequence shown as SEQ ID NO: 10, VH-CDR1 as shown in SEQ ID NO: 16 and VH-CDR2 as shown in SEQ ID NO: 22 of VH-CDR 3; and comprising the amino acid sequence as set forth in SEQ ID NO: 28, VL-CDR1 as set forth in SEQ ID NO: 34 and VL-CDR2 as shown in SEQ ID NO: 40 of VL-CDR 3; and/or

4) Comprises the amino acid sequence shown as SEQ ID NO: 11, VH-CDR1 as shown in SEQ ID NO: 17 and VH-CDR2 as shown in SEQ ID NO: 23, the heavy chain variable domain of VH-CDR 3; and comprising the amino acid sequence as set forth in SEQ ID NO: 29, VL-CDR1 as set forth in SEQ ID NO: 35 and VL-CDR2 as shown in SEQ ID NO: 41 of VL-CDR 3; and/or

5) Comprises the amino acid sequence shown as SEQ ID NO: 12, VH-CDR1 as shown in SEQ ID NO: 18 and VH-CDR2 as shown in SEQ ID NO: 24 a heavy chain variable domain of VH-CDR 3; and comprising the amino acid sequence as set forth in SEQ ID NO: 30, VL-CDR1 as set forth in SEQ ID NO: 36 and VL-CDR2 as shown in SEQ ID NO: 42 of VL-CDR 3.

2. The antibody of claim 1, wherein the heavy chain variable domain comprises a sequence selected from the group consisting of SEQ ID NO: 44. 45, 46, 47 and 48, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99% identity thereto; and/or the light chain variable domain comprises a sequence selected from the group consisting of SEQ ID NOs: 50. 51, 52, 53 and 54, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99% identity thereto;

preferably, the antibody comprises:

1) as shown in SEQ ID NO: 44 and a heavy chain variable domain as set forth in SEQ ID NO: 50, a light chain variable domain; and/or

2) As shown in SEQ ID NO: 45 and a heavy chain variable domain as set forth in SEQ ID NO: 51; and/or

3) As shown in SEQ ID NO: 46 and a heavy chain variable domain as set forth in SEQ ID NO: 52, a light chain variable domain; and/or

4) As shown in SEQ ID NO: 47 and a heavy chain variable domain as set forth in SEQ ID NO: 53 with a light chain variable domain; and/or

5) As shown in SEQ ID NO: 48 and a heavy chain variable domain as set forth in SEQ ID NO: 54, a light chain variable domain;

more preferably, the antibody comprises: selected from the group consisting of SEQ ID NO: 56. 57, 58, 59, 60 and 61, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99% identity thereto.

3. The antibody of claim 1 or 2, wherein the antibody is a single chain antibody, scFv; more preferably, the heavy and light chain variable domains of the single chain antibody scFv are linked by a linker peptide chain rich in glycine and serine, and the order of the heavy and light chain variable domains is interchangeable.

4. A chimeric antigen receptor targeted to CD123 comprising: the antibody of any one of claims 1-3; a hinge region; a transmembrane domain; a co-stimulatory domain; and a CD3 ζ intracellular signaling domain;

preferably, the co-stimulatory domain is selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, B7-H3, or any combination thereof; more preferably, the co-stimulatory domain is selected from 4-1BB and CD28, said 4-1BB comprising the amino acid sequence as set forth in SEQ ID NO: 62;

preferably, the CD3 ζ intracellular signaling domain comprises a sequence as set forth in SEQ ID NO: 63;

preferably, the hinge and transmembrane domains are selected from the hinge and transmembrane domains of IgG1, IgG4, CD8 a, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1 or CD 34.

5. The chimeric antigen receptor of claim 4, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 65. 66, 67, 68, 69 and 70, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99% identical thereto.

6. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody of any one of claims 1-3 or the chimeric antigen receptor of any one of claims 4-5.

7. A vector comprising the nucleic acid molecule of claim 6; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.

8. A cell transfected with the vector of claim 7, thereby comprising the nucleic acid molecule of claim 6 or the vector of claim 7; preferably, the cell is a T cell.

9. A pharmaceutical composition comprising the antibody of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-5, the nucleic acid molecule of claim 6, the vector of claim 7, or the cell of claim 8, and a pharmaceutically acceptable carrier.

10. Use of the antibody of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-5, the nucleic acid molecule of claim 6, the vector of claim 7, or the cell of claim 8 for the manufacture of a medicament for treating a CD123 expressing tumor; preferably, the CD123 expressing tumor is Acute Myeloid Leukemia (AML).

Technical Field

The present invention relates to CD 123-targeting scFv sequences with different antigen recognition domains, Chimeric Antigen Receptor (CAR) and CAR-T cells designed based on these scFv sequences, and their use for the treatment of CD 123-expressing tumors, in particular acute myeloid leukemia.

Background

Acute Myeloid Leukemia (AML) is a malignant tumor with an increasing incidence, mainly affecting the elderly with a median age of 66 years. The National Cancer Institute estimates that new cases in 2018 will reach 19520, where 10670 people may die from the disease [1 ]. Although the survival rate of patients 65-74 years of age has increased from 20% to 30% at 12 months in the past thirty consecutive years, the therapeutic effect of AML is still unsatisfactory at present, and the 5-year survival rate of these patients is < 5% [2 ]. For younger AML patients, the primary treatment is intensive chemotherapy, and for patients with a higher risk of relapse (cure rate 35-40%), Allogeneic Hematopoietic Cell Transplantation (AHCT) is used, while the prognosis for elderly patients remains poor [2 ]. Relapsed AML patients have a probability of achieving a second remission of less than 50%, whereas only 16% of patients achieve disease control after a second rescue chemotherapy [3 ]. Anthracyclines and arabidopsis have been the main treatment since the introduction of combined chemotherapy in the 70 s of the 20 th century [4, 5 ]. Therefore, new therapeutic strategies are needed for this disease.

The concept of chimeric antigen receptor T cells (CAR-T cells) was first proposed in the late 80 s of the 20 th century, combining antibody specificity and T cell killing effects, forming an adoptive immune effect [6, 7 ]. The molecular structure of the Chimeric Antigen Receptor (CAR) mainly comprises an antibody single-chain variable fragment (scFv), an intracellular signal domain, a hinge region and a transmembrane region which are used for connecting the scFv and the intracellular signal domain. In recent years, good results have been achieved with anti-CD 19 and anti-CD 20 chimeric antigen receptor T cells (CAR-T cells) for the treatment of lymphoid malignancies (B-cell acute lymphoblastic leukemia and non-hodgkin's lymphoma) [8 ]. At present, CAR-T targets for AML treatment are CD123, CD33, CLL1, FLT3, etc.; among these, CD 123-targeted CAR-T has been the most studied.

CD123 is the IL-3 receptor a chain (IL-3 Ra). CD123 and CD131 together constitute a high-affinity IL-3 receptor, and after being combined with IL-3, the high-affinity IL-3 receptor can promote cell proliferation and differentiation and inhibit hematopoietic apoptosis [9, 10 ]. High expression of CD123 is associated with activation of the signaling and activator of transcription 5(STAT5) signaling pathway. The STAT5 activation pathway is implicated in a variety of hematological malignancies [11 ]. CD123 is predominantly expressed in the hematopoietic system, with low or no expression of CD123 in normal CD34+ CD 38-cells [12 ]. Furthermore, CD123 is normally highly expressed in myeloid progenitors, B-lymphoid progenitors, while erythroid progenitors and multipotent progenitors are poorly expressed or not expressed [13, 14 ]. CD123 is highly expressed in more than 60% -90% of AML blasts [15-18 ]. Bras AE et al [19] examined 455 AML patients, of which about 55% of the AML cells expressed CD123 with a positive rate of over 80%.

Although CAR-T cells have achieved good results in tumor cell therapy, CAR-T efficacy and safety remain major problems, mainly off-target effects, cytokine storm, neurotoxicity, etc. [20 ]. The optimal therapeutic efficacy and safety of CAR-T cells depends on a number of factors, among which efficient activation of CAR-T cells plays an important role. The degree of activation of CAR-T cells is influenced by a number of factors, such as the affinity of the scFv of the CAR molecule, the length of the hinge region, the costimulatory signal domain, the density of antigens on the surface of the target cell. The scFv of the CAR molecule acts as a region that binds directly to the target cell surface antigen, the affinity of which is important for the activation of the whole CAR-T cell. When the affinity of the scFv of the CAR molecule is low, the CAR-T cells cannot obtain enough antigen recognition, so that the optimal activation state cannot be reached; when the affinity of the scFv of the CAR molecule is high, the CAR-T cell can recognize normal cells with relatively low antigen density while recognizing tumor cells, so that a serious off-target effect is generated.

Therefore, in order to further improve the safety and effectiveness of CAR-T cells, ensure efficient activation of CAR-T cells, avoid off-target effects, there is still a need to screen for scfvs with moderate affinity for the target.

Disclosure of Invention

In a first aspect, the present invention provides an antibody that specifically binds CD123 comprising a heavy chain variable domain VH and a light chain variable domain VL, said heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 (GYX)₁X₂X₃X₄(D)YX₅X₆X₇) The VH-CDR1 amino acid sequence of SEQ ID NO: 2

(X₈X₉X₁0(X₁₁)X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈NX₁₉X₂₀X₂₁KX₂₂X₂₃X₂₄) And the VH-CDR2 amino acid sequence of SEQ ID NO: 3

(AX₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁(YD)X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀) The VH-CDR3 amino acid sequence of (a); wherein X₁Selected from S, T; x₂Selected from F, I; x₃Selected from T, S, M; x₄Selected from S, D, T, G; x₅Selected from W, Y, V, A, N; x₆Selected from M, V, I, W; x₇Selected from N, H; x₈Selected from Y, R; x₉Selected from I, C; x₁₀Selected from D, N, Y, S; x₁₁Selected from P, C; x₁₂Selected from Y, G; x₁₃Selected from D, N, S; x₁₄Selected from S, N, D, G; x₁₅Selected from E, A, G, S; x₁₆Selected from T, I, S; x₁₇Selected from H, S, N; x₁₈Selected from Y, S; x₁₉Selected from Q, E, P; x₂₀Selected from K, N, S; x₂₁Selected from F, L; x₂₂Selected from D, G, S; x₂₃Selected from K, R; x₂₄Selected from A, I; x₂₅Selected from R, G; x₂₆Selected from G, S, E; x₂₇Selected from E, P, R; x₂₈Selected from G, S; x₂₉Selected from N, Y, L, W; x₃₀Selected from W, Y, L; x₃₁Selected from G, D; x₃₂Selected from R, E; x₃₃Selected from Y, S, T; x₃₄Selected from D, Y, G; x₃₅Selected from G, Y; x₃₆Selected from Y, L; x₃₇Selected from A, G, P; x₃₈Selected from M, L; x₃₉Selected from D, A; x₄₀Selected from Y, C; the light chain variable domain comprises SEQ ID NO: 4 (X)₄₁SX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉(QK)X₅₀X₅₁X₅₂X₅₃W), the VL-CDR1 amino acid sequence of SEQ ID NO: 5 (X)₅₄SX₅₅X₅₆X₅₇X₅₈X₅₉) And the amino acid sequence of VL-CDR2 of SEQ ID NO: 6 (X)₆₀X₆₁X₆₂X₆₃X₆₄(P)X₆₅TF) VL-CDR3 amino acid sequence; wherein X₄₁Selected from A, S; x₄₂Selected from Q, K, S; x₄₃Selected from S, D; x₄₄Selected from I, V, L; x₄₅Selected from S, N, D, L; x₄₆Selected from K, Y, S, D, N; x₄₇Selected from D, S; x₄₈Selected from G, S; x₄₉Selected from D, N; x₅₀Selected from S, N; x₅₁Selected from D, Y; x₅₂Selected from L, I, M; x₅₃Selected from A, N, H; x₅₄Selected from G, T, A; x₅₅Selected from T, N, Q; x₅₆Selected from L, S, R; x₅₇Selected from Q, E, A, I, D; x₅₈Selected from S, P; x₅₉Selected from G, E; x₆₀Selected from Q, N; x₆₁Selected from H, Y, S, F, G; x₆₂Selected from N, D, H, Y; x₆₃Selected from K, L, E, R, S; x₆₄Selected from Y, L, D, S, F, T; x₆₅Selected from Y, R, W; preferably, the antibody is a scFv.

In a second aspect, the present invention provides a chimeric antigen receptor targeting CD123 comprising: the antibody of the first aspect; a hinge region; a transmembrane domain; a co-stimulatory domain; and a CD3 ζ intracellular signaling domain.

In a third aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the first aspect or the chimeric antigen receptor of the second aspect.

In a fourth aspect, the present invention provides a vector comprising the nucleic acid molecule of the third aspect.

In a fifth aspect, the invention provides a cell transfected with the vector of the fourth aspect, thereby comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect.

In a sixth aspect, the invention provides a pharmaceutical composition comprising an antibody of the first aspect, a chimeric antigen receptor of the second aspect, a nucleic acid molecule of the third aspect, a vector of the fourth aspect or a cell of the fifth aspect, and a pharmaceutically acceptable carrier.

In a seventh aspect, the invention provides the use of an antibody of the first aspect, a chimeric antigen receptor of the second aspect, a nucleic acid molecule of the third aspect, a vector of the fourth aspect or a cell of the fifth aspect for the manufacture of a medicament for the treatment of a CD123 expressing tumor.

According to the invention, the optimal scFv sequence of the target CD123 is obtained by performing affinity screening on scFv of the CAR molecule of the target CD123 and comparing killing effects of the CAR-T cell, so that the CAR-T cell has better safety and effectiveness. Compared with the prior art, the invention can effectively improve the killing efficiency of the CD 123-targeted CAR-T cells on tumor cell lines and primary tumor cells, and improve the cell proliferation capacity.

Drawings

Figure 1 shows the transduction efficiency of CD123 CAR molecules of different scfvs in T cells.

FIG. 2 shows the total cell proliferation of CD123 CAR-T during culture.

FIG. 3 shows the effect on CD123 CAR-T cell differentiation upon stimulation by target cells AML primary cells and THP-1 cells, respectively.

FIG. 4 shows the killing efficiency of CD123 CAR-T on CD123 positive cell line (THP-1) and CD123 negative cell line (K562).

FIG. 5 shows the killing efficiency of CD123 CAR-T on AML primary cells.

Figure 6 shows cytokine expression of CD123 CAR-T5 hours after AML primary cell stimulation.

Detailed Description

The present invention provides an antibody that specifically binds to CD123 comprising a heavy chain variable domain comprising VH-CDR1, VH-CDR2 and VH-CDR3, and a light chain variable domain comprising VL-CDR1, VL-CDR2 and VL-CDR3, VH and VL-CDR 3.

In a specific embodiment, the VH-CDR1 is selected from the group consisting of SEQ ID NOs: 8. 9, 10, 11, 12; the VH-CDR2 is selected from the group consisting of SEQ ID NO: 14. 15, 16, 17, 18; the VH-CDR3 is selected from the group consisting of SEQ ID NO: 20. 21, 22, 23, 24; the VL-CDR1 is selected from the group consisting of SEQ ID NO: 26. 27, 28, 29, 30; the VL-CDR2 is selected from the group consisting of SEQ ID NO: 32. 33, 34, 35, 36; the VL-CDR3 is selected from the group consisting of SEQ ID NO: 38. 39, 40, 41, 42, or a pharmaceutically acceptable salt thereof.

In particular, antibodies of the invention that specifically bind to CD123 include:

In another specific embodiment, said heavy chain variable domain in the antibody of the invention comprises a sequence selected from the group consisting of SEQ ID NO: 44. 45, 46, 47 and 48, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99% identity thereto; and/or the light chain variable domain comprises a sequence selected from the group consisting of SEQ ID NOs: 50. 51, 52, 53 and 54, or comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99% identity thereto.

In particular, the invention provides an antibody that specifically binds to CD123, comprising:

1) as shown in SEQ ID NO: 44 and a heavy chain variable domain as set forth in SEQ ID NO: 50, a light chain variable domain; and/or

2) As shown in SEQ ID NO: 45 and a heavy chain variable domain as set forth in SEQ ID NO: 51; and/or

3) As shown in SEQ ID NO: 46 and a heavy chain variable domain as set forth in SEQ ID NO: 52, a light chain variable domain; and/or

4) As shown in SEQ ID NO: 47 and a heavy chain variable domain as set forth in SEQ ID NO: 53 with a light chain variable domain; and/or

5) As shown in SEQ ID NO: 48 and a heavy chain variable domain as set forth in SEQ ID NO: 54, a light chain variable domain;

in another embodiment, the invention provides an antibody that specifically binds to CD123, comprising: selected from the group consisting of SEQ ID NO: 56. 57, 58, 59, 60 and 61, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99% identity thereto.

Preferably, the antibody of the invention is a single chain antibody scFv; more preferably, the heavy and light chain variable domains of the single chain antibody scFv are linked by a linker peptide chain rich in glycine and serine, and the order of the heavy and light chain variable domains is interchangeable.

The present invention also provides a chimeric antigen receptor targeting CD123 comprising: an antibody according to the present invention; a hinge region; a transmembrane domain; a co-stimulatory domain; and a CD3 ζ intracellular signaling domain.

In specific embodiments, the co-stimulatory domain is selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, B7-H3, or any combination thereof; preferably, the co-stimulatory domain is selected from 4-1BB or CD28, said 4-1BB comprising the amino acid sequence as set forth in SEQ ID NO: 62, or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the CD3 ζ intracellular signaling domain comprises a sequence as set forth in SEQ ID NO: 63.

In a particular embodiment, the hinge and transmembrane domains are selected from the hinge and transmembrane domains of IgG1, IgG4, CD8 a, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, or CD 34.

Preferably, the chimeric antigen receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 65. 66, 67, 68, 69 and 70, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99% identical thereto.

The invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or chimeric antigen receptor of the invention.

The invention also provides a vector comprising a nucleic acid molecule of the invention; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.

The invention also provides a cell transfected with a vector of the invention, thereby comprising a nucleic acid molecule or vector of the invention; preferably, the cell is a T cell.

The invention also provides the use of an antibody, chimeric antigen receptor, nucleic acid molecule, vector or cell of the invention for the preparation of a medicament for the treatment of a CD123 expressing tumor; preferably, the CD123 expressing tumor is Acute Myeloid Leukemia (AML).

The technical solutions described herein may be combined in any combination, unless otherwise specified.

The invention is further illustrated by the following examples.

Example 1: CD123 CAR molecule design and CAR-T production

This example exemplifies the design of CD123 CAR molecules with different antigen recognition domains, and CAR-T cell preparation.

Antigen recognition domain design principle: CAR-T-1 was a control group whose scFv sequence for CD123 was reported from university of pennsylvania [21], designated scFv-01 herein. CAR-T-2-7 is a modified group, and the scFv of the modified group is derived from a monoclonal antibody sequence obtained by immunizing a mouse with a CD123 extracellular region fusion protein, and is named as scFv-02-07 in the specification.

The nucleotide sequences of the CD123 scFv of the different antigen recognition domains were synthesized by gene synthesis and then linked by homologous recombination with the CD8 α hinge region, CD8 α transmembrane region, 4-1BB and CD3 ζ intracellular region sequences to form the complete CAR molecule, i.e., CAR 1-7. The CAR nucleic acid fragment was constructed into the lentiviral transfer plasmid pLenti6.3/V5(Thermo Fisher, Waltham, MA, USA) by restriction endonuclease cleavage and ligase ligation. The lentivirus packaging plasmids pLP/VSVG, pLP1/MDK, pLP2/RSK (Thermo Fisher, Waltham, MA, USA) and the transfer plasmids obtained above were transfected into HEK293T cells using Lipofectamine 3000(Thermo Fisher, Waltham, MA, USA), the medium was collected after 48 hours, and after removing cell debris by centrifugation at 300g, it was centrifuged at 25000rpm for 3 hours using an ultracentrifuge. Dissolving the precipitate with 1mL of physiological saline to obtain the required lentiviral vector.

T cells were isolated from peripheral blood mononuclear cells of healthy volunteers using CD3/CD28 beads (Thermo Fisher, Cat # 40203D). Separating and purifying T cell at 1.0 × 10⁶Each cell/mL was inoculated into X-VIVO 15 medium (Lonza, Switzerland) containing 500IU/mL of IL-2 (Shandong Jintai bioengineering Co., Ltd., China). After 48 hours of culture, T cells were infected with lentiviral vectors bearing CD123 CAR molecules at MOI 0.5 to obtain CAR-T-1-7. After the lentivirus is infected for 24 hours, cell change is carried out, and the cells are inoculated into a fresh culture system X-VIVO 15 and added with 500IU/mL IL-2 for continuous culture. After 5 days of lentivirus infection, the cells in the culture system were repeatedly blown out using a pipette gun and collected into centrifuge tubes, and placed on a magnetic rack to remove CD3/CD28 beads. T cells were centrifuged and counted, and a portion of the cells were examined for CAR molecule transduction efficiency in each group of cells using a flow cytometer (Novocyte 2060R, ACEA Biosciences, San Diego, Calif., USA).

FIG. 1 shows the CAR molecule transduction efficiency of each group of cells after 5, 7, 9, and 12 days of T cell infection with lentivirus. Compared with the control group CAR-T-1, the CAR-T-2, CAR-T-3 and CAR-T-7 groups have the CAR molecule transduction efficiency close to that of the control group CAR-T-1, and the CAR-T-4, CAR-T-5 and CAR-T-6 groups are obviously higher than the control group, which shows that the CAR protein expression of the CAR-T cells of each group is better.

Example 2: in vitro proliferative capacity of engineered CD123 CAR-T

This example illustrates the detection of the proliferative capacity of CD123 CAR-T with different antigen recognition domains.

In the process of culturing CD123 CAR-T cells, lentivirus infected T cells are used as culture for the next day, then every 2 to 3 days, CAR-T cells are collected and centrifuged, 1-2 ml of culture system is added for resuspension, 10 microliter of cells are diluted by a certain fold, trypan blue (Solarbio, Cat # C0040) is used for staining, the total number of living cells and the total number of dead cells are counted under an inverted microscope (Ts2-FL, Nikon, Japan), and the proliferation condition of each group of cells is calculated.

FIG. 2 shows a significant increase in total cell proliferation of CAR-T-2 and CAR-T-7 having different antigen recognition domains compared to control CAR-T-1. Demonstrates that the scFv design of CAR-T-2 and CAR-T-7 can improve the proliferative capacity of CD123 CAR-T cells.

Example 3: cellular differentiation of engineered CD123 CAR-T under target cell stimulation

This example illustrates the detection of CD123 CAR-T with different antigen recognition domains, whose T cell differentiation is stimulated by AML primary cells or THP-1 cells (both expressing CD123), respectively, as two target cells.

CAR-T cells were cultured to day 5, and each group was divided into two 5X 10 portions⁵Cells were plated in 12-well plates at an effective target ratio (E: T) ═ 5: 1 target cells, AML primary cells or THP-1 cells, respectively, are added. After 6 days of target cell stimulation, i.e., when CAR-T cells were cultured to day 11, 1X 10 cells were collected from each group⁶Cells were centrifuged and 100 microliters of DPBS (HyClone, CAT # SH30028.02) resuspended; each set of CAR-T cells was then labeled with a fluorescent antibody associated with T cell differentiation and detected using a full spectrum flow cytometer (N7-00008-0A, Cytek Biosciences, Inc. Fremont).

In the results of fig. 3, the CD45RA + CD62L + cell population represents the combination of the naive T cell population and the dry memory T cell population, and the CD45RA + CD 62L-cell population is the effector T cell population. The CD45RA + CD62L + cell population ratios of CAR-T-2 and CAR-T-7 with different antigen recognition domains are higher than the control CAR-T-1, and the CD45RA + CD 62L-cell population ratios are lower than the control CAR-T-1. The modified CD123 antigen recognition domain has better CAR-T cell durability.

Example 4: killing efficiency of engineered CD123 CAR-T on cell lines in vitro

This example illustrates the detection of the killing efficiency of CD123 CAR-T with different antigen recognition domains against CD123 expressing and CD123 non-expressing tumor cell lines.

The cell line THP-1 expressing CD123 and the cell line K562 not expressing CD123 were collected and centrifuged, respectively, and the mixture was washed with 1ml of physiological saline (hebei tiancheng pharmaceutical co,china), add 5 microliters of Calcein-AM (concentration 1. mu.g/. mu.L, Cat # C3100MP, ThermoFisher, USA), mix gently, incubate for 30 minutes in an incubator at 37 ℃ to label the target cells. After the incubation, the cells were washed 2 times with physiological saline, resuspended in X-VIVO 15 and counted. To a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA), 1X 10 per well was added⁵And (c) labeled target cells as described above, and labeled with E: t is 10: 1 into each group of CAR-T cells, and placing at 37 deg.C and 5% CO₂Incubate in incubator for 5 hours. After incubation was complete, the positive control group, 1X 10, was lysed by addition of 2% Triton-X-100(Cat # T8787-100ML, Sigma, Germany)⁵A labeled target cell as described above; 100. mu.l of the supernatant of the killer system was placed in an ELISA plate for each well, and the fluorescence value (excitation wavelength: 495nm, emission wavelength: 515nm) was measured using a multifunctional microplate reader (Varioscan Lux, ThermoFisher).

FIG. 4 shows that for the CD123 positive cell line THP-1, the killing efficiency of CAR-T-3 and CAR-T-5 in CD123 CAR-T with different antigen recognition domains was significantly increased compared to the control group CAR-T-1. Meanwhile, the killing efficiency of each group of the modified CD123 CAR-T and the control group CAR-T-1 on a K562 cell line which does not express CD123 is consistent with that of a T cell group. The result shows that each group of CD123 CAR-T cells has certain killing effect on target cells and better specificity, and the scFv design of CAR-T-3 and CAR-T-5 is more beneficial to killing the tumor cells by CAR-T.

Example 5: killing efficiency of engineered CD123 CAR-T on AML primary cells in vitro

This example illustrates the detection of the killing efficiency of CD123 CAR-T with different antigen recognition domains against AML primary cells.

Taking 3 CD123 positive AML patient marrow blood samples (antigen density level is defined as follows, high antigen density: Median Fluorescence Intensity (MFI) is more than 10000), antigen density: MFI is more than 6000 and less than 10000, low antigen density: MFI small antigen density: MFI is less than 10000) with high antigen density (AML 1#, CD123 expression rate is 99.87% and MFI is 13298), antigen density (AML 2#, CD123 expression rate is 75.4% and MFI is 7518) and low antigen density (AML 3#, CD123 expression rate is 94.58% and MFI is 5252) respectivelyAt 6000), mononuclear cells were separated from a bone marrow blood sample of AML patients, and CD34 positive AML primary cells were sorted using CD34 MicroBeads (Cat # 130-. After the incubation, the cells were washed 2 times with physiological saline, resuspended in X-VIVO 15 and counted. To a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA), 1X 10 per well was added⁵Adding CD123 CAR-T cells into the above labeled AML primary cells at E: T ratio of 10: 1, placing at 37 deg.C and 5% CO₂Incubate in incubator for 5 hours. After incubation was complete, the positive control group was lysed by adding 2% Triton-X-100, i.e., 1X 10⁵An AML primary cell marked as described above; 100. mu.l of the supernatant of the killer system was placed in an ELISA plate for each well, and the fluorescence value (excitation wavelength: 495nm, emission wavelength: 515nm) was measured using a multifunctional microplate reader (Varioscan Lux, ThermoFisher).

Figure 5 shows that CAR-T-3 with different antigen recognition domains has significantly enhanced killing efficiency on 3 primary cells of CD123 positive AML patients with high (i.e., AML # 1), medium (i.e., AML # 2), and low (i.e., AML # 3) antigen density compared to control CAR-T-1. The CD123 CAR-T cell with CAR-T-3scFv is shown to be capable of effectively improving killing efficiency on primary cells.

Example 6: cytokine expression of engineered CD123 CAR-T under target cell stimulation

This example illustrates the detection of cytokine expression levels of CD123 CAR-T with different antigen recognition domains under stimulation of target cell AML primary cells.

To a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA), 1X 10 per well was added⁵Target cell AML primary cells having a CD123 expression rate of 99.87%, MFI of 13298, and a ratio of E: t is 10: 1 into each group of CAR-T cells, and placing at 37 deg.C and 5% CO₂Incubate in incubator for 5 hours. Cell supernatants were removed using CBA kit (BD Biosciences, San Jose, Calif., USA) and flow cytometryThe expression levels of various cytokines were measured by a cytometer (Novocyte 2060R, ACEA Biosciences, San Diego, Calif., USA).

FIG. 6 shows that the CAR-T-6 group with different antigen recognition domains showed significantly increased expression levels of IL-2, TNF, IFN- γ upon stimulation of target cells, compared to the control group CAR-T-1; the cytokine expression of the CAR-T cells of the remaining groups was similar to that of the control group CAR-T-1. Demonstrates that scFv design of CAR-T-6 favors expression of CD123 CAR-T cytokines.

Specific amino acid sequences involved in the present invention are shown in tables 1 and 2 below.

TABLE 1 CDR and scFv sequences of exemplary antibodies

Table 2 exemplary chimeric antigen receptor sequences

Reference documents:

[1]Surveillance,Epidemiology,and End Results(SEER)Program.Research Data(1973–2015),National Cancer Institute,Dccps,Surveillance Research Program,Released April 2018,Based on the November 2017Submission.2019.(www.seer.cancer.gov).

[2]Thein MS,Ershler WB,Jemal A,Yates JW,Baer MR.Outcome of older patients with acute myeloid leukemia:an analysis of SEER data over 3decades.Cancer 2013；119(15):2720–7.

[3]Kantarjian HM,DiNardo CD,Nogueras-Gonzalez GM,Kadia TM,Jabbour E,Bueso-Ramos CE,et al.Results of second salvage therapy in 673adults with acute myelogenous leukemia treated at a single institution since 2000.Cancer 2018；124(12):2534–40.

[4]Jang J,Lee J,Jang JH,Jung CW,Park S.Anti-leukemic effects of simvastatin on NRAS(G12D)mutant acute myeloid leukemia cells.Mol Biol Rep.2019；46:5859–66.[5]Roboz GJ.Novel approaches to the treatment of acute myeloid leukemia.Hematology Am Soc Hematol Educ Program.2011；2011:43–50.

[6]Benmebarek,M.R.；Karches,C.H.；Cadilha,B.L.；Lesch,S.；Endres,S.；Kobold,S.Killing mechanisms of chimeric antigen receptor(CAR)T cells.Int.J.Mol.Sci.2019,20,1283.

[7]Gross,G.；Waks,T.；Eshhar,Z.Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity.Proc.Natl.Acad.Sci.USA 1989,86,10024–10028.

[8]Khalil DN,Smith EL,Brentjens RJ,Wolchok JD.The future of cancer treatment:immunomodulation,CARs and combination immunotherapy.Nat Rev Clin Oncol 2016；13(6):394.

[9]L,Nomdedéu JF,López O,et al.Interleukin-3 receptorα chain(CD123)is widely expressed in hematologic malignancies.Haematologica.2001；86:1261–9.

[10]Blalock WL,Weinstein-Oppenheimer C,Chang F,et al.Signal transduction,cell cycle regulatory,and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells:possible sites for intervention with anti-neoplastic drugs.Leukemia Aug.1999；13:1109–66.

[11]Testa U,Riccioni R,Militi S,et al.Elevated expression of IL-3Ralpha in acute myelogenous leukemia is associated with enhanced blast proliferation,increased cellularity,and poor prognosis.Blood.2002；100:2980–8.

[12]Jordan CT,Upchurch D,Szilvassy SJ,et al.The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.Leukemia,2000,14(10),1777–1784.

[13]Wognum AW,De Jong MO,Wagemaker G.Differential Expression of Receptors for Hemopoietic Growth Factors on Subsets of CD34+Hemopoietic Cells.Leukemia&Lymphoma,1996,24(1-2),11–25.

[14]Huang S,Chen Z,Yu JF,et al.Correlation Between IL-3 Receptor Expression and Growth Potential of Human CD34+Hematopoietic Cells from Different Tissues.Stem Cells,1999,17(5),265–272.

[15]Munoz L,Nomdedeu JF,Lopez O,et al.Interleukin-3 Receptor Alpha Chain(CD123)Is Widely Expressed In Hematologic Malignancies.Haematologica,2001 86:1261-1269

[16]Xie LH,Biondo M,Busfield SJ,et al.CD123 target validation and preclinical evaluation of ADCC activity of anti-CD123 antibody CSL362 in combination with NKs from AML patients in remission.Blood Cancer Journal,2017,7(6),e567.

[17]Baroni ML,Sanchez Martinez D,Gutierrez Aguera F,et al.(2020).41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo.Journal for ImmunoTherapy of Cancer,2020,8(1),e000845.

[18]Testa U,Pelosi E,Frankel A.CD123 is a membrane biomarker and a therapeutic target in hematologic malignancies.Biomarker Research,2014,2(1),4.

[19]Bras AE,de Haas V,van Stigt A,et al.CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping.Cytometry Part B:Clinical Cytometry.2018.

[20]Gauthier J,Turtle CJ.Insights into cytokine release syndrome and neurotoxicity after CD19-specific CAR-T cell therapy.Curr Res Transl Med 2018；66(2):50–2.

[21]Cummins K,Frey N,Nelson A,Schmidt A,Luger S,Hexner E,et al.Treating Relapsed/Refractory.(RR)AML With Biodegradable Anti-CD123 CAR Modified T Cells(2017)Atlanta,GA:Blood ASH.

sequence listing

<110> Beijing Art Miao Shenzhou pharmaceutical science and technology Co., Ltd

<120> CD 123-targeted chimeric antigen receptor and use thereof

<160> 70

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11

<212> PRT

<213> synthetic sequence

<220>

<223> the 3 rd amino acid is selected from Ser, Thr; the 4 th amino acid is selected from Phe and Ile; the 5 th amino acid is selected from Thr, Ser and Met; the 6 th amino acid is selected from Ser, Asp, Thr and Gly; the 9 th amino acid is selected from Trp, Tyr, Val, Ala and Asn; the 10 th amino acid is selected from Met, Val, Ile and Trp; the 11 th amino acid is selected from Asn and His;

<400> 1

Gly Tyr Xaa Xaa Xaa Xaa Asp Tyr Xaa Xaa Xaa

1 5 10

<210> 2

<211> 19

<212> PRT

<213> synthetic sequence

<220>

<223> the first amino acid is selected from Tyr, Arg; the 2 nd amino acid is selected from Ile and Cys; the 3 rd amino acid is selected from Asp, Asn, Tyr and Ser; the 4 th amino acid is selected from Pro and Cys; the 5 th amino acid is selected from Tyr and Gly; the amino acid at the 6 th position is selected from Asp, Asn and Ser; the 7 th amino acid is selected from Ser, Asn, Asp and Gly; the 8 th amino acid is selected from Glu, Ala, Gly and Ser; the 9 th amino acid is selected from Thr, Ile and Ser; the 10 th amino acid is selected from His, Ser and Asn; the 11 th amino acid is selected from Tyr and Ser; the 13 th amino acid is selected from Gln, Glu and Pro; the 14 th amino acid is selected from Lys, Asn, Ser; the 15 th amino acid is selected from Phe and Leu; the 17 th amino acid is selected from Asp, Gly and Ser; the 18 th amino acid is selected from Lys, Arg; amino acid 19 is selected from Ala, Ile;

<400> 2

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Lys

1 5 10 15

Xaa Xaa Xaa

<210> 3

<211> 19

<212> PRT

<213> synthetic sequence

<220>

<223> amino acid at position 2 selected from Arg, Gly; the 3 rd amino acid is selected from Gly, Ser and Glu; the 4 th amino acid is selected from Glu, Pro and Arg; the 5 th amino acid is selected from Gly and Ser; the 6 th amino acid is selected from Asn, Tyr, Leu and Trp; the 7 th amino acid is selected from Trp, Tyr and Leu; the 8 th amino acid is selected from Gly and Asp; the 11 th amino acid is selected from Arg and Glu; the 12 th amino acid is selected from Tyr, Ser and Thr; the 13 th amino acid is selected from Asp, Tyr and Gly; the 14 th amino acid is selected from Gly and Tyr; the 15 th amino acid is selected from Tyr and Leu; amino acid 16 is selected from Ala, Gly, Pro; the 17 th amino acid is selected from Met and Leu; amino acid 18 is selected from Asp and Ala; the 19 th amino acid is selected from Tyr and Cys;

<400> 3

Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Asp Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa

<210> 4

<211> 17

<212> PRT

<213> synthetic sequence

<220>

<223> amino acid 1 selected from Ala and Ser; the 3 rd amino acid is selected from Gln, Lys and Ser; the 4 th amino acid is selected from Ser and Asp; the 5 th amino acid is selected from Ile, Val and Leu; the 6 th amino acid is selected from Ser, Asn, Asp and Leu; the 7 th amino acid is selected from Lys, Tyr, Ser, Asp and Asn; the 8 th amino acid is selected from Asp and Ser; the 9 th amino acid is selected from Gly and Ser; the 10 th amino acid is selected from Asp and Asn; the 13 th amino acid is selected from Ser and Asn; the 14 th amino acid is selected from Asp and Tyr; the 15 th amino acid is selected from Leu, Ile and Met; the 16 th amino acid is selected from Ala, Asn and His;

<400> 4

Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Lys Xaa Xaa Xaa Xaa

1 5 10 15

Trp

<210> 5

<211> 7

<212> PRT

<213> synthetic sequence

<220>

<223> amino acid at position 1 selected from Gly, Thr, Ala; the 3 rd amino acid is selected from Thr, Asn and Gln; the 4 th amino acid is selected from Leu, Ser and Arg; the 5 th amino acid is selected from Gln, Glu, Ala, Ile and Asp; the 6 th amino acid is selected from Ser and Pro; the 7 th amino acid is selected from Gly and Glu;

<400> 5

Xaa Ser Xaa Xaa Xaa Xaa Xaa

1 5

<210> 6

<211> 9

<212> PRT

<213> synthetic sequence

<220>

<223> amino acid at position 1 selected from Gln, Asn; the 2 nd amino acid is selected from His, Tyr, Ser, Phe and Gly; the 3 rd amino acid is selected from Asn, Asp, His and Tyr; the 4 th amino acid is selected from Lys, Leu, Glu, Arg and Ser; the 5 th amino acid is selected from Tyr, Leu, Asp, Ser, Phe and Thr; the 7 th amino acid is selected from Tyr, Arg and Trp;

<400> 6

Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr Phe

1 5

<210> 7

<211> 10

<212> PRT

<213> synthetic sequence

<400> 7

Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn

1 5 10

<210> 8

<211> 10

<212> PRT

<213> synthetic sequence

<400> 8

Gly Tyr Ser Phe Thr Asp Tyr Tyr Val His

1 5 10

<210> 9

<211> 10

<212> PRT

<213> synthetic sequence

<400> 9

Gly Tyr Thr Phe Met Thr Tyr Val Ile His

1 5 10

<210> 10

<211> 11

<212> PRT

<213> synthetic sequence

<400> 10

Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn

1 5 10

<210> 11

<211> 10

<212> PRT

<213> synthetic sequence

<400> 11

Gly Tyr Thr Phe Ser Ser Tyr Asn Met His

1 5 10

<210> 12

<211> 10

<212> PRT

<213> synthetic sequence

<400> 12

Gly Tyr Ser Phe Thr Gly Tyr Tyr Met His

1 5 10

<210> 13

<211> 19

<212> PRT

<213> synthetic sequence

<400> 13

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

1 5 10 15

Asp Lys Ala

<210> 14

<211> 19

<212> PRT

<213> synthetic sequence

<400> 14

Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro Lys Phe Lys

1 5 10 15

Gly Lys Ala

<210> 15

<211> 19

<212> PRT

<213> synthetic sequence

<400> 15

Tyr Cys Asn Pro Tyr Asn Asp Gly Ile Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly Lys Ala

<210> 16

<211> 18

<212> PRT

<213> synthetic sequence

<400> 16

Tyr Ile Ser Tyr Ser Gly Ser Ser Asn Ser Asn Pro Ser Leu Lys Ser

1 5 10 15

Arg Ile

<210> 17

<211> 19

<212> PRT

<213> synthetic sequence

<400> 17

Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly Lys Ala

<210> 18

<211> 19

<212> PRT

<213> synthetic sequence

<400> 18

Arg Ile Asn Pro Tyr Asn Asn Ala Thr Ser Tyr Asn Gln Asn Phe Lys

1 5 10 15

Asp Lys Ala

<210> 19

<211> 8

<212> PRT

<213> synthetic sequence

<400> 19

Ala Arg Gly Asn Trp Asp Asp Tyr

1 5

<210> 20

<211> 16

<212> PRT

<213> synthetic sequence

<400> 20

Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr

1 5 10 15

<210> 21

<211> 17

<212> PRT

<213> synthetic sequence

<400> 21

Ala Arg Ser Pro Ser Tyr Tyr Gly Arg Ser Tyr Tyr Tyr Gly Met Asp

1 5 10 15

Tyr

<210> 22

<211> 6

<212> PRT

<213> synthetic sequence

<400> 22

Ala Arg Gly Met Asp Tyr

1 5

<210> 23

<211> 19

<212> PRT

<213> synthetic sequence

<400> 23

Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala

1 5 10 15

Met Asp Tyr

<210> 24

<211> 12

<212> PRT

<213> synthetic sequence

<400> 24

Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys

1 5 10

<210> 25

<211> 11

<212> PRT

<213> synthetic sequence

<400> 25

Ala Ser Lys Ser Ile Ser Lys Asp Leu Ala Trp

1 5 10

<210> 26

<211> 11

<212> PRT

<213> synthetic sequence

<400> 26

Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp

1 5 10

<210> 27

<211> 15

<212> PRT

<213> synthetic sequence

<400> 27

Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp

1 5 10 15

<210> 28

<211> 12

<212> PRT

<213> synthetic sequence

<400> 28

Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp

1 5 10

<210> 29

<211> 11

<212> PRT

<213> synthetic sequence

<400> 29

Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp

1 5 10

<210> 30

<211> 17

<212> PRT

<213> synthetic sequence

<400> 30

Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala

1 5 10 15

Trp

<210> 31

<211> 7

<212> PRT

<213> synthetic sequence

<400> 31

Gly Ser Thr Leu Gln Ser Gly

1 5

<210> 32

<211> 7

<212> PRT

<213> synthetic sequence

<400> 32

Thr Ser Thr Leu Gln Pro Gly

1 5

<210> 33

<211> 7

<212> PRT

<213> synthetic sequence

<400> 33

Ala Ser Asn Leu Glu Ser Gly

1 5

<210> 34

<211> 7

<212> PRT

<213> synthetic sequence

<400> 34

Thr Ser Asn Leu Ala Ser Gly

1 5

<210> 35

<211> 7

<212> PRT

<213> synthetic sequence

<400> 35

Ala Ser Gln Ser Ile Ser Glu

1 5

<210> 36

<211> 7

<212> PRT

<213> synthetic sequence

<400> 36

Ala Ser Thr Arg Asp Ser Gly

1 5

<210> 37

<211> 9

<212> PRT

<213> synthetic sequence

<400> 37

Gln His Asn Lys Tyr Pro Tyr Thr Phe

1 5

<210> 38

<211> 8

<212> PRT

<213> synthetic sequence

<400> 38

Gln Tyr Asp Asp Leu Tyr Thr Phe

1 5

<210> 39

<211> 9

<212> PRT

<213> synthetic sequence

<400> 39

Gln Ser Asn Glu Asp Pro Tyr Thr Phe

1 5

<210> 40

<211> 9

<212> PRT

<213> synthetic sequence

<400> 40

Gln Phe His Arg Ser Pro Arg Thr Phe

1 5

<210> 41

<211> 9

<212> PRT

<213> synthetic sequence

<400> 41

Asn Gly His Ser Phe Pro Tyr Thr Phe

1 5

<210> 42

<211> 9

<212> PRT

<213> synthetic sequence

<400> 42

Gln His Tyr Ser Thr Pro Trp Thr Phe

1 5

<210> 43

<211> 115

<212> PRT

<213> synthetic sequence

<400> 43

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 44

<211> 123

<212> PRT

<213> synthetic sequence

<400> 44

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

1 5 10 15

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

20 25 30

Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 45

<211> 124

<212> PRT

<213> synthetic sequence

<400> 45

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

1 5 10 15

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

20 25 30

Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Phe

35 40 45

Gly Tyr Cys Asn Pro Tyr Asn Asp Gly Ile Asn Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Pro Ser Tyr Tyr Gly Arg Ser Tyr Tyr Tyr Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 46

<211> 113

<212> PRT

<213> synthetic sequence

<400> 46

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

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

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ser Ser Asn Ser Asn Pro Ser Leu

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

85 90 95

Ala Arg Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser

<210> 47

<211> 126

<212> PRT

<213> synthetic sequence

<400> 47

Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala

100 105 110

Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210> 48

<211> 119

<212> PRT

<213> synthetic sequence

<400> 48

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 49

<211> 107

<212> PRT

<213> synthetic sequence

<400> 49

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

1 5 10 15

Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp

20 25 30

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

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser 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 Glu Pro

65 70 75 80

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 50

<211> 106

<212> PRT

<213> synthetic sequence

<400> 50

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 51

<211> 111

<212> PRT

<213> synthetic sequence

<400> 51

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys

100 105 110

<210> 52

<211> 108

<212> PRT

<213> synthetic sequence

<400> 52

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Val Phe Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

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

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Phe His Arg Ser Pro

85 90 95

Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 53

<211> 107

<212> PRT

<213> synthetic sequence

<400> 53

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

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Glu Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Phe Cys Gln Asn Gly His Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 54

<211> 113

<212> PRT

<213> synthetic sequence

<400> 54

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly

1 5 10 15

Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

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

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln

85 90 95

His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 55

<211> 237

<212> PRT

<213> synthetic sequence

<400> 55

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

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

130 135 140

Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser

145 150 155 160

Lys Asp Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu

165 170 175

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

180 185 190

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

195 200 205

Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr

210 215 220

Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230 235

<210> 56

<211> 244

<212> PRT

<213> synthetic sequence

<400> 56

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

1 5 10 15

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

20 25 30

Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln

130 135 140

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

145 150 155 160

Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His

165 170 175

Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu

180 185 190

Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gly Asp

195 200 205

Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr

210 215 220

Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr Phe Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys

<210> 57

<211> 250

<212> PRT

<213> synthetic sequence

<400> 57

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

1 5 10 15

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

20 25 30

Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Phe

35 40 45

Gly Tyr Cys Asn Pro Tyr Asn Asp Gly Ile Asn Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Pro Ser Tyr Tyr Gly Arg Ser Tyr Tyr Tyr Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr

130 135 140

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

145 150 155 160

Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met

165 170 175

Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile Tyr

180 185 190

Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu

210 215 220

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

225 230 235 240

Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys

245 250

<210> 58

<211> 236

<212> PRT

<213> synthetic sequence

<400> 58

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

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

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ser Ser Asn Ser Asn Pro Ser Leu

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

85 90 95

Ala Arg Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Val Phe Leu Gly

130 135 140

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

145 150 155 160

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

165 170 175

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

180 185 190

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

195 200 205

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Phe His Arg Ser Pro

210 215 220

Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230 235

<210> 59

<211> 248

<212> PRT

<213> synthetic sequence

<400> 59

Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala

100 105 110

Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

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

145 150 155 160

Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp

165 170 175

Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala

180 185 190

Ser Gln Ser Ile Ser Glu Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val

210 215 220

Gly Val Tyr Phe Cys Gln Asn Gly His Ser Phe Pro Tyr Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Lys

245

<210> 60

<211> 247

<212> PRT

<213> synthetic sequence

<400> 60

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ala Met Ser Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser Ser

145 150 155 160

Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser

180 185 190

Thr Arg Asp Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly

195 200 205

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

210 215 220

Asp Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 61

<211> 244

<212> PRT

<213> synthetic sequence

<400> 61

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

115 120 125

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

130 135 140

Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Tyr Val His Trp Val Arg Gln

145 150 155 160

Ser His Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn

165 170 175

Gly Ala Thr Asn Tyr Asn Pro Lys Phe Lys Gly Lys Ala Thr Phe Thr

180 185 190

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

195 200 205

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr

210 215 220

Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

225 230 235 240

Thr Val Ser Ser

<210> 62

<211> 42

<212> PRT

<213> synthetic sequence

<400> 62

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 63

<211> 112

<212> PRT

<213> synthetic sequence

<400> 63

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 64

<211> 501

<212> PRT

<213> synthetic sequence

<400> 64

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

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

35 40 45

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

50 55 60

Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln

65 70 75 80

Arg Pro Asp Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp

85 90 95

Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr

100 105 110

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

115 120 125

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Asn Trp Asp Asp

130 135 140

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

145 150 155 160

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Ile Thr

165 170 175

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

180 185 190

Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp Leu Ala Trp Tyr Gln

195 200 205

Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr

210 215 220

Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

225 230 235 240

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met

245 250 255

Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr Thr Phe Gly Gly Gly

260 265 270

Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

275 280 285

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

290 295 300

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

305 310 315 320

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

325 330 335

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

340 345 350

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

355 360 365

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

370 375 380

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

385 390 395 400

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

405 410 415

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

420 425 430

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

435 440 445

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

450 455 460

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

465 470 475 480

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

485 490 495

Ala Leu Pro Pro Arg

500

<210> 65

<211> 508

<212> PRT

<213> synthetic sequence

<400> 65

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

35 40 45

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

50 55 60

Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Tyr Val His Trp Val Arg Gln

65 70 75 80

Ser His Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn

85 90 95

Gly Ala Thr Asn Tyr Asn Pro Lys Phe Lys Gly Lys Ala Thr Phe Thr

100 105 110

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

115 120 125

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr

130 135 140

Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

145 150 155 160

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

165 170 175

Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala

180 185 190

Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile

195 200 205

Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg

210 215 220

Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg

225 230 235 240

Phe Ser Gly Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn

245 250 255

Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp

260 265 270

Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr

275 280 285

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

290 295 300

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

305 310 315 320

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

325 330 335

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

340 345 350

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

355 360 365

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

370 375 380

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

385 390 395 400

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu

405 410 415

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

420 425 430

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

435 440 445

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

450 455 460

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

465 470 475 480

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

485 490 495

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 66

<211> 514

<212> PRT

<213> synthetic sequence

<400> 66

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

35 40 45

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

50 55 60

Ala Ser Gly Tyr Thr Phe Met Thr Tyr Val Ile His Trp Val Lys Gln

65 70 75 80

Lys Pro Gly Gln Gly Leu Glu Trp Phe Gly Tyr Cys Asn Pro Tyr Asn

85 90 95

Asp Gly Ile Asn Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr

100 105 110

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

115 120 125

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Pro Ser Tyr Tyr

130 135 140

Gly Arg Ser Tyr Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

145 150 155 160

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala

180 185 190

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser

195 200 205

Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro

210 215 220

Gly Gln Pro Pro Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser

225 230 235 240

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

245 250 255

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

260 265 270

Gln Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

275 280 285

Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

290 295 300

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro

305 310 315 320

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

325 330 335

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

340 345 350

Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu

355 360 365

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

370 375 380

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

385 390 395 400

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

405 410 415

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

420 425 430

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

435 440 445

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

450 455 460

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

465 470 475 480

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

485 490 495

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

500 505 510

Pro Arg

<210> 67

<211> 500

<212> PRT

<213> synthetic sequence

<400> 67

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

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

35 40 45

Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr

50 55 60

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

65 70 75 80

Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser

85 90 95

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

100 105 110

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

115 120 125

Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Gly Met Asp Tyr Trp

130 135 140

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser

165 170 175

Pro Ala Ile Met Ser Val Phe Leu Gly Glu Arg Val Thr Met Thr Cys

180 185 190

Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Tyr Cys His Gln Phe His Arg Ser Pro Arg Thr Phe Gly Gly Gly Thr

260 265 270

Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

275 280 285

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

290 295 300

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

305 310 315 320

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

325 330 335

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys

340 345 350

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

355 360 365

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

370 375 380

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

385 390 395 400

Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

405 410 415

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

420 425 430

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

435 440 445

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

450 455 460

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

465 470 475 480

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

485 490 495

Leu Pro Pro Arg

500

<210> 68

<211> 512

<212> PRT

<213> synthetic sequence

<400> 68

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

35 40 45

Gly Ala Glu Leu Val Arg Ser Gly Ala Ser Val Lys Met Ser Cys Lys

50 55 60

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

65 70 75 80

Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Gly Asn

85 90 95

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

100 105 110

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

115 120 125

Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Glu Arg Gly Leu Tyr

130 135 140

Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

145 150 155 160

Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

165 170 175

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Thr

180 185 190

Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser

195 200 205

Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu

210 215 220

Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Glu Ile

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser

245 250 255

Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Phe Cys Gln Asn

260 265 270

Gly His Ser Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

275 280 285

Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

290 295 300

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

305 310 315 320

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

325 330 335

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

340 345 350

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

355 360 365

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

370 375 380

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

385 390 395 400

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

405 410 415

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

420 425 430

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

435 440 445

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

450 455 460

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

465 470 475 480

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

485 490 495

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 69

<211> 511

<212> PRT

<213> synthetic sequence

<400> 69

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

35 40 45

Gly Pro Glu Leu Val Lys Ser Gly Ala Ser Val Lys Ile Ser Cys Lys

50 55 60

Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Gln

65 70 75 80

Ser His Val Lys Ser Leu Glu Trp Ile Gly Arg Ile Asn Pro Tyr Asn

85 90 95

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

100 105 110

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

115 120 125

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Glu Gly Trp Leu

130 135 140

Leu Pro Leu Ala Cys Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

165 170 175

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

180 185 190

Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser

195 200 205

Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser

210 215 220

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

225 230 235 240

Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

245 250 255

Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln His

260 265 270

Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

275 280 285

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

290 295 300

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

305 310 315 320

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

325 330 335

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

340 345 350

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

355 360 365

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

370 375 380

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

385 390 395 400

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln

405 410 415

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

420 425 430

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

435 440 445

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

450 455 460

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

465 470 475 480

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

485 490 495

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 70

<211> 508

<212> PRT

<213> synthetic sequence

<400> 70

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser

35 40 45

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

50 55 60

Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys

65 70 75 80

Pro Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln

85 90 95

Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gly Asp Tyr

100 105 110

Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr

115 120 125

Cys Leu Gln Tyr Asp Asp Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu

130 135 140

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Thr

165 170 175

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

180 185 190

Asp Tyr Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu

195 200 205

Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro

210 215 220

Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr

225 230 235 240

Ala Tyr Met Gln Phe Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

245 250 255

Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met

260 265 270

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr

275 280 285

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

290 295 300

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

305 310 315 320

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

325 330 335

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

340 345 350

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

355 360 365

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

370 375 380

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

385 390 395 400

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu

405 410 415

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

420 425 430

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

435 440 445

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

450 455 460

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

465 470 475 480

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

485 490 495

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

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