RU2785993C2 - HYBRID PROTEINS OF sPD-1-Fc VARIANT - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, namely to hybrid proteins based on protein-1 of programmed cell death (hereinafter – PD-1); it can be used in medicine for the treatment, reduction, or prevention of metastasis or invasion of tumor in an object with cancer and for the treatment of an object with an infection. Hybrid protein of sPD-1-Fc protein is proposed, containing a domain of a variant of soluble PD-1 (hereinafter – sPD-1), including an amino acid substitute compared to SEQ ID NO: 1, where the specified amino acid substitute is in a position selected from a group consisting of 120, 112, 107, 104, 67, 69, 96, and 42; and where the specified domain of sPD-1 variant includes a set of amino acid G104S/S107V/A112I/A120V compared to SEQ ID NO: 1; an optional linker, and Fc domain.

EFFECT: invention provides obtainment of hybrid proteins of sPD-1-Fc variants, which bind to and antagonize PD-1 ligands.

38 cl, 42 dwg, 10 tbl, 11 ex

Description

Cross-reference to related applications

This application claims priority over U.S. Provisional Application No. 62/731,488, filed September 14, 2018, and U.S. Provisional Application No. 62/888,320, filed August 16, 2019, which are incorporated herein by reference in their entirety.

The technical field to which the invention belongs

This invention relates to sPD-1-Fc variant fusion proteins, polynucleotides encoding sPD-1-Fc variant fusion proteins, methods for making sPD-1-Fc variant fusion proteins, and methods for using sPD-1-Fc variant fusion proteins, for example, in treatment of diseases such as cancer, infections, etc.

Prior Art

PD-1 (programmed cell death protein-1) is an important immune checkpoint receptor expressed by activated T cells and B cells. The protein acts as an immunosuppression mediator. PD-1 is expressed on activated T cells, B cells, and natural killer (NK) cells. The ligands for PD-1 are programmed cell death protein-1 ligand 1 (PD-L1, alternatively B7-H1) and programmed cell death protein-1 ligand 2 (PD-L2, alternatively B7-DC), which are expressed on many tumor cells. and antigen presenting cells such as monocytes, dendritic cells (DC) and macrophages.

PD-1 is a member of the immunoglobulin (Ig) superfamily, which includes a single Ig V-like domain in the extracellular region. The cytoplasmic domain of PD-1 includes two tyrosines, with the most proximal to the membrane tyrosine located within the immunoreceptor tyrosine inhibitory motif (ITIM). PD-1 attenuates antigen receptor signaling by recruiting cytoplasmic phosphatases through its cytoplasmic domain. Human and mouse PD-1 proteins share about 60% amino acid identity, retaining four potential N-glycosylation sites and residues that define the Ig-V domain.

PD-1 acts to deliver a negative immune response signal when it is induced in T cells. Activation of PD-1 through selective binding to one of its ligands activates an inhibitory immune response that reduces T cell proliferation and/or the intensity and/or duration of the T cell response. PD-1 also regulates the activity of effector T cells in peripheral tissues in response to infection or tumor progression (Pardoll, Nat Rev Cancer, 2012, 12 (4): 252-264).

Endogenous immune checkpoints, such as the PD-1 signaling pathway, which normally interrupt immune responses to mitigate damage to collateral tissues, may be used by tumors to avoid destruction by the immune system. The interaction between PD-L1 and PD-1 in cancer may reduce the number of tumor-infiltrating immune cells and inhibit the immune response to malignant tumor cells. Suppression of T cell activation and cytokine secretion by binding to PD-1 has been observed in several human cancers (Freeman et al., J Exp Med, 2000, 192(7): 1027-34; Latchman et al, Nat Immunol, 2001, 2(3):261-8). In addition, the PD-1 ligand, PD-L1, is overexpressed in many cancers, including breast cancer, colon cancer, esophageal cancer, gastric cancer, glioma, leukemia, lung cancer, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer. glands, renal cell carcinoma, and urothelial cancer. Cancer patients have also been shown to have a limited or reduced adaptive immune response due to increased PD-1/PD-L1 interactions in immune cells. This increase in activated PD-1 signaling has also been observed in patients with viral infections. For example, hepatitis B and C viruses can induce overexpression of PD-1 ligands on hepatocytes and activate PD-1 signaling in effector T cells. This in turn leads to T cell depletion and immune tolerance to viral infection (Boni et al., J Virol, 2007, 81: 4215-4225; Golden-Mason et al, J Immunol, 2008, 180: 3637- 3641).

Current PD-1 antagonists such as pidilizumab, pembrolizumab (Keytruda®) and nivolumab (Opdivo®) are antibodies that target PD-1 on all lymphatic cells in the body. These antibodies have nanomolar affinities for PD-1 that are weaker than the interaction between PD-1 and its ligands at the immune synapse, for example, at the interface between an antigen-presenting cell and a lymphocyte.

There is a need in the art for an effective protein-based therapeutic treatment that can alleviate or reverse the inhibition of adaptive immunity in patients with cancer or infection. The present invention satisfies this and other needs.

It is an object of the present invention to provide sPD-1-Fc variant fusion proteins having improved properties (e.g., increased binding affinity for PD-L1 and/or PD-L2, and increased protein stability, etc.) as well as methods for designing and the use of such sPD-1-Fc variant fusion proteins in the treatment of patients with cancers and/or infections.

Brief description of the invention

The present invention provides, inter alia, sPD-1-Fc variant fusion proteins, polynucleotides encoding sPD-1-Fc variant fusion proteins, methods for generating sPD-1-Fc variant fusion proteins, and methods for using sPD-1-Fc variant fusion proteins. in particular for the treatment of a disease or disorder in which the adaptive immune system is suppressed or an increase in the magnitude or level of the immune response is required. In some embodiments, sPD-1-Fc variant fusion proteins can be used to treat cancer or a chronic viral infection. In other embodiments, the sPD-1-Fc variant fusion protein described herein can be used as an adjuvant therapy for the treatment of cancer.

In one aspect, the invention provides an sPD-1-Fc variant fusion protein comprising:

a) a soluble PD-1 variant (sPD-1) domain comprising an amino acid substitution compared to SEQ ID NO: 1, wherein said amino acid substitution is at a position selected from the group consisting of 120, 112, 107, 104, 67, 69, 96 and 42;

b) an optional linker; and

c) Fc domain.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the Fc fusion protein comprises from N- to C-terminus:

a) sPD-1 domain variant;

b) an optional linker; and

c) Fc domain.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the Fc fusion protein comprises from N- to C-terminus:

a) Fc domain;

b) an optional linker; and

c) sPD-1 domain variant.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the sPD-1 domain variant shares at least 95% identity with SEQ ID NO: 1.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the sPD-1 domain variant has amino acid substitution(s) at one of said positions, two of said positions, three of said positions, four of said of the provisions, five of the specified provisions, six of the specified provisions, seven of the specified provisions, or eight of the specified provisions.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the sPD-1 domain variant comprises an amino acid substitution selected from the group consisting of: A120V, A112I, S107V, G104S, S67G, P69L, N96S, and S42G.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the sPD-1 domain variant comprises a set of amino acid substitutions selected from the group consisting of S42G/S67G/P69L/G104S/S107V./A112I/A120V , S42G/S67G/P69L/N96S/G104S/S107V/A112I/A120V, P69L/G104S/S107V/A112I/A120V /A120V and G104S/S107V/A112I.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the sPD-1 domain variant comprises a set of S42G/S67G/P69L/G104S/S107V/A112I/A120V amino acid substitutions.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the sPD-1 domain variant comprises a set of S42G/S67G/P69L/N96S/G104S/S107V/A112I/A120V amino acid substitutions.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein said sPD-1 domain variant comprises the P69L/G104S/S107V/A112I/A120V amino acid substitution set.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein said sPD-1 domain variant comprises the S67G/G104S/S107V/A112I/A120V amino acid substitution set.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein said sPD-1 domain variant comprises the S67G/P69L/G104S/S107V/A112I/A120V amino acid substitution set.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein said sPD-1 domain variant comprises the G104S/S107V/A112I/A120V amino acid substitution set.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein said sPD-1 domain variant comprises the G104S/S107V/A112I amino acid substitution set.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described herein, wherein the sPD-1 domain variant has SEQ ID NO: 2.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described herein, wherein the sPD-1 domain variant has SEQ ID NO: 3.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described herein, wherein the sPD-1 domain variant has SEQ ID NO: 72.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described herein, wherein the sPD-1 domain variant has SEQ ID NO: 110.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described herein, wherein the sPD-1 domain variant has SEQ ID NO: 130.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described herein, wherein the sPD-1 domain variant has SEQ ID NO: 131.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described herein, wherein the sPD-1 domain variant has SEQ ID NO: 138.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the Fc domain is a human IgG Fc domain or a human IgG variant Fc domain.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the Fc domain of human IgG comprises a human IgG4 hinge-CH2-CH3.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the Fc domain is a variant Fc domain from human IgG.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the human IgG variant Fc domain comprises a human IgG4 hinge-CH2-CH3 with amino acid substitution S228P according to the EU numbering index.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the linker is selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the linker is GGGGS.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the Fc fusion protein has SEQ ID NO: 5.

In a further aspect, the invention provides an sPD-1-Fc variant fusion protein as described above, wherein the Fc fusion protein has SEQ ID NO: 6.

In a further aspect, the invention provides a nucleic acid encoding an sPD-1-Fc variant fusion protein as described above.

In a further aspect, the invention provides an expression vector comprising a nucleic acid as described above.

In an additional aspect, the invention relates to a host cell containing a nucleic acid or expression vector, as described above.

In a further aspect, the invention provides a method for producing an sPD-1-Fc variant fusion protein, comprising: a) culturing a host cell as described above under conditions in which the Fc fusion protein is expressed; and b) isolating the Fc fusion protein.

In a further aspect, the invention provides a method of treating, reducing, or preventing tumor metastasis or invasion in a cancer subject, the method comprising administering to the subject a therapeutically effective dose of one or more of said sPD-1-Fc variant fusion proteins as described above.

In a further aspect, the invention provides a method of treating, reducing or preventing tumor metastasis or invasion in a cancer subject as described above, wherein the cancer is selected from the group consisting of melanoma, glioma, lymphoma, myeloma, head and neck cancer, esophageal cancer, kidney cancer, lung cancer, breast cancer, liver cancer, colorectal cancer, gallbladder cancer, stomach cancer, pancreatic cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer and any other solid malignant tumor.

In a further aspect, the invention provides a method of treating a subject with an infection, wherein the method comprises administering to the subject a therapeutically effective dose of one or more of said sPD-1-Fc variant fusion proteins as described above.

In a further aspect, the invention provides a method of treating an object with an infection as described above, wherein the infection is a fungal infection, a bacterial infection, or a viral infection.

In a further aspect, the invention provides a method of treating a subject with an infection as described above, wherein the viral infection is selected from the group consisting of hepatitis B virus infection, hepatitis C virus infection, human papillomavirus infection, human immunodeficiency virus (HIV) infection, T infection - human lymphotrophic virus (HTLV), Epstein-Barr virus infection, herpes virus infection, cytomegalovirus infection and any other chronic viral infection.

In a further aspect, the invention provides a method of treating, reducing or preventing metastasis or tumor invasion in a subject with a cancer, as described above, or a method of treating a subject with an infection, as described above, wherein the effective dose of one or more sPD-1 variant fusion proteins is Fc inhibit, reduce or modulate signaling mediated by wild-type PD-1 in the subject.

In a further aspect, the invention provides a method of treating, reducing or preventing metastasis or tumor invasion in a subject with a cancer, as described above, or a method of treating a subject with an infection, as described above, wherein an effective dose of one or more Fc fusion proteins enhance T-cell object's response.

Brief description of the drawings

In FIG. 1 schematically shows the polypeptide domains of wild-type PD-1 (PD1 WT), sPD-1 variant version 1 (PD1 V1), sPD-1 variant version 2 (PD1 V2), and IgG4. The signal peptide region consists of residues 1 to 20 and is in italics, and the extracellular domain (ECD) is from residues 21 to 170. The mutated N-glycosylation site is in bold and underlined. Other mutated residues are in bold. Linkers are double underlined. Sequence numbering starts from the first amino acid of the signal peptide region.

In FIG. 2 shows the result of sequence alignment of human PD-1, chain B from 4ZQK and chain A from 5IUS. Identical residues are marked in blue, residues missing in both crystal structures are marked in red, residues missing only in 4ZQK are marked in blue.

Fig. 3 is an overlay of the human PD-1 model and the human PD-1/PD-L1 crystal structure (PDBID: 4ZQK). The crystal structures of PD-1 and PD-L1 in 4ZQK are shown in blue and green, respectively. The 7 residues (Ser62, Ser87, Pro89, Gly124, Ser127, Ala132 and Ala140 with position numbering starting from the signal region) on PD-1 for mutation are shown with orange sticks, the N-glycosylation site (Asn116) is shown with purple sticks. The position numbering starts from the first amino acid of the signal peptide region.

Fig. Figure 4 shows the temporal evolution of the RMSD backbone positions for WT PD-1/PD-L1 (marked in blue), PD-1/PD-L1 with 3 interface mutations (marked in red), and PD-1/PD-L1 with all 8 interface mutations. mutations (marked in green).

Fig. 5A shows sequence alignment results for human PD-L2 and mouse PD-L2 (PDBID: 3RNQ, 3BP6 and 3BP5). The sequence identity of human PD-L2 and mouse PD-L2 is about 72%. Fig. 5B shows a human PD-L2 consensus model. Identical remains are marked in blue. Remains in the missing loop are marked in red.

Fig. 6 shows a comparison of the PD-1 binding interaction region between the human PD-L2 model and the mouse PD-L2 crystal structure (PDBID: 3BP5). Residuals are shown as lines. The remnants of the interaction region are marked in green in the human PD-L2 model (shown in dark blue). The crystal structure of the PD-L2 mouse is shown in pink.

Fig. 7 is an overlay of a human PD-1/PD-L2 model and a mouse PD-1/PD-L2 crystal structure (PDBID: 3BP5). Human PD-1 and PD-L2 models are shown in cyan and dark blue, mouse PD-1 and PD-L2 structures are shown in yellow and pink, respectively.

Fig. 8 shows the binding affinity of the sPD-1-Fc variant fusion proteins to PD-L1 and PD-L2. The sPD-1-Fc variant fusion protein shows about a 10,000-fold improvement in PD-L1 binding compared to the WT PD-1-Fc fusion protein ("parent Fc fusion protein") and about a 200-fold improvement in PD-L2 binding compared to original hybrid protein Fc.

In FIG. 9A shows the binding activity of sPD-1-Fc variant fusion proteins (ie, sPD-1-Fc variant version 1 fusion protein and sPD-1-Fc variant version 2 fusion protein) on hPD-L1 MC38 knockout cells. Both sPD-1-Fc variant fusion proteins bind much better than the wild-type group (ie, the wild-type PD-1-Fc fusion protein group) to knockout MC38-PD-L1 cells. In FIG. 9B shows the binding activity of sPD-1-Fc variant fusion proteins (ie, sPD-1-Fc variant version 1 fusion protein and sPD-1-Fc variant version 2 fusion protein) on parental MC38 cells. The sPD-1-Fc variant fusion proteins also bind to parental MC38 cells, but to a lesser extent.

Fig. 10 shows T cell activation in the presence of Hep3B-hPD-L1 cells upon incubation with various concentrations of wild-type sPD-1, sPD-1 and hIgG4 mutants. T cell activity is measured by IFN-β. Error bars are the mean and standard deviation for three technical repetitions. The experiment was carried out twice independently with PBMC isolated from different donors.

Fig. 11A shows the sPD-1-Fc variant version 2 fusion protein showing better anti-tumor activity compared to the anti-PD-L1 antibody, as evidenced by the reduction in tumor volume in the PD-1-ECD-Fc group (i.e., in the fusion group). sPD-1-Fc variant version 2 protein) compared to a positive control group (i.e., an anti-PD-L1 antibody group). Fig. 11B shows the sPD-1-Fc variant version 2 fusion protein showing superior anti-tumor activity compared to anti-PD-L1 antibody, as evidenced by enhanced tumor growth inhibition in the PD-1-ECD-Fc group (i.e., in the fusion protein version 2 of the sPD-1-Fc variant) compared to a positive control group (i.e., an anti-PD-L1 antibody group). Fig. 11C shows the sPD-1-Fc variant version 2 fusion protein showing superior antitumor activity compared to the anti-PD-L1 antibody, as evidenced by increased survival in the PD-1-ECD-Fc group (i.e., the fusion protein version 2 sPD-1-Fc variants) compared to a positive control group (i.e., an anti-PD-L1 antibody group). In FIG. 11D shows the relative changes in body weight in the vehicle control, PD-1-ECD-Fc (i.e., variant sPD-1-Fc fusion protein version 2 groups) and positive control (i.e., anti-PD-L1- antibodies).

Fig. 12 shows the hinge sequences of human IgG1, IgG2, IgG3 and IgG4.

Fig. 13 shows that human PD-L2 is overexpressed in Hep3B and MC38 cell lines. Positive clones were selected by fluorescence activated cell sorting (FACS).

In FIG. 14 shows that the sPD-1-Fc variant 2 fusion protein binds strongly to Hep3b-hPDL2 cells in the PD1 protein and tab1 PDL2 binding assay on Hep3b-hPDL2 cells.

Fig. 15 shows T cell activation test in co-culture of PBMC and Hep3B-OS8-PDL2 4B9.

In FIG. 16 shows tumor volumes on different days after administration of vehicle control, sPD-1-Fc variant 2 fusion protein, or pembrolizumab in an in vivo tumor model using the MC38-hPDL2 colorectal tumor cell line.

In FIG. 17A-17O show the amino acid sequences of the mature extracellular domain (ECD) from WT PD1 (SEQ ID NO: 11) and various sPD-1 variants (SEQ ID NO: 12-139) compared to WT PD1. A signal peptide region, such as the sequence shown in SEQ ID NO: 7, may be added before each of the sequences shown in FIG. 17A-17O. A linker domain, such as GGGGS, may be added after each of the sequences, as shown in FIG. 17A-17O. The mutated N-glycosylation site is in bold and underlined. Other mutated residues are in bold. Mutations in each colony are summarized in Table 10 with sequence numbering starting from its mature region. In FIG. 17A shows the amino acid sequences of WT PD1 (SEQ ID NO: 11) and various sPD-1 variants (SEQ ID NO: 12-19). In FIG. 17B shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 20-28). In FIG. 17C shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 29-37). In FIG. 17D shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 38-46). In FIG. 17E shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 47-55). In FIG. 17F shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 56-64). In FIG. 17G shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 65-73). In FIG. 17H shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 74-82). In FIG. 17I shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 83-91). In FIG. 17J shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 92-100). In FIG. 17K shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 101-109). In FIG. 17L shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 110-118). In FIG. 17M shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 119-127). In FIG. 17N shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 128-136). In FIG. 17O shows the amino acid sequences of various sPD-1 variants (SEQ ID NOs: 137-139).

In FIG. 18A-18F show T-cell expression of PD-L2, PD-L1, and CD8+ in human microarray tissues from ovarian, esophageal, gastric, and brain tumors. Fig. 18A shows that representative microarray images of tissues from ovarian cancer containing both normal and malignant specimens (N = 156) were stained with antibodies against human PD-L2 (gray), against human PD-L1 (Latchman, Wood et al. ) and cytokeratin (red) by fluorescent immunohistochemistry. The staining intensity of each sample was scored and quantified according to tumor grade (upper right, PD-L2; lower right, PD-L1). Fig. 18B shows representative images of normal and malignant esophageal tissues (N=72) stained with fluorescent immunohistochemistry against human PD-L2 (grey), against human PD-L1 (Latchman, Wood et al.) and cytokeratin (red). Each sample was evaluated and quantified to show PD-L2 (upper right) and PD-L1 (lower right) expression according to tumor classification. Fig. 18C shows representative microarray images of gastric cancer tissues (N=76) stained and then quantified for both PD-L2 (upper right) and PD-L1 (lower right) expression based on tumor classification. Fig. 18D shows representative microarray images of glioblastoma tissues (N = 152) stained and then quantified for expression of PD-L2 (upper right) and PD-L1 (lower right) based on tumor classification, DAPI staining indicates the presence of nuclei. Fig. 18E shows a microarray of ovarian cancer tissues containing the same samples examined in FIG. 18A stained for anti-human CD8 (red) and DAPI (blue) antibodies with representative images shown herein. Positive for CD8 cells are marked with arrows, and the scale mark is 100 μm. Fig. 18F shows a microarray of esophageal cancer tissues containing the same samples examined in FIG. 18B stained with anti-human CD8 (red) and DAPI (blue) antibodies with representative images shown. Positive for CD8 cells were marked with arrows, and the scale mark is 100 μm. Each sample was quantified showing the number of positive nuclei per field and plotted according to tumor classification (right). All quantitative data were plotted with the score or number of positive nuclei per patient sample with the calculated mean and standard deviation. Statistical analysis was performed using one-way ANOVA to compare treatment groups. P-value *=<0.05, **=<0.01 and ***=<0.001.

In FIG. 19A-19D show sPD-1 mutants with excellent binding affinity for PD-L1 and PD-L2. Fig. 19A shows a graphical illustration of three different strategies for inhibiting PD-1 signaling. Fig. 19B shows representative flow cytometry dot plots demonstrating yeast display binding of the sPD-1 mutant library to PD-L1. Clones with the strongest binding to PD-L1 were selected by successively decreasing the concentration of PD-L1 (grade 1 - grade 4) followed by increasing incubation time (grade 5 and grade 6). Fig. 19C shows that the kinetics of sPD-1 mutant version 1 (Topalian, Hodi et al. 2012, N Engl. J. Med. 366 (26): 2443-2454, hereby incorporated by reference in its entirety), and wild-type PD-1 binding ( bottom) with human PD-L1 was determined using the BIAcore Surface Plasma Resonance System. Each curve represents a separate analyte concentration. Fig. 19D shows that the kinetics of sPD-1 mutant version 1 (Topalian, Hodi et al. 2012, N Engl. J. Med. 366 (26): 2443-2454, incorporated herein by reference in its entirety) and wild-type PD-1 binding (bottom) with human PD-L2 was determined using the BIAcore Surface Plasma Resonance System. Each curve represents a separate analyte concentration.

In FIG. 20A-20C show computer simulation-based structural analysis of sPD-1 mutants co-complexed with human PD-L1 and PD-L2. Fig. 20A shows in the left panel that the G124S sPD-1 mutation (orange) forms a hydrogen bond with PD-L1 Tyr123 (green and red rods). The structure of wild-type PD-1 is shown in blue. The right panel shows the A132I mutation forming another hydrogen bond to PD-L1 Gln166. Wild type PD-1 structure (blue), PD-1 mutant structure (orange). The crystal structure of PD-L1 is shown in pink and the co-complex of PD-L1 with the sPD-1 mutant is shown in green. In FIG. 20B shows a comparison of the surface complementarity of the A132I mutation. The PD-L1 binding site is labeled to reveal the electrostatic potential surface. Red indicates negative electrostatic potential, blue indicates positive electrostatic potential, and gray indicates hydrophobic regions. Wild-type PD-1 is depicted in cyan pattern and balls (left panel); mutant PD-1 shown in orange pattern and balls (right panel). In FIG. 20C shows a surface complementarity comparison of the A132I mutation with PD-L2 with the same color annotation as in FIG. 20b.

In FIG. 21A-21C demonstrate that sPD-1 mutants showed superior ability to block both PD-L1 and PD-L2 mediated activities in a ligand-dependent manner without affecting T cell viability. In FIG. 21A shows a cellular receptor blocking assay demonstrating percent inhibition of Hep3B-hPD-L1 binding to wild-type biotin-conjugated PD-1 in competition with wild-type PD-1, hIgG4, and sPD-1 mutants. One of two independent experiments shown in this document with single points representing the average of two technical replicates. Fig. 21B shows a cell-based receptor blocking assay showing percent inhibition of Hep3B-hPD-L2 cell binding to biotin-conjugated wild-type PD-1 in competition with wild-type sPD-1, hIgG4, and sPD-1 version 1 and 2 mutants. One of of the two independent experiments shown herein, with single points representing the average of two technical replicates. Fig. 21C shows T cell proliferation over time in the presence of sPD-1 version 2 mutant and aPD-1 antibody with or without recombinant PD-L1 added. T cells treated with CD3/CD28 antigen were used as a positive control and untreated T cells served as a negative control. Each data point shows the mean and standard deviation of the technical repetition. The experiment was repeated with T cells isolated from another donor. Statistical analysis was performed using one-way ANOVA to compare between treatment groups and repeated ANOVA to detect changes over time. P-value *=<0.05, **=<0.01.

In FIG. 22A-22H show that the sPD-1 mutant inhibits tumor growth in mouse models of colorectal, melanoma, and ovarian cancer tumors. In FIG. 22A shows the biodistribution of the Alexa Fluor® 488 labeled sPD-1 mutant plotted over time. In FIG. 22B shows the biodistribution of the sPD-1 mutant in mouse serum after a single dose of the 10 mg/kg molecule detected by anti-human IgG4 ELISA. Each data point is the average of two animals collected at the same time point. Fig. 22C shows tumor growth over time in C57B/6 mice inoculated with MC39-hPD-L2 colorectal cancer then correlated with vehicle control, sPD-1 mutant 10 mg/kg, or pembrolizumab 10 mg/kg. Each data point represents the mean and SEM for an individual tumor measured over time. Fig. 22D shows tumor growth over time in C57B/6 mice inoculated with B16/OVA melanoma cells then treated with vehicle control or 10 mg/kg sPD-1 mutant. The structure of wild-type PD-1 is shown in blue. Fig. 22E shows a Kaplan-Meier survival plot in C57B/6 mice orthotopically inoculated with ID8 mouse ovarian tumor cells treated with vehicle control, anti-mouse αPD-1 blocking antibody 10 mg/kg, and sPD-1 mutant 10 mg/kg. Animals were sacrificed when ascites developed. In FIG. 22F shows representative dot plots of flow cytometry of CD4+ and CD8+ cytotoxic T cells isolated from tumor (upper panel) and spleen (lower panel) of B16/OVA melanoma tumors treated with vehicle control, anti-mouse αPD-1 blocking antibody 10 mg/kg and sPD-1 mutant 10 mg/kg. In FIG. 22G shows CD8+ T cells isolated and analyzed from MC38-hPD-L2 tumors treated with vehicle control, anti-mouse PD-1 blocking antibody 10 mg/kg, and sPD-1 mutant 10 mg/kg. In FIG. 22H shows CD4+ T cells isolated and analyzed from MC38-hPD-L2 tumors treated with vehicle control, anti-murine PD-1 blocking antibody 10 mg/kg, and sPD-1 mutant 10 mg/kg. Statistical analysis was performed using one-way ANOVA to compare treatment groups and repeated ANOVA to detect changes over time. For survival curves, the Kaplan-Meier score was calculated. P-value *=<0.05, **=<0.01. ***=<0.001.

In FIG. 23A-23E show that the sPD-1 mutant is able to suppress tumor growth in a PD-L2 dependent ovarian cancer model. In FIG. 23A shows that ID8 PD-L1 CRSIPR KO tumors were inoculated into PD-L1 KO mice and stained for PD-L1 and PD-L2 expression. Scale mark 100 μm. Fig. 23B shows subcutaneous tumor growth over time in C57B/6 PD-L1 KO mice inoculated with ID8 PD-L1 CRSIPR KO tumors then treated with vehicle control, anti-mouse αPD-L1 blocking antibody 10 mg/kg and sPD-1 mutant 10 mg/kg. Fig. 23C shows the total mass of ID8 PD-L1 CRISPR KO tumors at the time of abort. In FIG. 23D shows Kaplan-Meier survival analysis of C57B/6 PD-L1 KO mice orthotopically inoculated with ID8 PD-L1 CRISPR KO mouse ovarian tumor cells treated with vehicle control, anti-mouse PD-L1 antibody 10 mg/kg, and sPD-1 10 mutant. mg/kg. Animals were sacrificed when ascites developed. In FIG. 23E shows IHC staining for PD-L1 and PD-L2 in ID8 tumors treated with vehicle control, anti-mouse αPD-L1 blocking antibody 10 mg/kg, and sPD-1 mutant 10 mg/kg. Scale mark 100 μm. Statistical analysis was performed using one-way ANOVA to compare between treatment groups and repeated ANOVA to detect changes over time. For survival curves, the Kaplan-Meier score was calculated. P-value *=<0.05, **=<0.01. ***=<0.001.

In FIG. 24A-24E show that PD-L1 and PD-L2 expression are positively correlated in patient tumor samples. In FIG. 24A shows a Pearson correlation plot of PD-L1 and PD-L2 expression in ovarian cancer. In FIG. 24B shows a Pearson correlation plot of PD-L1 and PD-L2 expression in esophageal cancer. In FIG. 24C shows a Pearson correlation plot of PD-L1 and PD-L2 expression in gastric cancer. In FIG. 24D shows a Pearson correlation plot of PD-L1 and PD-L2 expression in glioblastoma. In FIG. 24E shows IHC staining of human PD-L1 and PD-L2 on normal tonsil tissue showing a specific staining pattern.

In FIG. 25A-25B show CD8 expression in ovarian and esophageal cancer patient samples. In FIG. 25A shows that ovarian cancer samples have been quantified by the number of CD8 positive nuclei per field and plotted according to tumor classification. In FIG. 25B shows that esophageal cancer samples were quantified by the number of CD8 positive nuclei per field and plotted according to tumor classification.

Fig. 26 shows directed evolution sorting of mutant clones showing excellent binding characteristics. The first two sorts were performed by sequentially reducing the amount of PD-L1 incubated with the library. Sort three - sort six were screens based on a combination of reduced ligand concentration and a dissociation kinetics sorting strategy in which clones were isolated based on the ability to bind PD-L1 in the presence of lower ligand concentration and longer incubation time in the presence of competitors. The percentages in each panel correspond to the closed subpopulation collected.

In FIG. 27A and 27B show a kinetic analysis of the binding of the sPD-1 V2 mutant to PD-L1 and PD-L2. In FIG. 27A shows a BIAcore T200 binding analysis of sPD-1 version 2 mutant with PD-L1 binding kinetics at 25°C. In FIG. 27B shows BIAcore T200 binding analysis of sPD-1 version 2 mutant with PD-L2 binding kinetics at 25°C.

In FIG. 28A-28F show production of recombinant sPD-1 mutant protein. Fig. 28A shows SDS-PAGE of purified wild-type sPD-1-Fc, lane 1 under reducing conditions and lane 2 under non-reducing conditions. Fig. 28B shows SEC-HPLC analysis of purified wild-type sPD-1 Fc and endotoxin levels shown in the table below. Fig. 28C shows SDS-PAGE of the purified version 1 sPD-1 mutant, lane 1 under reducing conditions and lane 2 under non-reducing conditions. Fig. 28D shows SEC-HPLC analysis of purified sPD-1 version 1 mutant and endotoxin levels shown in the table below. Fig. 28E shows SDS-PAGE of the purified version 2 sPD-1 mutant, lane 1 under reducing conditions and lane 2 under non-reducing conditions. Fig. 28F shows SEC-HPLC analysis of purified sPD-1 version 2 mutant and endotoxin levels shown in the table below.

In FIG. 29 shows the protein interaction between the sPD-1 mutant and PD-L1 and the surface complementarity and hydrogen bond analysis for each of the three mutations at the PD-1 and PD-L1 binding interface.

Fig. 30 compares protein interactions for a wild-type and mutated human PD-1/PD-L2 model and shows the results of surface complementarity and hydrogen bond analysis for 2 mutations at the PD-1 and PD-L2 binding interface.

In FIG. 31 shows FACs-based analysis between wild-type sPD-1, PD-1 mutant version 2 and IgG4 and Hep3B cells overexpressing PD-L2.

In FIG. 32A-32E show confirmation of human PD-L2 overexpression in MC38 cells. In FIG. 32A is a table showing the percentage and mean fluorescence intensity of MC38 cells expressing 21 individual human PD-L2 clones. Fig. 32B shows MC38-hPDL2 cells showing less than 30% of the hPD-L2 positive population. Fig. 32C shows MC38-hPD-L2 cells showing less than 30%-50% of the hPD-L2 positive population. Fig. 32D shows MC38-hPD-L2 cells showing 50%-60% hPD-L2 positive population. Fig. 32E shows MC38-hPD-L2 cells showing over 60% of the hPD-L2 positive population.

In FIG. 33A and 33B show confirmation of human PD-L2 expression in Hep3B-OS8 cells and MC38 cells. Fig. 33A shows a table showing the mean fluorescence intensity and percentage of Hep3B-OS8 cells transfected with 16 cDNA clones positive for human PD-L2 overexpression. The positive clones were sorted to the right from lowest efficiency to highest efficiency. Ab1 is an anti-human PD-L2 antibody and Ab2 is a control IgG. Fig. 33B shows PD-L2 expression on original Hep3B cells, Hep3B-OS8 with vector only, Hep3B-OS8-hPD-L1 and Hep3B-OS8-hPD-L2 cells (upper panel); and PD-L2 expression on parental MC38, MC38-hPD-L and MC38-hPD-L2 cells (bottom panel). The orange histogram shows positive expression of PD-L2.

In FIG. 34 shows the total body weight of mice with MC38-hPD-L1 tumors treated with vehicle control, sPD-1 mutant, and atezoluzumab during the course of the experiment. N = 10 for each treatment group. The error bar represents the mean and standard deviation.

In FIG. 35A-35B show the immune profile of NK tumor cells and macrophages in parental MC38 tumor models treated with anti-mouse PD-1 antibody and sPD-1 mutant. Fig. 35A shows the percentage of positive NK cells in tumors of each animal treated with vehicle control (N=8), anti-mouse PD-1 antibody (N=8), and sPD-1 mutant antibody (N=10). Individual data points, mean and standard deviation are shown. Fig. 35B shows the percentage of positive macrophages in tumors of each animal treated with vehicle control (N=8), anti-mouse PD-1 antibody (N=8), and sPD-1 mutant antibody (N=10). Individual data points, mean and standard deviation are shown.

In FIG. 36A and 36B show the isolation of PD-L1 negative cells after CRISPR transfection with PD-L1. In FIG. 36A shows PD-L1 negative ID8 cells sorted and harvested after PD-L1 CRISPR transfection with clone 4 (left) and clone 5 (right). In FIG. 36B shows PD-L1-negative MC38 PD-L2 overexpressing cells sorted and harvested after PD-L1 CRISPR transfection with clone 4 (left) and clone 5 (right).

In FIG. 37 shows sPD-1 enriched library pool sequencing from each successive sorting round. In FIG. 37 shows sPD-1 enriched library pool sequencing from each successive sorting round.

In FIG. 38A shows an illustrated map of the amino acid mutations present in the original PD-1 mutant clone. All mutations were used to create a library of 128 sPD-1 mutant clones that includes all possible permutations of the 7 mutations. In FIG. 38B shows a list of the top 5 mutant clones selected from the 128 sPD-1 mutant clone library for further binding analysis.

Fig. 39A shows a dose-response curve of binding and Kd of the original sPD-1 mutant to PD-L1. Fig. 39B shows a dose dependent binding curve and Kd of clone #1 with PD-L1. Fig. 39C shows a dose dependent binding curve and Kd of clone #2 with PD-L1. Fig. 39D shows a dose dependent binding curve and Kd of clone #3 with PD-L1. Fig. 39E shows the dose dependent binding and Kd curve of clone #4. Fig. 39F shows a dose dependent binding curve and Kd of clone #5 with PD-L1.

In FIG. 40A shows an illustrated map of the amino acid mutations present in the original PD-1 mutant clone. All mutations were used to create a library of 128 sPD-1 mutant clones that includes all possible permutations of the 7 mutations. In FIG. 40B shows a list of the top 5 mutant clones selected from the 128 sPD-1 mutant clone, indicating Kd against human PD-L1.

Fig. 41A shows a dose-dependent curve of binding, Kd and Bmax of the original sPD-1 mutant to PD-L2. Fig. 41B shows a dose dependent binding curve, Kd and Bmax of clone #1 with PD-L2. Fig. 41C shows a dose-dependent curve of binding, Kd and Bmax of clone #2 to PD-L2. Fig. 41D shows a dose dependent binding curve, Kd and Bmax of clone #3 with PD-L2. Fig. 41E shows a dose dependent binding curve, Kd and Bmax of clone #4 with PD-L2. FIG. 41F shows a dose dependent binding curve, Kd and Bmax of clone #5 with PD-L2.

Fig. 42A shows an illustrated map of the amino acid mutations present in the original PD-1 mutant clone. All mutations were used to create a library of 128 sPD-1 mutant clones that includes all possible permutations of the 7 mutations. FIG. 42B shows a list of the top 5 mutant clones selected from sPD-1 mutant clone 128, with Kd against human PD-L2 listed.

Detailed description of the invention

Introduction

PD-1 is an inhibitory cell surface receptor involved in the control of T cell function during immunity and tolerance. After binding to its ligand, for example, PD-L1 or PD-L2, PD-1 inhibits the effector functions of T cells. Structure PD-1 is a type 1 single-pass membrane protein. PD-1 is encoded by the programmed cell death receptor 1 gene (Entrez Gene ID: 5133). The sequence (coding) of human PD-1 mRNA is given, for example, in Genbank accession no. NM 005018. The sequence of the human PD-1 polypeptide is set forth, for example, in Genbank Accession No. NP_005009 or UniProt No. Q15116. PD-1 is also known as programmed cell death 1, PDCD1, PD1, CD279, SLEB2, hPD-1 and hSLE-1. The wild-type human PD-1 polypeptide consists of 288 amino acids. The signal peptide sequence consists of residues 1 to 20 (Fig. 1), the ECD consists of residues 21 to 170 (Fig. 1), the transmembrane domain consists of residues 171 to 191, and the intracellular domain consists of residues 192 to 288 ( this document uses a numbering that starts with the signal peptide region, as will be understood by those skilled in the art, the numbering can also be based on using the first amino acid of the mature sequence as the first amino acid position, as shown in SEQ ID NO: 1 - SEQ ID NO : 6 from table 1, i.e. the ECD domain can also be from 1 to 150, etc.).

As is known in the art, there are a number of therapeutic antibodies that bind to PD-1 to block PD-1 from binding to the PD-L1 or PD-L2 receptor, resulting in reduced immune suppression to affect immune activation. These antibodies include KEYTRUDA® and OPDIVO®, as well as a number of others tested in the clinic. Similarly, there are anti-PD-L1 antibodies that are approved to lead to a similar mechanism and therapeutic effect, such as TECENTRIQ®.

The present invention is directed to a new mechanism for using ECD from human PD-1, including variants, to achieve a similar biological function and therapeutic effect. Thus, the present invention provides fusion proteins. The fusion proteins described herein contain two common functional components. The first component includes soluble ECD variants from human PD-1 (hereinafter "sPD-1 variant"). Variants of sPD-1 serve to increase binding affinity for PD-L1 and/or PD-L2 and/or protein stability. The second component is an Fc domain from a human IgG protein, eg human IgG4, to provide a significant increase in the half-life of the sPD-1 fusion protein variant. These two components or domains are typically linked using a domain linker, such as a glycine-serine linker, as described herein, to form the sPD-1-Fc variant fusion protein of the present invention.

The sPD-1 variant domain herein binds to and blocks/antagonizes PD-1 ligands, i.e. PD-L1 and/or PD-L2, and is linked to the Fc domain (optionally including the hinge domain and optionally other linkers) of the human IgG protein. sPD-1 variants can act as competitive PD-L1 and/or PD-L2 antagonists, block the PD-1 immune checkpoint pathway, and prevent signaling through the PD-1 receptor. Thus, the compositions and methods provided herein block T cell inhibitory signals and result in immune-mediated antitumor activity. In addition, the compositions and methods can activate, enhance, or enhance the immune response in a subject suffering from an infection, such as a chronic infection. The present invention provides compositions and methods for stimulating a T cell response, such as stimulating T cell proliferation, increasing T cell activation, and/or decreasing T cell inhibition signals in patients with cancer or infection.

Definitions

As used in this document, the following terms have the meanings assigned to them, unless otherwise indicated.

As used herein, the singular forms include the plural unless the context clearly indicates otherwise. Thus, for example, reference to "cell" includes a plurality of such cells, and reference to "agent" includes reference to one or more agents known to those skilled in the art, and so on.

Used in this document, the term "protein" means at least two covalently linked amino acids, which includes proteins, polypeptides, oligopeptides and peptides.

The term "isolated" refers to a molecule that is essentially free from its natural environment. For example, an isolated protein contains substantially no cellular material or other proteins from the cell or tissue source from which it is derived. The term "isolated" also refers to formulations in which the isolated protein is pure enough to be administered as a pharmaceutical composition, or at least about 70-80%, 80-90%, or 90-95% (w/w). ), or at least about 95%, 96%, 97%, 98%, 99%, or 100% (w/w).

The term "ligand" refers to a biomolecule that is capable of binding to and complexing with a second biomolecule, such as a receptor present on the surface of target cells, to accomplish a biological purpose. The ligand is typically an effector molecule that binds to a site on the target protein, for example, via intermolecular forces such as ionic bonds, hydrogen bonds, hydrophobic interactions, dipole-dipole bonds, or van der Waals forces. The sPD-1 variant of the present invention can bind to and complex with a PD-1 ligand such as PD-L1 and/or PD-L2.

The term "receptor" refers to a biomolecule present on the surface of a target cell that is capable of binding to and complexing with a second biomolecule, such as a ligand. The term "receptor" refers to a biomolecule present on the surface of a target cell that is capable of binding to and complexing with a second biomolecule, such as a ligand. PD-L1 and PD-L2 are examples of cell surface receptors.

By "position" in this description refers to the location in the sequence of the protein. The provisions may be numbered sequentially or according to a fixed format, such as the EU index. In some embodiments of the present invention, the positions are numbered sequentially, starting with the first amino acid of the mature protein.

By "amino acid modification" as used herein is meant an amino acid substitution, insertion and/or deletion in a polypeptide sequence.

By "amino acid substitution" or "substitution", as used herein, is meant the replacement of an amino acid at a particular position in the sequence of the parent polypeptide with another amino acid. In particular, in some embodiments, the substitution is with an amino acid that does not occur naturally at a particular position, or does not occur naturally in an organism or in any organism. For example, substitution S228P refers to a polypeptide variant, in this case a human IgG4 Fc variant, in which the serine at position 228 is replaced by a proline. To be clear, a protein that has been designed to change the coding sequence of a nucleic acid, but without changing the original amino acid (e.g., changing CGG (coding for arginine) to CGA (still encoding for arginine) to increase expression levels in the host) is not an "amino acid replacement"; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position it started at, then it is not an amino acid substitution.

By "amino acid insertion" or "insertion" in the context of the present description means the addition of an amino acid sequence at a specific position in the sequence of the original polypeptide. For example, -233E or 233E denotes an insertion of glutamic acid after position 233 and before position 234. In addition, -233ADE or A233ADE denotes an insertion of AlaAspGlu after position 233 and before position 234.

By "amino acid deletion" or "deletion" as used herein, is meant the removal of an amino acid sequence at a particular position in the original polypeptide sequence. For example, E233- or E233#, E233() or E233del denotes a deletion of glutamic acid at position 233. In addition, EDA233- or EDA233# denotes a deletion of the GluAspAla sequence that starts at position 233.

Under the "original polypeptide" in the context of the present description refers to the original polypeptide, which is subsequently modified to create a variant. The parent polypeptide may be a naturally occurring polypeptide or a variant or modified version of a naturally occurring polypeptide. The parent polypeptide may refer to the polypeptide itself, compositions that contain the parent polypeptide, or the amino acid sequence that encodes it. Accordingly, as used herein, the term "parent immunoglobulin" means an unmodified immunoglobulin polypeptide that has been modified to create a variant. In this context, "original Fc domain" will refer to the given variant; thus, a “human IgG variant Fc domain” is compared to the original human IgG Fc domain, e.g., a “human IgG4 variant Fc domain” is compared to the original human IgG4 Fc domain, and so on.

By "wild type or WT" as used herein is meant an amino acid sequence or nucleotide sequence that occurs naturally, including allelic variants. The WT protein has an amino acid or nucleotide sequence that has not been intentionally modified.

By “variant protein,” “protein variant,” or “variant,” as used herein, is meant a protein that differs from that of the parent protein based on at least one amino acid modification. In some embodiments, the parent proteins are human wild-type sequences. In some embodiments, the parent proteins are variant human sequences. A protein variant may refer to the protein itself, the composition containing the protein, or the amino acid sequence that encodes it. Preferably, the protein variant has at least one amino acid modification compared to the original protein, for example, from about one to about twenty amino acid modifications, and preferably from about one to about eight amino acid modifications compared to the original. The sequence of a protein variant provided herein will preferably have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the original protein sequence, and most preferably at least about 90% identity, more preferably at least about 95%/97%/98%/99% identity. Sequence identity between two similar sequences (eg, sPD-1 variable domains) can be measured using algorithms such as that of Smith, T.F. & Waterman, M.S. (1981) "Comparison Of Biosequences," Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S.B. & Wunsch, CD. (1970) "A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins," J. Mol. Biol.48: 443 [homology alignment algorithm], Pearson, W.R. & Lipman, D.J. (1988) "Improved Tools For Biological Sequence Comparison," Proc. Natl. Acad. sci. (USA) 85: 2444 [similarity search method]; or Altschul, S.F. et al, (1990) "Basic Local Alignment Search Tool," J. Mol. Biol. 215: 403-10, "BLAST" algorithm, see https://blast.ncbi.nlm.nih.gov/Blast.cgi. When using any of the above algorithms, default parameters are used (for window length, gap opening penalty, etc.). In one embodiment, sequence identity is performed using the BLAST algorithm using default parameters.

"IgG variant" or "IgG variant" in the context of the present description means an antibody that differs from the original IgG (again, in many cases, from the human IgG sequence) by at least one amino acid modification.

"Fc variant" or "Fc variant" in the context of the present description means a protein containing at least one amino acid modification compared to the original Fc domain. In some embodiments, the parent Fc domain is a human wild-type Fc sequence, such as an Fc region from IgG1, IgG2, IgG3, or IgG4. Thus, a "variant human IgG4 Fc domain" is a domain that contains amino acid modifications (usually amino acid substitutions) compared to a human IgG4 Fc domain. For example, S241P or S228P is a hinge variant with a proline change at position 228 relative to the parent IgG4 hinge polypeptide, where S228P is EU numbered and S241P is Kabat numbered. The EU suffix or EU suffix, as in the Kabat or EU numbering scheme, refers to the EU numbering (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al., incorporated herein in their entirety by reference, and see also Edelman et al., 1969, Proc Natl Acad Sci USA 63: 78-85, incorporated herein by reference in their entirety). In some embodiments, the parent Fc domains are variant human Fc sequences. For all positions discussed in the present invention that refer to the Fc domain of human IgG, unless otherwise indicated, the amino acid position numbering corresponds to the EU index. The modification may be an addition, a deletion, a substitution, or any combination thereof, as described herein. Alternatively, Fc domain variants can be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20 amino acid modifications compared to the original Fc domain. In addition, as discussed herein, the Fc domain variants herein still retain the ability to form a dimer with another Fc domain, as well as to bind to the FcRn receptor, as measured using the known methods described herein, such as non-denaturing gel- electrophoresis.

The term "soluble PD-1" or "sPD-1" as used herein means the soluble portion of a programmed cell death protein 1 (PD-1) polypeptide containing an extracellular domain (ECD) or fragment or truncated version thereof, but not a transmembrane domain or cytoplasmic (intracellular) domain of PD-1. The ECD from wild-type human PD-1 is shown from residues 21-170 in FIG. 1 and is also shown as SEQ ID NO: 1 in Table 1. In some embodiments, the original wild-type sPD-1 domain may have N-terminal and/or C-terminal truncations, as long as the truncated wild-type sPD-1 retains biological activity. eg binding to PD-L1 and/or PD-L2.

The term "sPD-1 variant" refers to wild type sPD-1 variant. The sPD-1 variant retains specific binding to a PD-1 ligand such as PD-L1 and/or PD-L2, but has amino acid substitutions and may have N- or C-terminal truncations compared to wild-type sPD-. 1. Specific binding in this case is determined using a standard binding assay such as ELISA, Biacore, Sapidyne KinExA or binding assay by flow cytometry, which can also be used to determine the binding affinity. As discussed herein, sPD-1 variants may, in some cases, have increased binding affinity compared to wild-type sPD-1.

The term "binding affinity" refers to the ability of a ligand or variant thereof to form coordinated bonds with a protein, such as a receptor or variant thereof. The binding affinity between a ligand and a protein can be represented by the equilibrium dissociation constant (KD), the koff/kon ratio between a ligand and a protein (eg, a receptor or a variant thereof). KD and binding affinity are inversely related. For example, a KD value refers to the concentration of the sPD-1 variant required to bind to the PD-1 ligand, and a lower KD value (lower concentration of the PD-1 variant) corresponds to a higher binding affinity for the PD-1 ligand. A high binding affinity corresponds to a greater intermolecular force between the ligand and the protein. A low binding affinity corresponds to a lower intermolecular force between the ligand and the protein. In some cases, an increase in ligand binding affinity can be represented as a decrease in the dissociation rate, for example, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, according to at least 200 times, at least 500 times or more.

The ability of the sPD-1 variant to bind to PD-L1 and/or PD-L2 can be determined, for example, by the ability of the putative ligand to bind to PD-L1 and/or PD-L2 coated on a test plate. In one embodiment, the binding activity of sPD-1 variants to PD-L1 and/or PD-L2 can be assayed either by immobilizing a ligand, eg, PD-L1 and/or PD-L2, or an sPD-1 variant. For example, the assay may involve immobilizing PD-L1 and/or PD-L2 fused to a His tag on Ni-activated NTA resin beads. Agents can be added to an appropriate buffer and the beads are incubated for a specified period of time at a given temperature. After washes to remove unbound material, bound protein can be released, for example, with SDS, high pH buffers, and the like. and analyzed.

Alternatively, the binding affinity of the sPD-1 variant to PD-L1 and/or PD-L2 can be determined by presenting the sPD-1 variant on the surface of microbial cells, such as the surface of yeast cells, and detecting the bound complex using, for example, flow cytometry. . The binding affinity of sPD-1 to PD-1 ligands can be measured using any known method recognized in the art, including but not limited to the method described in the examples, radioactive ligand binding assays, non-radioactive (fluorescent) ligand binding assays, surface plasmon resonance (SPR) such as Biacore™, Octet™, Plasmon Waveguide Resonance (PWR), thermodynamic binding assays, whole-cell ligand binding assays, and structure-based ligand binding assays.

"Specific binding" or "specifically binds to" or "specific to" a particular ligand or variant means a binding that is markedly different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a similarly structured molecule that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target. In some embodiments, binding affinity is measured using assays in the art as discussed above, such as the standard Biacore assay.

Specific binding to a particular ligand or variant thereof may occur, for example, to a protein having a KD for another ligand protein of at least about 10 ^-4 M, at least about 10 ^-5 M, at least about 10 ^{- 6} M, at least about 10 ^-7 M, at least about 10 ^-8 M, at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, at least about 10 ^-11 M, at least about 10 ^-12 M, or higher, where KD refers to the rate of dissociation of a particular protein-ligand interaction. Typically, a protein that specifically binds a ligand will have a KD of 20, 50, 100, 200, 500, 1000, 5000, 10,000 or more times the control molecule compared to the protein.

By "residue" as used herein is meant a position in a protein and associated amino acid identities. For example, asparagine 297 (also referred to as Asn297 or N297) is a residue at position 297 in a human IgG1 antibody.

By "hinge" or "hinge region" or "antibody hinge region" or "hinge domain" is meant herein a flexible polypeptide containing amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 215, and the IgG CH2 domain begins at EU residue position 231. Thus, for IgG, an antibody hinge is defined herein as comprising positions 216 (E216 in IgG1) to 230 (p230 in IgG1), where the numbering corresponds to the EU index, as in Kabat. In some cases, a "hinge fragment" is used that contains fewer amino acids at one or both of the N- and C-termini of the hinge domain. As stated herein, in some cases, Fc domains including a hinge are used, with the hinge typically being used as a flexible linker. (Also, as further described herein, additional flexible linker components can be used with or without a hinge).

By "Fc", "Fc region", or "Fc domain", as used herein, is meant a polypeptide containing the CH2-CH3 domains of an IgG molecule, and in some cases including a hinge. In the EU numbering for human IgG1, the CH2-CH3 domain consists of amino acids 231 to 447, and the hinge is 216 to 230. Thus, the definition of "Fc domain" includes both amino acids 231-447 (CH2-CH3) and 216- 447 (hinge-CH2-CH3), or fragments thereof. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and in some cases includes a flexible hinge located at the N-terminus relative to these domains. For IgA and IgM, Fc may include a J chain. For IgG, the Fc domain includes the Cγ2 and Cγ3 immunoglobulin domains, and in some cases includes the lower hinge region between Cγ1 and Cγ2. An "Fc fragment" in this context may contain fewer amino acids at one or both of the N- and C-termini, but still retain the ability to form a dimer with another Fc domain or Fc fragment, which can be detected by standard methods, usually depending on size ( eg, non-denaturing chromatography, size exclusion chromatography, etc.) Fc domains from human IgG are of particular use in the present invention and can be an Fc domain from human IgG1, IgG2, IgG3 or IgG4. As a rule, IgG1, IgG2 and IgG4 are used more often than IgG3. In some embodiments, amino acid modifications are made in the Fc region, for example, to change binding to one or more FcγR receptors or to the FcRn receptor, and/or to increase the in vivo half-life.

By “IgG subclass modification” or “isotype modification”, as used herein, is meant an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of another aligned IgG isotype. For example, since IgG1 contains tyrosine and IgG2 contains phenylalanine at EU positions 296, the F296Y substitution in IgG2 is considered a modification of the IgG subclass. Similarly, since IgG1 has a proline at position 241 and IgG4 contains a serine there, the IgG4 molecule with S241P is considered to be a modification of the IgG subclass. Note that subclass modifications are considered amino acid substitutions in this document.

By "non-natural modification" in this description is meant a modification of an amino acid that is not isotypic. For example, since none of the IgGs contains an asparagine at position 297, a substitution of N297A in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification.

By "amino acid" and "amino acid identity" as used herein, is meant one of the 20 naturally occurring amino acids encoded by DNA and RNA.

By "effector function", as used herein, is meant a biochemical event that results from the interaction of an Fc region of an antibody with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. In many cases, it is desirable to eliminate most or all of the effector functions using either different IgG isotypes (eg, IgG4) or amino acid substitutions in the Fc domain; however, retention of binding to the FcRn receptor is desirable, as this tends to increase the half-life of the fusion protein in human serum.

By "gamma receptor Fc", "FcγR", or "FcgammaR", as used herein, is meant any member of a family of proteins that bind to the Fc region of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including the FcγRIa, FcγRIb, and FcγRIc isoforms; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including the isoforms FcγRIIIa (including the V158 and F158 allotypes) and FcγRIIIb (including the FcγRIIb-NA1 and FcγRIIb-NA2 allotypes) (Jefferis et al., 2002, Immunol Lett 82: 57-65, incorporated herein in their entirety reference), as well as any undiscovered human FcγR or FcγR isoforms or allotypes. FcγR can be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγR, isoform or allotype of FcγR.

By "FcRn" or "neonatal Fc receptor", as used herein, is meant a protein that binds the Fc region of an IgG antibody and is encoded at least in part by the FcRn gene.

The terms "linker" or "linker peptide" in the context of the present description have a length sufficient to link two molecules in such a way that they take the correct conformation relative to each other, so that they retain the desired activity. The linker or linker peptide may advantageously include the following amino acid residues: Gly, Ser, Ala or Thr. In one embodiment, the linker is about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers from 1 to 20 amino acids in length can be used, and in some embodiments, from about 4 to about 10 amino acids can be used. Useful linkers include glycine-serine polymers including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer equal to at least one (and usually from 3 to 4), glycine-alanine polymers, alanine-serine polymers and other flexible linkers. Alternatively, a variety of non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers.

In some embodiments, the linker is a "domain linker" used to link any two domains as described herein together, for example, to link an sPD-1 variant domain to an Fc (variant) domain. While any suitable linker may be used, in many embodiments a glycine-serine polymer is used, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1 (and usually 3 to 4 to 5), as well as any peptide sequence that allows recombinant attachment of two domains with sufficient length and flexibility to allow each domain to retain its biological function. As discussed herein, a particularly useful domain linker is the GGGGS linker attached to the IgG4 hinge domain.

Under the "target cell" in the context of the present description refers to a cell that expresses the target polypeptide or protein.

By "host cell" in the context of producing sPD-1-Fc variant fusion proteins of the present invention is meant a cell that contains exogenous nucleic acids encoding components of an sPD-1-Fc variant fusion protein and is capable of expressing such Fc fusion protein under suitable conditions. . Suitable host cells are described below.

By "improved activity" or "improved function", as used herein, is meant the desired change in at least one biochemical property. Improved function in this context can be measured as a percentage increase or decrease in a particular activity, or as a "fold" change with an increase in properties sought (e.g., increased binding affinity and/or specificity for PD-L1 and/or PD-L2, increased stability of the fusion protein , increased half-life in vivo, etc.). In general, percentage changes are used to describe changes in biochemical activity of less than 100%, and fold changes are used to describe changes in biochemical activity of more than 100% (compared to the original protein). In the present invention, percentage changes (typically an increase) in biochemical activity of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% can be made. , 98% and 99%. In the present invention, a "fold increase" (or decrease) compared to the original protein is measured. In many embodiments, the improvement is at least 1.1 times (1.1), 1.5 times (1.5 times), 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times , 9 times, 10 times, 50 times, 100 times, 200 times or more. For example, as shown in FIG. 8, the sPD-1-Fc variant fusion protein showed about a 10,000-fold improvement in PD-L1 binding compared to the original WT PD-1-Fc fusion protein ("fusion protein with original Fc") and about a 200-fold improvement in PD binding. -L2 compared to the original Fc fusion protein.

Soluble fusion proteins of the PD-1-Fc variant

As described herein, the soluble PD-1-Fc variant fusion proteins ("sPD-1-Fc variant fusion protein") of the invention comprise an sPD-1 variant domain, an Fc domain, and optionally a linker binding the sPD-1 variant domain. with the Fc domain.

As described herein, the fusion protein format can take several configurations with the switching order of the component domains in the protein (from N- to C-terminus). In one embodiment, the fusion protein comprises an N- to C-terminal variant sPD-1 domain linker domain-Fc domain. In some embodiments, the fusion protein comprises an N- to C-terminal domain of an Fc-linker domain variant of the sPD-1 domain. In some embodiments, no linker is used, in which case the fusion protein contains, N- to C-terminally, either an sPD-1 domain variant-Fc domain or an Fc domain-sPD-1 domain variant. Note that in some cases the same fusion protein may be labeled slightly differently. For example, in the case where the Fc domain includes a hinge domain, the fusion protein containing the variant sPD-1 domain-Fc domain still includes a linker in the form of a hinge domain. Otherwise, the same protein may not have a hinge domain included in the Fc domain, in which case the fusion protein contains the sPD-1-CH2-CH3 domain variant.

Otherwise, the same protein may not have a hinge domain included in the Fc domain, in which case the fusion protein contains the sPD-1-CH2-CH3 domain variant.

In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins as described above, wherein the Fc domain contains a hinge domain and the sPD-1 variant is linked to the Fc domain via an additional linker as described herein. Thus, the fusion protein can be, N- to C-terminal, a sPD-1 domain variant -linker domain-hinge domain -CH2-CH3; sPD-1 domain variant -CH2-CH3 linker domain; hinge domain-CH2-CH3-linker domain-sPD-1 domain variant or CH2-CH3-linker domain-sPD-1 variant.

In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins as described above, wherein the Fc domain does not contain a hinge domain and the sPD-1 domain variant is linked to the Fc domain via a linker domain (e.g., without a hinge), as described in this document.

Variants of sPD-1 domains

Variant sPD-1 domains of the invention include variant human PD-1 soluble ECD. sPD-1 variants serve to increase binding affinity and/or specificity for PD-L1 and/or PD-L2 over wild type PD-1, as determined by art binding affinity assays such as the Biacore assay.

In some embodiments, the sPD-1 variants of the present invention are antagonists that bind to and block a PD-1 ligand (eg, PD-L1 and/or PD-L2) and thereby prevent the ligand from binding to its PD-1 receptor. Antagonists can enhance the immune response by inhibiting the PD-1 signaling pathway by reducing the amount of ligand available to bind the PD-1 receptor. Thus, the object can elicit a more robust immune response.

In some cases, a useful sPD-1 domain variant binds specifically to PD-L1 and/or PD-L2 on a target cell, such as a cancer cell, and thereby reduces (eg, blocks, prevents, etc.) the interaction between PD-L1/PD-L2 and PD-1 (eg, wild-type PD-1 on an immune cell, eg, on a T cell). Thus, the sPD-1 variant provided herein can act as an engineered decoy receptor for PD-L1 and/or PD-L2. By reducing the interaction between PD-L1 and/or PD-L2 and wild-type PD-1, the sPD-1 domain variant can reduce the immune inhibitory signals produced by the PD-L/PD-1 interaction and therefore can increase the immune response (e.g. , by increasing T-cell activation). A suitable sPD-1 domain variant may include a portion of the PD-1 that is sufficient to bind the PD-1 ligand with recognizable affinity, e.g., high affinity, which is typically located between the signal sequence and the transmembrane domain or fragment thereof that retains binding activity.

The names of the designated proteins/protein domains and the corresponding amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 10 are listed in Table 1, respectively.

Table 1. SEQ ID numbers, descriptions and corresponding amino acid sequences

SEQID NO
(Description) Amino acid sequence SEQID NO:1
(sPD-1 WT ECD) PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSSPSPRPAGQFQTLV SEQ ID NO:2
(ECD version 1 version sPD-1) PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSEGFVLNWYRMSPSNQTDKLAAFPEDRGQLGQDCRFRVTQLPNGRDFHMSVVRARRSDSGTYLCSAIVLAPKIQIKESLRVELRVTERRAEVPTAHPSSPSPRPAGQFQTLV SEQ ID NO:3
(ECD version 2 variant sPD-1) PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSEGFVLNWYRMSPSNQTDKLAAFPEDRGQLGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCSAIVLAPKIQIKESLRVELRVTERRAEVPTAHPSSPSPRPAGQFQTLV SEQ ID NO:4
(PD-1 WT ECD + domain linker + Fc fusion protein) PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:5
(ECD version 1 variant sPD-1 + domain linker + Fc fusion protein) PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSEGFVLNWYRMSPSNQTDKLAAFPEDRGQLGQDCRFRVTQLPNGRDFHMSVVRARRSDSGTYLCSAIVLAPKIQIKESLRVELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:6
(ECD version 2 variant sPD-1 + domain linker + Fc fusion protein) PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSEGFVLNWYRMSPSNQTDKLAAFPEDRGQLGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCSAIVLAPKIQIKESLRVELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:7
(signal area PD-1 WT) MQIPQAPWPVVWAVLQLGWR SEQ ID NO:8
(signaling region PD-1 WT + PD-1 WT ECD + domain linker + Fc fusion protein) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:9
(PD-1 WT signaling region + sPD-1 variant version 1 ECD + domain linker + Fc fusion protein) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSEGFVLNWYRMSPSNQTDKLAAFPEDRGQLGQDCRFRVTQLPNGRDFHMSVVRARRSDSGTYLCSAIVLAPKIQIKESLRVELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:10
(PD-1 WT signaling region + sPD-1 variant version 2 ECD + domain linker + Fc fusion protein) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSEGFVLNWYRMSPSNQTDKLAAFPEDRGQLGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCSAIVLAPKIQIKESLRVELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

In some embodiments, sPD-1 variants include amino acid substitutions, deletions or insertions, or any combination thereof, in the PD-1 domain of the WT as indicated in SEQ ID NO: 1, which increases or enhances its binding activity to PD-L1, PD-L2, or both compared to wild-type PD-1.

The present invention provides sPD-1 variant domains containing at least one amino acid substitution at one or more (e.g., multiple) positions corresponding to positions 120, 112, 107, 104, 67, 69, 96, and 42, compared to wild type of the original PD-1 domain of SEQ ID NO: 1 using numbering starting from the mature region. In some embodiments, the sPD-1 variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% , at least 98% or at least 99% but less than 100% sequence identity with the original PD-1 domain. In some embodiments, the parent PD-1 domain is SEQ ID NO: 1. In a preferred embodiment, the variant sPD-1 domain is at least 95%, at least 96%, at least 97%, at least , 98% or at least 99% but less than 100% sequence identity with SEQ ID NO: 1. In some embodiments, as noted herein, the sPD-1 domain variant may have N-terminal and/or C- terminal truncation compared to wild-type sPD-1, as long as the truncated sPD-1 variant retains biological activity (eg, binding to PD-L1 and/or PD-L2). For clarity, the sPD-1 variants of the invention contain at least one amino acid substitution and therefore do not have SEQ ID NO: 1.

The present invention provides sPD-1 variant domains containing at least one amino acid substitution at one or more (e.g., multiple) positions corresponding to positions 42, 67, 69, 96, 104, 107, 112, and 120, compared with wild-type parent domain PD-1 ECD SEQ ID NO: 1.

In some embodiments, the sPD-1 domain variant has amino acid substitutions at one of the specified positions, two of the specified positions, three of the specified positions, four of the specified positions, five of the specified positions, six of the specified positions, seven of the specified positions, or eight of the specified provisions as shown in Table 10.

In some embodiments, the present invention provides sPD-1 variant domains containing one or more amino acid substitutions selected from the group consisting of: A120V, A112I, S107V, G104S, S67G, P69L, N96S, and S42G compared to SEQ ID NO: 1 .

In some embodiments, the present invention provides sPD-1 variant domains comprising a set of amino acid substitutions selected from the group consisting of S42G/S67G/P69L/G104S/S107V/A112I/A120V, S42G/S67G/P69L/N96S/G104S/S107V/ A112I/A120V, S42G/S67G, S42G/P69L, S42G/G104S, S42G/S107V, S42G/A112I, S42G/A120V, S67G/P69L, S67G/G104S, S67G/S107V, S67G/A112I, S67G/A1 P69L/S107V S67G/S107V S42G/S67G/A112I S42G/S67G/A120V S42G/P69L/G104S S42G/P69L/S107V S42G/P69L/A112I S42G/P69L/A120V10 S42G/S42G S42G/G104S/A112I S42G/G104S/A120V S42G/S107V/A112I S42G/S107V/A120V S42G/A112I/A120V S67G/P69L/G104S A120V S67G/G104S/S107V S67G/G104S/A112I S67G/G104S/A120V S67G/S107V/A112I S67G/S107V/A120V G104S/120V, P69L /S107V/A11 2I G104G P69L/S107V S42G/S67G/P69L/A112I S42G/S67G/P69L/A120V S42G/S67G/G104S/S107V S42G/S67G/G104S/A112I /A120V67/S42G A112I S42G/S67G/S107V/A120V S42G/S67G/A112I/A120V S42G/P69L/G104S/S107V S42G/P69L/G104S/A112I /PV69 S42G/P69L/S107V/A120V S42G/P69L/A112I/A120V S42G/G104S/S107V/A112I S42G/G104S/S107V/A120V /S107V, S67G/P69L/G104S/A112I, S67G/P69L/G104S/A120V S107V/A120V, S67G/G104S/A112I/A120V, S67G/S107V/A112I/A120V, P69L/G104S/S107V/A112I, P69L/G104S/S104S69/A120V A120V, P69L/S107V/A112I/A120V, G104S/S107V/A112I /A120V, S42G/S67G/P69L/G104S/S107V, S42G/S67G/P69L/G104S/A112I, S42G/S67G/P69 L/G104S/A120V, S42G/S67G /P69L/S107V/A112I, S42G/S67G/P69L/S107V/A120V, S42G/S67G/P69L/A112I/A120V, S42G/S67G/G104S/S107V/A112I67, S107V/A112I67 G104S/S1G6, S107V G104S/A112I/A120V S42G/S67G/S107V/A112I/A120V S42G/P69L/G104S/S107V/A112I , S42G/G104S/S107V/A112I/A120V, S67G/P69L/G104S/S107V/A112I, S67G/P69L/G104S/S107V/A1 S107V/A112I/A120V, P69L/G104S/S107V/A112I/A120V, S42G/S67G/P69L/G104G/A112 /P69L/S107V/A112I/A120V, S42G/S67G/G104S/S107V/A112IG/A120 G104S/S107V/A112I/A120V and S67G/P69L/G104S/S107V/A112I/A120V compared to SEQ ID NO: 1. In some In embodiments, the invention provides sPD-1 variant Fc fusion protein(s) comprising sPD-1 variant domain(s) as described herein, an optional linker, and an Fc domain.

In some embodiments, the present invention provides sPD-1 variant domains comprising a set of amino acid substitutions selected from the group consisting of S42G/S67G/P69L/G104S/S107V/A112I/A120V, S42G/S67G/P69L/N96S/G104S/S107V/ A112I/A120V, P69L/G104S/S107V/A112I/A120V, S67G/G104S/S107V/A112I/A120V, S67G/P69L/G104S/S107V/A112I/A120V, G104S/A107V20V/S107V/S102 to compared to SEQ ID NO: 1. In some embodiments, the present invention provides variant sPD-1-Fc fusion proteins comprising a variant sPD-1 domain as described herein, an optional linker and an Fc domain.

In some embodiments, the present invention provides sPD-1 variant domains containing amino acid substitutions S42G/S67G/P69L/G104S/S107V/A112I/A120V compared to SEQ ID NO: 1; and sPD-1-Fc variant fusion proteins containing the sPD-1 domain variant as disclosed herein, an optional linker, and an Fc domain.

In some embodiments, the present invention provides sPD-1 variant domains comprising amino acid substitutions S42G/S67G/P69L/N96S/G104S/S107V/A112I/A120V compared to SEQ ID NO: 1; and sPD-1-Fc variant fusion proteins containing the sPD-1 domain variant as disclosed herein, an optional linker, and an Fc domain.

In some embodiments, the present invention provides sPD-1 variant domains comprising amino acid substitutions P69L/G104S/S107V/A112I/A120V compared to SEQ ID NO: 1; and sPD-1 variant Fc fusion proteins comprising a sPD-1 variant domain as disclosed herein, an optional linker, and an Fc domain.

In some embodiments, the present invention provides sPD-1 variant domains comprising amino acid substitutions S67G/G104S/S107V/A112I/A120V compared to SEQ ID NO: 1; and sPD-1 variant Fc fusion proteins comprising a sPD-1 variant domain as disclosed herein, an optional linker, and an Fc domain.

In some embodiments, the present invention provides sPD-1 variant domains comprising amino acid substitutions S67G/P69L/G104S/S107V/A112I/A120V compared to SEQ ID NO: 1; and sPD-1 variant Fc fusion proteins comprising a sPD-1 variant domain as disclosed herein, an optional linker, and an Fc domain.

In some embodiments, the present invention provides sPD-1 variant domains containing G104S/S107V/A112I/A120V amino acid substitutions compared to SEQ ID NO: 1; and sPD-1 variant Fc fusion proteins comprising a sPD-1 variant domain as disclosed herein, an optional linker, and an Fc domain.

In some embodiments, the present invention provides sPD-1 variant domains containing G104S/S107V/A112I amino acid substitutions compared to SEQ ID NO: 1; and sPD-1 variant Fc fusion proteins comprising the sPD-1 variant domain as disclosed herein, an optional linker, and an Fc domain.

In some embodiments, the present invention provides an sPD-1 domain variant having SEQ ID NO: 2 and sPD-1 variant Fc fusion proteins comprising SEQ ID NO: 2, an optional linker and an Fc domain.

In some embodiments, the present invention provides an sPD-1 domain variant having SEQ ID NO: 3 and sPD-1 variant Fc fusion proteins comprising SEQ ID NO: 3, an optional linker and an Fc domain.

In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins having SEQ ID NO: 5.

In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins having SEQ ID NO: 6.

In some embodiments, the sPD-1 domain variant contains an amino acid substitution for serine at position 42 with position numbering starting from the mature region. In some embodiments, the substitution is made to any other of the 19 naturally occurring amino acids, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan , valine and tyrosine, in some embodiments cysteine (due to possible disulfide formation) or proline (due to steric effects) is not used. In some embodiments, the amino acid substitution is S42G. In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins comprising an sPD-1 variant domain as described herein, an optional linker, and an Fc domain.

In some embodiments, the sPD-1 domain variant contains an amino acid substitution for serine at position 67, with positions numbered starting from the mature region. In some embodiments, the substitution is made to any other of the 19 naturally occurring amino acids, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan , valine and tyrosine, in some embodiments cysteine (due to possible disulfide formation) or proline (due to steric effects) is not used. In some embodiments, the amino acid substitution is S67G. In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins comprising an sPD-1 variant domain as described herein, an optional linker, and an Fc domain.

In some embodiments, the sPD-1 domain variant contains a proline amino acid substitution at position 69 with position numbering starting from the mature region. In some embodiments, the substitution is made to any other of the 19 naturally occurring amino acids, serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine , tryptophan, valine and tyrosine, in some embodiments cysteine is not used (due to the possible formation of disulfide). In some embodiments, the amino acid substitution is P69L. In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins comprising an sPD-1 variant domain as described herein, an optional linker, and an Fc domain.

In some embodiments, the sPD-1 domain variant contains the amino acid substitution of asparagine at position 96, with position numbering starting from the mature region. In some embodiments, the substitution is made to any other of the 19 naturally occurring amino acids, serine, threonine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan , valine and tyrosine, in some embodiments cysteine (due to possible disulfide formation) or proline (due to steric effects) is not used. In some embodiments, the amino acid substitution is N96S. In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins comprising an sPD-1 variant domain as described herein, an optional linker, and an Fc domain.

In some embodiments, the sPD-1 domain variant contains the glycine amino acid substitution at position 104 with position numbering starting from the mature region. In some embodiments, the replacement is any other of the 19 naturally occurring amino acids, serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, in some embodiments cysteine (due to possible disulfide formation) or proline (due to steric effects) is not used. In some embodiments, the amino acid substitution is G104S. In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins comprising an sPD-1 variant domain as described herein, an optional linker, and an Fc domain.

In some embodiments, the sPD-1 domain variant contains an amino acid substitution for serine at position 107 with position numbering starting from the mature region. In some embodiments, the substitution is made to any other of the 19 naturally occurring amino acids, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan , valine and tyrosine, in some embodiments cysteine (due to possible disulfide formation) or proline (due to steric effects) is not used. In some embodiments, the amino acid substitution is S107V. In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins comprising an sPD-1 variant domain as described herein, an optional linker, and an Fc domain.

In some embodiments, the sPD-1 domain variant contains an alanine amino acid substitution at position 112 with position numbering starting from the mature region. In some embodiments, the replacement is any other of the 19 naturally occurring amino acids, serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, in some embodiments cysteine (due to possible disulfide formation) or proline (due to steric effects) is not used. In some embodiments, the amino acid substitution is A112I. In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins comprising an sPD-1 variant domain as described herein, an optional linker, and an Fc domain.

In some embodiments, the sPD-1 domain variant contains an alanine amino acid substitution at position 120, with positions numbered starting from the mature region. In some embodiments, the replacement is any other of the 19 naturally occurring amino acids, serine, threonine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine, methionine, proline, phenylalanine, tryptophan, valine and tyrosine, in some embodiments cysteine (due to possible disulfide formation) or proline (due to steric effects) is not used. In some embodiments, the amino acid substitution is A120V. In some embodiments, the present invention provides sPD-1-Fc variant fusion proteins comprising an sPD-1 variant domain as described herein, an optional linker, and an Fc domain.

In some embodiments, the sPD-1 protein variant is shorter than the full length ECD. In some embodiments, sPD-1 variants may contain a truncated version of the ECD as long as the truncated form retains the ability to bind human PD-L1 and/or PD-L2 as measured by one of the binding assays described herein. As is known in the art, both N- and C-terminal truncations are possible, for example, from about 1, 5, 10, 15, 20, 25, 30, 33, 35, 40, 45, or 50 residues to about 130, 135, 140, 145, 149 or 150 residues from SEQ ID NO: 1, for example 33-150 residues. In some cases, only a few amino acids (eg, 1, 2, 3, 4, 5, or 6) are removed from one or both of the N- and C-terminus while the activity is maintained.

In some embodiments, the sPD-1 variant described herein has a binding affinity for a PD-1 ligand (i.e., PD-L1 and/or PD-L2) that is better than the wild-type PD-1 polypeptide/domain. In some embodiments, sPD-1 variants have a binding affinity for PD-L1 and/or PD-L2 that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10- fold, 50 fold, 100 fold, 200 fold, or more than wild-type PD-1. In some embodiments, sPD-1 variants may have a binding affinity for PD-L1 that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold , 100-fold, 200-fold or more than wild-type PD-1. In some embodiments, sPD-1 variants may have a binding affinity for PD-L2 that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold , 100-fold, 200-fold or more than wild-type PD-1.

In some embodiments, the binding affinity of the sPD-1 variant to PD-L1, PD-L2, or both is increased by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared to wild-type PD-1. In other embodiments, the sPD-1 variants of the present invention have a binding affinity of less than about 1 x 10 ^-8 M, 1 x 10 ^-9 M, 1 x 10 ^-10 M, or 1 x 10 ^-12 M for PD-L1 and/or PD-L2. The sPD-1 variants of the present invention may have a binding affinity of less than about 1 x 10 ^-8 M, 1 x 10 ^-9 M, 1 x 10 ^-10 M, or 1 x 10 ^-12 M for PD-L1. The sPD-1 variants of the present invention may have a binding affinity of less than about 1 x 10 ^-8 M, 1 x 10 ^-9 M, 1 x 10 ^-10 M, or 1 x 10 ^-12 M for PD-L2. In other embodiments, sPD-1 variants inhibit or compete for binding of wild-type PD-1 to PD-L1 and/or PD-L2 in vivo or in vitro, or both.

In some embodiments, the sPD-1 variant has a dissociation half-life for PD-L1 and/or PD-L2 that is 2-fold or greater (e.g., 5-fold or greater, 10-fold or greater, 100-fold or greater, 500-fold or more, 1000-fold or more, 5000-fold or more, 10,000-fold or more, etc.), greater than the half-life of dissociation from PD-L1 for wild-type PD-1.

Fc domains

As discussed herein, in addition to the sPD-1 variant domains described above, the fusion proteins of the present invention also include antibody Fc domains that are typically based on the IgG class, which has several subclasses including, but not limited to, IgG1, IgG2, IgG3 and IgG4. As described herein, the Fc domain optionally includes the hinge domain of an IgG antibody.

Fc domains from human IgG are particularly suitable for the present invention and can be an Fc domain from human IgG1, IgG2, IgG3 or IgG4. As a rule, IgG1, IgG2 and IgG4 are used more often than IgG3.

The Fc domain of the human IgG protein included in the fusion protein of the present invention provides a significant increase in the half-life of the fusion protein and provides additional binding or interaction with Ig molecules. In some embodiments, the sPD-1-Fc variant fusion protein can assist in the purification, multimerization, binding, and neutralization of other molecules compared to the monomeric sPD variant polypeptide.

The Fc domains that find use in the invention may also contain Fc variants to change function as needed. However, any Fc variants will typically retain both the ability to form dimers and the ability to bind FcRn. Thus, while many of the embodiments herein rely on the use of a human IgG4 domain to avoid effector function, Fc variants can be created that enhance or abolish function in other IgG domains. Thus, for example, ablation variants that reduce or abolish effector function in IgG1 or IgG2 can be used, and/or Fc variants that provide stronger binding to FcRn can be used, as will be appreciated by those skilled in the art.

Fc domains from IgG4

The IgG4 subclass differs from other IgG subclasses in that it exhibits little binding to the C1q protein complex and cannot activate the classical complement pathway (A. Nirula et al., 2011, Current Opinion in Rheumatology 23: 119-124, incorporated herein in its entirety by reference ). As a result, IgG4 finds use in the present invention because it does not have a significant effector function and is thus used to block receptor-ligand binding without cell depletion.

In another embodiment, the Fc domains of the present invention are Fc domains from human IgG4.

In some embodiments, the Fc domain of the present invention comprises a CH2-CH3 hinge from human IgG4.

In some embodiments, the Fc domain of the present invention comprises CH2-CH3 from human IgG4.

In another embodiment, the Fc domains of the present invention are variant Fc domains from human IgG4. However, the Fc domain variants in this document still retain the ability to form a dimer with another Fc domain, as measured using known ones, as well as the ability to bind to FcRn, as this contributes significantly to the increase in serum half-life of the fusion proteins described herein. .

The IgG4 Fc domain variant may include an addition, deletion, substitution, or any combination thereof, compared to the original human IgG4 Fc domain.

In some embodiments, the variant human IgG4 Fc domains of the present invention may have at least 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identity with the corresponding parental IgG4 Fc domain. human (using the identification algorithms described above, with one implementation using the BLAST algorithm as known in the art using default parameters).

In some embodiments, the Fc domain variants of human IgG4 of the present invention may be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12 , 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the original human IgG4 Fc domains.

In some embodiments, the variant human IgG4 Fc domain contains the amino acid substitution of serine at position 228 for proline according to the EU numbering index.

Other Fc domains from IgG

In some embodiments, the Fc domains of the present invention may be Fc domains from IgGs other than IgG4, such as human IgG1, IgG2, or IgG3. As a rule, IgG1 and IgG2 are used more often than IgG3.

In some embodiments, the Fc domain of the present invention is an Fc domain from human IgG1.

In some embodiments, the Fc domain of the present invention is an Fc domain from human IgG2.

In some embodiments, the Fc domain of the present invention is a variant Fc domain from human IgG1.

In some embodiments, the Fc domain of the present invention is a variant Fc domain from human IgG2.

In some embodiments, the human IgG1 Fc domain variants of the invention may have at least 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identity with the corresponding parental human IgG1 Fc domain (with using the identification algorithms described above with one implementation using the BLAST algorithm as known in the art using default parameters).

In some embodiments, variants of human IgG2 Fc domains of the invention may have at least 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identity with the corresponding parental human IgG2 Fc domain ( using the identification algorithms described above, with one implementation using the BLAST algorithm as known in the art using default parameters).

In some embodiments, the human IgG3 Fc domain variants of the invention may have at least 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identity with the corresponding parental human IgG3 Fc domain (with using the identification algorithms described above, one implementation uses the BLAST algorithm known in the art using default parameters).

In some embodiments, variants of human IgG1 Fc domains of the present invention may be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12 , 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the original Fc domains from human IgG1.

In some embodiments, the human IgG2 Fc domain variants of the present invention may be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications compared to the original Fc domains from human IgG2.

In some embodiments, the human IgG3 Fc domain variants of the present invention may be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications compared to the original Fc domains from human IgG3.

Linkers

The two domains described above (ie, a variant sPD-1 domain and an Fc domain) are typically linked using a linker domain as described herein. In the context of the invention, it is important that the two domains are connected by a flexible linker in such a way that the two domains can act independently. This can be done in various ways using conventional linkers and/or a hinged linker.

While any suitable linker may be used, in many embodiments a glycine-serine polymer is used, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1 (and usually 3 to 4 to 5), as well as any peptide sequence that allows recombinant attachment of two domains with sufficient length and flexibility to allow each domain to retain its biological function.

In some embodiments, the hinge domain of a human IgG antibody is used. The hinge domains of human IgG1, IgG2, IgG3 and IgG4 are shown in FIG. 12. In some cases, the hinge domain may also contain amino acid substitutions. For example, as shown in FIG. 1 and Table 1 use a hinge domain from IgG4 containing the S228P variant.

In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In one embodiment, the linker is about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length.

In another embodiment, the linker is 1 to 20 amino acids in length, preferably about 5 to about 10 amino acids in length.

Private embodiments of the invention.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant exhibiting at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO: 1.

In some embodiments, the sPD-1-Fc variant fusion proteins of the present invention comprise: a) a soluble PD-1 variant (sPD-1) domain comprising an amino acid substitution compared to SEQ ID NO: 1, where said amino acid substitution is at position, selected from the group consisting of 42, 67, 69, 96, 104, 107, 112 and 120 b) a linker and c) an Fc domain. In some embodiments, the sPD-1-Fc variant fusion protein contains from N- to C-terminus: a) said sPD-1 domain variant; b) said linker; and c) said Fc domain. In some embodiments, the sPD-1-Fc variant fusion protein contains from N- to C-terminus: a) said Fc domain; b) said linker; and c) said sPD-1 domain variant. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG Fc variant domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion proteins of the present invention comprise a soluble PD-1 variant (sPD-1) domain comprising an amino acid substitution compared to SEQ ID NO: 1, wherein said amino acid substitution is at position number, selected from the group consisting of 42, 67, 69, 96, 104, 107, 112 and 120 and an Fc domain. In some embodiments, the sPD-1-Fc variant fusion protein contains from N- to C-terminus: a) said sPD-1 domain variant; and b) said Fc domain. In some embodiments, the sPD-1-Fc variant fusion protein contains from N- to C-terminus: a) said Fc domain; and b) said sPD-1 domain variant. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG Fc variant domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises: a) an sPD-1 domain variant comprising an amino acid substitution selected from the group consisting of S42G, S67G, P69L, N96S, G104S, S107V, A112I, and A120V by compared with SEQ ID NO: 1; b) a linker; and c) an Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG variant Fc domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises: an sPD-1 domain variant comprising an amino acid substitution selected from the group consisting of S42G, S67G, P69L, N96S, G104S, S107V, A112I, and A120V vs. with SEQ ID NO: 1; and the Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG Fc variant domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises: a) an sPD-1 domain variant comprising a set of amino acid substitutions S42G/S67G/P69L/G104S/S107V/A112I/A120V, S42G/S67G/P69L/N96S/ G104S/S107V/A112I/A120V, S42G/S67G, S42G/P69L, S42G/G104S, S42G/S107V, S42G/A112I, S42G/A120V, S67G/P69L, S67GV/G104, S67G/A112I, S67G/A12L0 G104S, P69L/S107V, P69L/A112I, P69L/A120V, G104S/S107V, G104S/A112I, G104S/A120V, S107V/A112I, S107V12/A120V, S107V12/A120V,/S67G/P69L /S67G/S107V S42G/S67G/A112I S42G/S67G/A120V S42G/P69L/G104S S42G/P69L/S107V S42G/P69L42/A112I/A120V S42G/G104S/S107V /G104S/A120V S42G/S107V/A112I S42G/S107V/A120V S42G/A112I/A120V S67G/P69L/S104V69 S67G/P69L/G104V69 S67G/P69L/A112I /S107V S67G/G104S/A112I S67G/G104S/A120V S67G/S107V/A112I S67G/S107V/A120V S67G20/A1/G104S/S107V P69L/G104S/A112I S107V/A112I, P69L/S107V/120V, P69L/A112 I/A120V G104S/S107V/A112I G104S/S107V/A120V G104S/A112I/A120V S107V/A112I/A120V S42SL/S67 S42G/S67G/P69L/S107V /P69L/A120V, S42G/S67G/G104S/S107V, S42G/S67G/G104S/A112I, S42G/S67G/G104S/S42G/S67G/G104S A112I/A120V, S42G/P69L/G104S/S107V, S42G/P69L/G104S/A112I, S42G/P69L/G104SG/A120 /S107V/A112I, S42G/G104S/S107V/A120V, S42G/G104S/A112I/A112I/S P69L/S107V/A112I, S67G/P69L/S107V/A120VL, S67V12/P69 A120VL, S67V12/P69 A112I/A120V, P69L/G104S/S107V/A112I, P69LV10, P69LV10/P10V4S/G10V4S G104S/A112I/A120V, P69L/S107V/A112I/A120V, G104S/S107V/A112I/A120V, S42G/S67G/P69L/G104S/S107V , S42G/S67G/P69L/G104S/A112I, S42G/S67G/P69L/G104S/A120V, S42G/S67G/P69L/S107V/A112I, S42G/S67G/P69 L/S107V/A120V, S42G/S67G/P69L/A112I/A120V, S42G/S67G10/G10V4S/A1 /A120V/S42G/P69L/G104S/S107V/A112I/S42G/P69L20V/G10 S42G/P69L/G104S/A112I/A120V/S42G/P69L/S107V/A112I/A120V P69L/G104S/S107V/A112I, S67G/P69L20V/G10 S67G/P69L/G104S/A112I/A120V /A120V, S42G/S107GV/P69 A112I, S42G/S67G/P69L/G104S/S107V/A120V, S42G/S67G/P69L/G104S/A112I/A120V, S42G/S67G/P69L/S107V/A1/12I/A120V, S42 G104V/A120/S107/S107 S42G/P69L/G104S/S107V/A112I/A120V and S67G/P69L/G104S/S107V/A112I/A120V versus SEQ ID NO: 1; b) an optional linker; and c) an Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG Fc variant domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant comprising a set of amino acid substitutions S42G/S67G/P69L/G104S/S107V/A112I/A120V as compared to SEQ ID NO: 1, an optional linker, and Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG variant Fc domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant comprising a set of amino acid substitutions S42G/S67G/P69L/N96S/G104S/S107V/A112I/A120V compared to SEQ ID NO: 1, optional linker and Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG variant Fc domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant comprising a set of P69L/G104S/S107V/A112I/A120V amino acid substitutions compared to SEQ ID NO: 1, an optional linker, and an Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG variant Fc domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant comprising a set of S67G/G104S/S107V/A112I/A120V amino acid substitutions compared to SEQ ID NO: 1, an optional linker, and an Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG variant Fc domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant comprising a set of amino acid substitutions S67G/P69L/G104S/S107V/A112I/A120V compared to SEQ ID NO: 1, an optional linker, and an Fc domain . In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG variant Fc domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant comprising a set of G104S/S107V/A112I/A120V amino acid substitutions compared to SEQ ID NO: 1, an optional linker, and an Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG Fc variant domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant comprising a G104S/S107V/A112I amino acid substitution set compared to SEQ ID NO: 1, an optional linker, and an Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is a human IgG Fc variant domain, wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human IgG variant Fc domain disclosed herein comprises a human IgG4 hinge-CH2-CH3 with the amino acid substitution S228P. In some embodiments, the linker is a domain linker selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group consisting of 1, 2, 3, 4 and 5. In some embodiments, the linker is GGGGS. In some embodiments, the domain linker is a combination of a hinge domain and a flexible linker, such as an IgG4 hinge with S228P, as well as a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant, an optional linker, and an Fc domain, wherein the sPD-1 domain variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO : 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO. : 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 ., SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO. : 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO : 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO : 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 , SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO : 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO : 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111 , SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118 , SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO : 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 and SEQ ID NO: 139.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 2, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 3, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 72, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 110, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 130, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 131, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 138, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 12, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 13, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 14, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 15, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 16, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 17, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 18, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 19, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 20, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 21, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 22, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 23, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 24, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 25, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 26, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 27, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 28, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 29, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 30, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 31, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 32, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 33, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 34, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 35, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 36, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 37, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 38, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 39, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 40, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 41, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 42, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 43, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 44, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 45, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 46, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 47, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 48, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 49, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 50, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 51, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 52, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 53, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 54, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 55, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 56, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 57, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 58, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 59, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 60, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 61, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 62, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 63, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 64, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 65, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 66, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 67, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 68, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 69, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 70, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 71, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 73, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 74, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 75, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 76, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 77, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 78, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 79, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 80, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 81, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 82, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 83, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 84, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 85, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 86, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 87, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 88, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 89, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 90, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 91, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 92, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 93, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 94, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 95, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 96, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 97, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 98, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 99, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 100, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 101, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 102, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 103, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 104, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 105, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 106, an optional linker, and an Fc domain.

In some embodiments, an sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 107, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 108, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 109, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 111, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 112, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 113, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 114, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 115, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 116, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 117, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 118, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 119, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 120, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 121, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 122, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 123, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 124, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 125, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 126, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 127, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 128, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 129, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 132, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 133, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 134, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 135, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 136, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 137, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises an sPD-1 domain variant as set forth in SEQ ID NO: 139, an optional linker, and an Fc domain.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises a human IgG Fc domain or a human IgG variant Fc domain.

In a preferred embodiment, the sPD-1-Fc variant fusion protein of the present invention comprises a variant Fc domain from human IgG.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises a human IgG Fc domain containing a human IgG4 hinge-CH2-CH3.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises a human IgG Fc domain containing CH2-CH3 from human IgG4.

In a preferred embodiment, the sPD-1-Fc variant fusion protein of the present invention comprises a human IgG Fc variant domain comprising a human IgG4 hinge-CH2-CH3 with amino acid substitution S228P according to the EU numbering index.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention comprises a linker that is selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is selected from the group, consisting of 1, 2, 3, 4 and 5.

In a preferred embodiment, the sPD-1-Fc variant fusion protein of the present invention contains a GGGGS linker.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention has the amino acid sequence shown in SEQ ID NO: 5.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention has the amino acid sequence shown in SEQ ID NO: 6.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention has a binding affinity for the PD-1 ligand that is at least about 1.1-fold or greater (e.g., 5-fold or greater, 10-fold or more, 100-fold or more, 500-fold or more, 1000-fold or more, 5000-fold or more, 10000-fold or more, etc.), compared with the original Fc fusion protein (e.g., fusion protein WT PD-1 - Fc, as indicated in SEQ ID NO: 4).

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention has a binding affinity for the PD-1 ligand that is at least about 100-fold greater than that of the parent fusion protein.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention has a binding affinity for the PD-1 ligand that is at least about 200 times stronger than the affinity of the original fusion protein.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention has a binding affinity for the PD-1 ligand that is at least about 10,000 times stronger than the affinity of the original fusion protein.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention has a binding affinity for a PD-1 ligand with a KD of 10 ^-8 M or higher affinity, where the affinity is measured by a standard Biacore assay.

In some embodiments, the sPD-1-Fc variant fusion protein of the present invention inhibits or prevents binding between a wild-type PD-1 polypeptide and a PD-1 ligand in vitro or in vivo.

The sPD-1 variant domain from the sPD-1-Fc variant fusion protein serves to increase binding affinity for PD-L1 and/or PD-L2. The (variant) Fc domain from the human IgG protein significantly increases the half-life of the fusion protein. The fusion proteins of the present invention are particularly useful in the treatment of a disease or disorder in which the adaptive immune system is suppressed or an enhanced immune response is required. In some embodiments, the sPD-1-Fc variant fusion proteins of the present invention can be used to treat cancers or infections.

Nucleic acids

The present invention also relates to compositions containing a nucleic acid encoding an sPD-1-Fc variant fusion protein of the present invention. Such nucleic acids may encode any of the sPD-1-Fc variant fusion proteins listed herein.

The nucleic acids of the present invention can be isolated and obtained substantially pure. Typically, nucleic acids, both DNA and RNA, will be prepared substantially free of other naturally occurring nucleic acid sequences, typically at least about 50%, typically at least about 90% pure, and typically are "recombinant", for example, flanked by one or more nucleotides to which they are not normally associated in the natural chromosome.

In some embodiments, the composition comprises a nucleic acid encoding an sPD-1-Fc variant fusion protein of SEQ ID NO: 5. In some embodiments, the composition comprises a nucleic acid encoding an sPD-1-Fc variant fusion protein of SEQ ID NO: 6.

In some embodiments, the nucleic acid encodes an sPD-1-Fc variant fusion protein, including a signal sequence. As is known in the art, signal sequences are used to direct the expression product to the outside of the cell. In this case, suitable signal sequences for expressing the fusion proteins of the invention are wild-type PD-1 signal sequences, as shown in FIG. 1 in italics and in SEQ ID NO: 7 of Table 1. As will be understood by those skilled in the art, suitable signal sequences for expression of the fusion proteins of the invention can be "matched" to the host cell used for expression. That is, when the fusion proteins of the invention are to be expressed in mammalian host cells, such as CHO cells, for example, signal sequences from CHO cells can be used.

In some embodiments, the composition comprises a nucleic acid encoding an sPD-1-Fc variant fusion protein of SEQ ID NO: 9. In some embodiments, the composition comprises a nucleic acid encoding an sPD-1-Fc variant fusion protein of SEQ ID NO: 10.

In some embodiments, the composition comprises a nucleic acid encoding an sPD-1-Fc variant fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO. : 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 , SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO : 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO : 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 , SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ I D NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO : 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 , SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139.

In some embodiments, the nucleic acid encoding the sPD-1-Fc variant fusion protein includes a codon-optimized version or variant.

"Codon-optimized" in this context refers to a particular host organism and its generally preferred amino acid codons; that is, the host organism, eg, an Aspergillus species, may produce higher translation and/or secretion using Aspergillus-preferred codons compared to the yeast producing organism.

Codon optimization can be used with any of the sPD-1-Fc variant fusion proteins of the present invention to optimize expression in the host cell used.

An sPD-1-Fc variant fusion protein can be typically generated by constructing genes encoding the fusion protein sequence using well known techniques including site-directed mutagenesis of the parent gene and synthetic gene construction.

Expression of the nucleic acids of the present invention may be regulated by their own or by other regulatory sequences known in the art.

Regulatory sequences

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding an sPD-1-Fc variant fusion protein of the present invention operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. The control sequence may include a promoter, a polynucleotide that is recognized by the host cell to express the polynucleotide. The promoter contains transcription control sequences that mediate the expression of the Fc fusion protein. A promoter can be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

In some embodiments, the control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant and directs the expressed variant phytase into the secretory pathway of the cell. The 5' end of the coding sequence of a polynucleotide may inherently contain a sequence encoding a signal peptide naturally linked in translation reading frame to a segment of the coding sequence that encodes a variant phytase. Alternatively, the 5' end of the coding sequence may contain a sequence encoding a signal peptide that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required if the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the sequence encoding the foreign signal peptide may simply replace the sequence encoding the native signal peptide to enhance secretion of the variant phytase. However, any signal peptide encoding sequence that directs the expressed variant into the secretory pathway of the host cell can be used.

Expression vectors

Also provided herein are expression vectors for in vitro or in vivo expression of one or more sPD-1-Fc variant fusion proteins of the present invention, either constitutively or under one or more regulatory elements. In some embodiments, the present invention provides expression vectors containing a polynucleotide encoding an sPD-1-Fc variant fusion protein, a promoter, and transcriptional and translational stop signals. Various nucleotide and control sequences may be joined together to form a recombinant expression vector, which may include one or more convenient restriction sites to allow the insertion or replacement of a polynucleotide encoding an Fc fusion protein at such sites. Alternatively, a polynucleotide can be expressed by inserting the polynucleotide or nucleic acid construct containing the polynucleotide into a suitable expression vector. When creating an expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked to the appropriate control sequences for expression.

The recombinant expression vector can be any vector (eg, plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, ie. a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates along with the chromosome(s) into which it has been integrated. In addition, one vector or plasmid, or two or more vectors or plasmids, which together contain all of the DNA to be introduced into the host cell's genome, or transposon, can be used. Vectors for use in the methods of the present invention include both integrating and non-integrating vectors.

Host cells and production strains

Those skilled in the art will recognize that there is a wide variety of producing host organisms for recombinant production of fusion proteins. The sPD-1-Fc variant fusion proteins of the present invention include, but are not limited to, bacterial cells, mammalian cells, and fungal cells, including yeast.

The nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art, such as polycationic transferrin mediated DNA transfer, naked or encapsulated nucleic acid transfection, liposome mediated DNA transfer, intracellular latex bead transport, DNA-coated, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.

Methods for obtaining hybrid proteins

The present invention also relates to methods for producing an sPD-1-Fc variant fusion protein, comprising: (a) culturing the host cell of the present invention under conditions suitable for expression of the sPD-1-Fc variant fusion protein; and (b) optionally recovering the sPD-1-Fc variant fusion protein.

Methods of treatment

Objects that can be treated

The enhanced T cell response achieved by using the sPD-1-Fc variant fusion protein of the present invention is sufficient to treat a disease or disorder including, but not limited to, cancer, viral infection, bacterial infection, fungal infection, and parasitic infection.

The methods of the present invention include administering to a subject in need of treatment a therapeutically effective amount of one or more of the sPD-1-Fc variant fusion proteins described herein. In some embodiments, the subject is suffering from a cancerous disease, and administration of a therapeutically effective amount of one or more sPD-1-Fc variant fusion proteins may treat, reduce, or prevent tumor metastasis or invasion in the subject. In other embodiments, the sPD-1-Fc variant fusion protein(s) of the present invention can reduce or inhibit solid tumor growth in a subject with cancer.

In some embodiments, a subject suffering from an infection, such as a local or systemic infection, is administered a therapeutically effective amount of one or more of the sPD-1 and Fc variant fusion proteins described herein to treat the infection. In other embodiments, the subject has a chronic infectious disease caused by a bacterium, virus, protozoan, parasite, or other microbial pathogen.

Therapeutic use

In some embodiments, a therapeutically effective composition or formulation comprising one or more sPD-1-Fc variant fusion proteins of the present invention may be administered systemically to an individual in need thereof, or by any other route of administration known in the art.

Dosing

In some embodiments, an effective dose of a therapeutic entity of the present invention, for example, for the treatment of metastatic cancer or infection, varies depending on many different factors, including routes of administration, target site, physiological state of the patient, whether the patient is human or animal, other drugs used, and whether the treatment is prophylactic or therapeutic. Treatment doses can be titrated to optimize safety and efficacy.

Examples

Example 1 Modeling of sPD1 Variant with Human PDL1 and PDL2 Complexes

Ways

The structure of the human PD-1/PD-L1 complex is available in the Protein Structure Database as PDBID - 4ZQK and 5IUS. However, in both PDB structures, some PD-1 residues are missing in the loop region, and some of the residues are mutated compared to wild-type PD-1. In order to better investigate the effect of mutations on binding affinity and protein stability, the missing loop was first fixed and the remainder mutated back to wild-type PD-1 based on the 4ZQK crystal structure. The mutated structure of PD-1 was modeled using the Schrodinger Suite Residue Scan module. Residues within 5.0 Å of the mutant residue were refined by side chain prediction and backbone sampling. Protein interaction analysis was performed for both the structure of the WT PD-1/PD-L1 complex and the structure of the sPD-1/PD-L1 variant complex. Three models of the PD-1/PD-L1 complex, model WT, model with 3 mutations in the interaction region, model with all 8 mutations, were subjected to molecular dynamics (MD) simulation for 10 ns using the OPLS3 force field for further refinement and optimization . Each complex was solvated in a periodic orthorhombic box with SPC water as a solvent. The minimum distance from any complex atom to the wall of the box was set to 10 Å. All three MD simulations were performed at a constant temperature of 300 K and a constant pressure of 1 atm. The trajectory coordinates stored at 1 ps intervals were used for the analysis.

Because the structure of human PD-L2 is not available in the protein databank, a PD-L2 homology model was constructed to investigate the interaction between human PD-1 and PD-L2. After looking for homology in a non-redundant PDB dataset, three mouse PD-L2 PDB structures were selected as templates. As described previously, protein interaction analysis was also performed for WT and the sPD-1 variant/PD-L2 model.

Modeling of the human PD-1/PD-L1 complex

The result of WT sequence alignment of PD-1, 4ZQK and 5IUS is shown in FIG. 2. Identical residues are marked in blue, residues missing in both crystal structures are marked in red, residues missing only in 4ZQK are marked in blue.

The PD-1 model with the fixed loop missing and residue 93 back mutated to Cys is shown in blue in FIG. 3. Crystal structures of PD-1 and PD-L1 in 4ZQK are shown in blue and green, respectively. 7 residues (Ser62, Ser87, Pro89, Gly124, Ser127, Ala132, and Ala140 with numbering starting at the signal region; which are equivalent to Ser42, Ser67, Pro69, Gly104, Ser107, Ala112, and Ala120, respectively, with numbering starting at the mature region) in PD-1, mutations are shown with orange sticks, the N-glycosylation site (Asn116) is shown with purple sticks.

The mutated structure of PD-1 was modeled using the Schrodinger Suite Residue Scan module. Residues within 5.0 Å of the mutant residue were refined by side chain prediction and backbone sampling. Protein interaction analysis was performed for both the structure of the WT PD-1/PD-L1 complex and the mutated structure of the PD-1/PD-L1 complex with all 8 mutated residues. The result of the analysis is presented in tables 2 and 3, respectively. Mutations G124S, S127V and A132I are considered as mutations in the region of interaction with the numbering of positions starting from the signal region. And mutations S62G, S86G, P89L and A140V are considered external mutations with position numbering starting from the signal region. The G124S mutation creates another hydrogen bond with PD-L1, the A132I mutation retains the hydrogen bond with PD-L1 and increases surface complementarity.

To further explore how mutations affect PD-1/PD-L1 binding, variance in binding affinity and protein stability was calculated before and after each mutation. The result is shown in Table 4. Calculation of binding affinity variation and protein stability was also performed for multiple mutations. The result is shown in Table 5. A more negative binding affinity or stability value indicates a greater increase. In general, mutations in the interaction region increase both the binding affinity and stability of the protein, while external mutations mainly affect stability. For all 8 mutations, binding affinity increases by 19.03 kcal/mol, and protein stability increases by 69.92 kcal/mol.

Three models of the PD-1/PD-L1 complex, model WT, model with 3 mutations in the interaction region, model with all 8 mutations, were subjected to molecular dynamics (MD) simulation for 10 ns using the OPLS3 force field for further refinement and optimization . Each complex was solvated in a periodic orthorhombic box with SPC water as a solvent. The minimum distance from any complex atom to the wall of the box was set to 10 Å. All three MD simulations were performed at a constant temperature of 300 K and a constant pressure of 1 atm. The trajectory coordinates stored at 1 ps intervals were used for the analysis. The time evolution of the RMSD positions of the backbone atoms is shown in FIG. 4. The MD result also shows that mutations increase the stability of the model.

Table 2. Interaction analysis of wild-type human PD-1/PD-L1 model proteins

Remainder Nearest Distance Specific Interactions #HB # salt # Pi # #vdW On a surface Submerged B:64:Val A:121:Ala 4.0 A 0 0 0 0 0 0.87 59.50% B:66:Asn A:121:Ala 2.9 A 1x hb with A:121:Ala one 0 0 0 0 0.76 98.20% B:68:Tyr A:122:Asp 2.8A 1x hb with A:122:Asp one 0 0 0 0 0.82 100.00% B:70:Met A:125:Arg 3.5A 0 0 0 0 0 0.84 33.00% B:73:Ser A:26:Asp 3.0A 1x hb with A:26:Asp one 0 0 0 0 0.69 32.90% B:74:Asn A:125:Arg 3.7A 0 0 0 0 0 0.86 18.10% B:75:Gln A:125:Arg A:26:Asp 3.0 A
3.0 A 1x hb with A:26:Asp 2x hb with A:125:Arg 3 0 0 0 0 0.8 89.60% B:76:Thr A:123:Tyr A:125:Arg 3.2 A
3.3A 1x hb with A:123:Tyr 2x hb with A:124:Lys 2 0 0 0 0 0.82 100.00% B:77:Asp A:124:lys 3.0A 1x hb with A:124:Lys one 0 0 0 0 0.69 24.40% B:78:lys A:19:Phe A:121:Ala 2.9 A
2.9A 1x hb with A:19:Phe 1x hb with A:121:Ala 2 0 0 0 0 0.67 78.30% B:84:Glu A:120:Gly A:18:Ala 3.5A
3.6 A 1x hb with A:19:Phe 1x hb with A:120:Gly 2 0 0 0 0 0.68 68.70% B:122:Leu A:123:Tyr 3.8 A 0 0 0 0 0 0.87 80.20% B:124:Gly A:123:Tyr 3.7A 0 0 0 0 0 0.93 100.00% B:126:!le A:123:Tyr 3.6A 0 0 0 0 0 0.92 99.90% B:127:Ser A:115:Met 3.7 A 0 0 0 0 0 0.89 4.60% B:128:Leu A:117:Ser 3.5A 0 0 0 0 0 0.85 83.00% B:130:Pro A:66:Gln 4.0 A 0 0 0 0 0 0.06 19.40% B:131:lys A:66:Gln 3.4A 0 0 0 0 0 0.87 43.10% B:132:Ala A:56:Tyr 3.4A 1x hb with A:66:Gln one 0 0 0 0 0.72 99.80% B:133:Gln A:56:Tyr 4.3 A 0 0 0 0 0 0.37 16.10% B:134:lle A:113:Arg 3.6 A 0 0 0 0 0 0.86 90.90% B:136:Glu A:123:Tyr A:125:Arg A:113:Arg 2.9A 2.9A 3.1A 1x hb, 1x salt bridge to A:113:Arg
1x hb with A:123:Tyr
1x hb, lx salt bridge to A:125:Arg 3 2 0 0 0 0.75 56.80% B:139:Arg A:125:Arg 3.3 A 0 0 0 0 0 0.72 11.40%

Table 3. Protein interaction analysis of the mutated PD-1 model (S62G, S87G, P89L, G124S, S127V, A132I, A140V and N116S with position numbering starting from the signal region)/human PD-L1

Remainder Nearest Distance Specific Interactions #HB # Salt #pi # #vdW On a surface Submerged 3:64:val A:121:Ala 3.7A 0 0 0 0 0 0.85 94.10% B:66:Asn A:121:Ala 2.9A 1x hb with A:121:Ala one 0 0 0 0 0.74 100.00% B:68:Tyr A:122:Asp 2.7A 1x hb with A:121:Asp one 0 0 0 0 0.75 100.00% 3:70:Met A:125:Arg 3.5A 0 0 0 0 0 0.82 18.10% B:73:Ser A:26:Asp 3.0A 1x hb with A:26:Asp one 0 0 0 0 0.28 32.90% 3:74:Asn A:125:Arg 3.7A 0 0 0 0 0 0.85 18.10% B:75:Gln A:125:Arg 3.0 A lx hb to A:26:Asp 3 0 0 0 0 0.8 89.60% B:76:Thr A:124:lys 2.9A 1x hb with A:124:Lys one 0 0 0 0 0.78 100.00% B:77:Asp A:124:lys 3.5 A 0 0 0 0 0 0.89 18.30% B:78:lys A:19:Phe 2.9 A 1x hb with A:19:Phe 2 0 0 0 0 0.69 76.10% 3:80:ala 0 0 0 0 0 0 10.20% 3:84:Glu A:19:Phe 3.1 A 1x hb with A:19:Phe 2 0 0 0 0 0.81 80.90% 3:85:Asp 0 0 0 0 0 0 8.10% 3:86:Arg A:120:Gly 3.7A 0 0 0 0 0 0.55 16.30% 3:92:Asp 0 0 0 0 0 0 0.90% B:122:Leu A:123:Tyr 3.5 A 0 0 0 0 0 0.8 78.80% B:124:ser A:123:Tyr 2.9 A 1x hb with A:123:Tyr one 0 0 0 0 0.71 100.00% B:126:lle A:115:Me 3.5 A 0 0 0 0 0 0.93 100.00% B:127:Val A:115:Met 4.4 A 0 0 0 0 0 0.33 0.00% B:128:Leu A:115:Me 3.5 A 0 0 0 0 0 0.93 75.00% B:129:Ala 0 0 0 0 0 0 1.50% B:130:Pro A:66:Gln 4.9 A 0 0 0 0 0 0 0.00% B:131:lys A:63:Asn 3.5A 0 0 0 0 0 0.75 36.00% B:132:lle A:66:Gln 2.8 A 1x hb with A:66:Gln one 0 0 0 0 0.86 98.60% B:133:Gln A:56:Tyr 3.0A 0 0 0 0 0 0.74 50.00% B:134:lle A:123:Tyr 3.5A 0 0 0 0 0 0.86 91.90% 3:136:glu A:123:Tyr 2.8 A lx hb, lx salt bridge to A:113:Arg 3 2 0 0 0 0.68 62.10% B:139:Arg A:125:Arg 3.1 A 0 0 0 0 0 0.79 10.70%

Table 4. Change in binding affinity and protein stability (in kcal/mol) of human PD-1/PD-L1 for each individual mutation

Remainder original mutated d affinity d Stability (solvated) 62 SER GLY -1.37 0.5 87 SER GLY 0.94 5.56 89 PRO LEU 0 -7.06 116 ASN SER -0.02 0.82 124 GLY SER -4.27 -10.24 127 SER VAL -0.12 -1.82 132 ALA ILE -10.94 -11.9 140 ALA VAL -0.77 -25.89

Table 5 Change in binding affinity and protein stability (in kcal/mol) of human PD-1/PD-L1 for multiple mutations. A more negative binding affinity or stability value indicates a greater increase.

Mutations d Affinities d Stability
(solvated) 8 mutations -19.03 -69.92 7 mutations (N116S excluded) -20.08 -66.99 4 external mutations -1.61 -30.66 4 Interaction Area Mutations -24.78 -30.58 2 mutations on lost loop 1.31 -12.08

Modeling of the human PD-1/PD-L2 complex

The sequence alignment results are shown in FIG. 5A. The sequence identity of human PD-L2 and mouse PD-L2 is about 72%. The human PD-L2 consensus model is shown in FIG. 5b. Identical remains are marked in blue. Remains in the missing loop are marked in red.

A comparison of the PD-1 binding interaction region between the human PD-L2 model and the mouse PD-L2 crystal structure (PDBID: 3BP5) is shown in FIG. 6. The residuals are shown as lines. The remnants of the interaction region are marked in green in the human PD-L2 model (shown in dark blue). The crystal structure of the PD-L2 mouse is shown in pink. The sequence identity at the interface is 66%. A model of the human PD-1/PD-L2 complex was constructed from the human PD-1 model (described in Part I) and the human PD-L2 consensus model by overlaying the mouse PD-1/PD-L2 crystal structure (PDBID: 3BP5) . An overlay of the human PD-1/PD-L2 model and mouse PD-1/PD-L2 (PDBID: 3BP5) crystal structure is shown in FIG. 7. Human PD-1 and PD-L2 models are shown in cyan and dark blue, mouse PD-1 and PD-L2 structures are shown in yellow and pink, respectively.

As described in the methods section, protein interaction analysis was also performed for the WT model and mutated human PD-1/PD-L2. The results are shown in Tables 6 and 7. G124S and A132I mutations slightly increase surface complementarity. Calculation of binding affinity variation and protein stability was also performed for each individual mutation and multiple mutations. The results are shown in Tables 8 and 9. In general, interaction region mutations increase binding affinity and external mutations increase stability. For all 8 mutations, binding affinity is increased by 10.62 kcal/mol and protein stability is increased by 10.7 kcal/mol. The variation in binding affinity and stability of PD-1/PD-L2 is not as significant as that of PD-1/PD-L1. This may be due to the difference in structural accuracy between the homology model and the crystal structure.

Table 6. Interaction analysis of wild-type human PD-1/PD-L2 model proteins

Remainder Nearest Distance Specific Interactions #HB # Salt # Pi # #vdw On a surface Submerged B:64:Val A:110:Trp 3.5 A 0 0 0 0 0 0.77 71.80% B:65:Leu A:110:Trp 3.9A 0 0 0 0 0 0.79 23.00% B:66:Asn A:110:Trp 2.8A 1x hb with A:110:Trp one 0 0 0 0 0.84 100.00% B:68:Tyr A:lll:Asp 2.8 A 1x hb with A:lll:Asp one 0 one 0 0 0.84 100.00% B:70:Met A:114:Tyr 4.2 A 0 0 0 0 0 0.9 14.10% B:73:Ser A:28:Glu 3.0 A 1x hb with A:28:Glu one 0 0 0 0 0.82 30.50% B:74:Asn A:99:Gln 2.8 A 1x hb with A:99:Gln one 0 0 0 0 0.88 49.60% B:75:Gln A:114:Tyr 2.8 A 1x hb with A:28:Glu 3 0 0 0 0 0.76 87.60% B:76:Thr A:113:lys 3.0A 1x hb with A:112:Tyr 2 0 0 0 0 0.87 100.00% B:77:Asp A:113:lys 4.6 A 0 0 0 0 0 0 13.00% B:78:lys A:110:Trp 2.9A 1x hb with A:21:Phe 2 0 0 0 0 0.7 68.10% B:83:Pro A:108:Val 3.5 A 0 0 0 0 0 0.73 34.50% B:84:Glu A:21:Phe 2.8A 1x hb with A:21:Phe one 0 0 0 0 0.72 99.90% B:85:Asp A:19:ala 3.3A 0 0 0 0 0 0.86 65.20% B:86:Arg A:108:Val 3.6A 0 0 0 0 0 0.97 12.70% B:92:Asp A:20:Leu 3.3A 0 0 0 0 0 0.89 38.70% B93:Cys A:20:Leu 4.3 A 0 0 0 0 0 0.91 22.90% B:122:Leu 0 0 0 0 0 0.49 100.00% B:124:Gly A:110:Trp 3.6A 0 0 0 0 0 0.8 100.00% B:125:Ala A:110:Trp 3.6 A 0 0 0 0 0 0.86 100.00% B:126:lle A:110:Trp 3.4 A 0 0 0 0 0 0.91 100.00% B:127:Ser A:105:lle 4.6 A 0 0 0 0 0 0.93 0.00% B:128:Leu A:105:lle 3.6 A 0 0 0 0 0 0.88 55.70% B:131:lys A:68:Pro 3.9 A 0 0 0 0 0 0.87 29.70% B:132:Ala A:60:Gln 2.7A 1x hb with A:60:Gln one 0 0 0 0 0.83 83.70% B:133:Gln A:67:Ser 2.8 A 1x hb with A:67:Ser one 0 0 0 0 0.87 53.60% B:134:lle A:103:lle 3.4 A 0 0 0 0 0 0.88 92.90% B:136:Glu A:112:Tyr 2.8 A 1x hb with A:112:Tyr 2 0 0 0 0 0.76 41.20% B:139:Arg A:114:Tyr 3.5A 0 0 0 0 0 0.89 10.20%

Table 7. Protein interaction analysis of the mutated model (S62G, S87G, P89L, G124S, S127V, A132I, A140V and N116S with position numbering starting from the signal region, which are equivalent to S42G, S67G, P69L, G104S, S107V, A112I, A120V and N96S , respectively, with position numbering starting from the mature region) human PD-1/PD-L2

Remainder Nearest Distance Specific Interactions #HB # Salt # Pi # #vdW On a surface Submerged B:64:Val A:110:Trp 3.3A 1x hb with A:110:Trp one 0 0 0 0 0.76 71.20% B:65:Leu A:110:Trp 4.0 A 0 0 0 0 0 0.29 13.20% B:66:Asn A:110:Trp 2.9A 1x hb with A:110:Trp one 0 0 0 0 0.83 100.00% B:68:Tyr A:111:Asp 2.9 A 1x hb with A:111:Asp one 0 one 0 0 0.86 100.00% B:70:Met A:114:Tyr 4.2 A 0 0 0 0 0 0.89 10.90% B:73:Ser A:28:Glu 3.0A 1x hb with A:28:Glu one 0 0 0 0 0.87 30.50% B:74:Asn A:99:Gln 2.8A 1x hb with A:99:Gln one 0 0 0 0 0.88 49.60% B:75:Gln A:114:Tyr 2.8 A 1x hb with A:28:Glu 3 0 0 0 0 0.77 87.70% B:76:Thr A:113:lys 3.0A 1x hb with A:112:Tyr 2 0 0 0 0 0.85 100.00% B:77:Asp A:113:lys 4.6 A 0 0 0 0 0 0 13.20% B:78:lys A:110:Trp 2.9 A 1x hb with A:21:Phe 2 0 0 0 0 0.68 68.10% B:81:Ala A:109:Ala 4.9 A 0 0 0 0 0 0.08 97.90% B:82:Phe A:109:Ala 4.8 A 0 0 0 0 0 0.29 3.00% B:83:Pro A:108:Val 3.5 A 0 0 0 0 0 0.77 37.80% B:84:Glu A:21:Phe 2.8 A 1x hb with A:21:Phe one 0 0 0 0 0.74 99.90% B:85:Asp A:19:ala 3.3 A 0 0 0 0 0 0.8 54.80% B:86:Arg A:108:Val 3.5 A 0 0 0 0 0 0.92 15.80% B:92:Asp A:20:Leu 3.4 A 0 0 0 0 0 0.89 39.50% B:93:Cys A:20:Leu 4.3A 0 0 0 0 0 0 22.20% B:124:ser A:110:Trp 3.5 A 0 0 0 0 0 0.85 100.00% B:125:Ala A:110:Trp 3.7 A 0 0 0 0 0 0.89 100.00% B:126:lle A:110:Trp 3.4 A 0 0 0 0 0 0.92 100.00% B:128:Leu A:105:lle 3.6 A 0 0 0 0 0 0.8 48.80% B:131:lys A:69:Hie 3.6 A 0 0 0 0 0 0.83 36.70% B:132:lle A:60:Gln 2.8 A 1x hb with A:60:Gln one 0 0 0 0 0.85 75.50% B:133:Gln A:67:Ser 3.0A 1x hb with A:67:Ser one 0 0 0 0 0.76 61.30% B:134:lle A:103:lle 3.4 A 0 0 0 0 0 0.9 96.00% B:135:lys 0 0 0 0 0 0 16.30% B:136:Glu A:112:Tyr 2.8A 1x hb with A:112:Tyr 2 0 0 0 0 0.79 38.50% B:139:Arg A:114:Tyr 3.4 A 0 0 0 0 0 0.89 12.10%

Table 8. Change in binding affinity and protein stability (in kcal/mol) of human PD-1/PD-L2 for each individual mutation

Remainder original mutated d Affinities d Stability (solvated) GLY 0.3 4.21 87 SER GLY 0 -0.68 89 PRO LEU 0.28 -4.71 116 ASN SER 0 0.85 124 GLY SER -3.06 6.47 127 VAL 1.65 -4.42 132 ALA ILE -9.47 -0.55 140 ALA VAL 0.25

Table 9 Change in binding affinity and protein stability (in kcal/mol) of human PD-1/PD-L2 for multiple mutations

Mutations d Affinities d Stability (solvated) 8 mutations -10.62 -10.7 7 mutations (N116S excluded) -10.21 -12.19 4 external mutations 0.59 -10.03 4 Interaction Area Mutations -11.69 8.59 2 mutations on lost loop 0.01 -4.95

Table 10. Colony tracking numbers, SEQ ID numbers and amino acid mutations in different colonies relative to wild type (SEQ ID NO: 11). The signal peptide region consists of residues 1 to 20, and the mature extracellular domain (ECD) starts from residues 21 to 170. The linker domain starts from residues 171 to 175. The numbering of amino acid mutations in each sequence starts from its mature region

colony tracking no. SEQID No. AK mutations relative to 1-WT
(SEQ ID NO:11) 2-7 mutations SEQ ID NO:12 S42G/S67G/P69L/G104S/S107V/
A112I/A120V 3-1 mutation SEQ ID NO:13 S42G 4-1 mutation SEQ ID NO:14 S67G 5-1 mutation SEQ ID NO:15 P69L 6-1 mutation SEQ ID NO:16 G104S 7-1 mutation SEQ ID NO:17 S107V 8-1 mutation SEQ ID NO:18 A112I 9-1 mutation SEQ ID NO:19 A120V 10-2 mutations SEQ ID NO:20 S42G/S67G 11-2 mutations SEQ ID NO:21 S42G/P69L 12-2 mutations SEQ ID NO:22 S42G/G104S 13-2 mutations SEQ ID NO:23 S42G/S107V 14-2 mutations SEQ ID NO:24 S42G/A112I 15-2 mutations SEQ ID NO:25 S42G/A120V 16-2 mutations SEQ ID NO:26 S67G/P69L 17-2 mutations SEQ ID NO:27 S67G/G104S 18-2 mutations SEQ ID NO:28 S67G/S107V 19-2 mutations SEQ ID NO:29 S67G/A112I 20-2 mutations SEQ ID NO:30 S67G/A120V 21-2 mutations SEQ ID NO:31 P69L/G104S 22-2 mutations SEQ ID NO:32 P69L/S107V 23-2 mutations SEQ ID NO:33 P69L/A112I 24-2 mutations SEQ ID NO:34 P69L/A120V 25-2 mutations SEQ ID NO:35 G104S/S107V 26-2 mutations SEQ ID NO:36 G104S/A112I 27-2 mutations SEQ ID NO:37 G104S/A120V 28-2 mutations SEQ ID NO:38 S107V/A112I 29-2 mutations SEQ ID NO:39 S107V/A120V 30-2 mutations SEQ ID NO:40 A112I/A120V 31-3 mutations SEQ ID NO:41 S42G/S67G/P69L 32-3 mutations SEQ ID NO:42 S42G/S67G/G104S 33-3 mutations SEQ ID NO:43 S42G/S67G/S107V 34-3 mutations SEQ ID NO:44 S42G/S67G/A112I 35-3 mutations SEQ ID NO:45 S42G/S67G/A120V 36-3 mutations SEQ ID NO:46 S42G/P69L/G104S 37-3 mutations SEQ ID NO:47 S42G/P69L/S107V 38-3 mutations SEQ ID NO:48 S42G/P69L/A112I 39-3 mutations SEQ ID NO:49 S42G/P69L/A120V 40-3 mutations SEQ ID NO:50 S42G/G104S/S107V 41-3 mutations SEQ ID NO:51 S42G/G104S/A112I 42-3 mutations SEQ ID NO:52 S42G/G104S/A120V 43-3 mutations SEQ ID NO:53 S42G/S107V/A112I 44-3 mutations SEQ ID NO:54 S42G/S107V/A120V 45-3 mutations SEQ ID NO:55 S42G/A112I/A120V 46-3 mutations SEQ ID NO:56 S67G/P69L/G104S 47-3 mutations SEQ ID NO:57 S67G/P69L/S107V 48-3 mutations SEQ ID NO:58 S67G/P69L/A112I 49-3 mutations SEQ ID NO:59 S67G/P69L/A120V 50-3 mutations SEQ ID NO:60 S67G/G104S/S107V 51-3 mutations SEQ ID NO:61 S67G/G104S/A112I 52-3 mutations SEQ ID NO:62 S67G/G104S/A120V 53-3 mutations SEQ ID NO:63 S67G/S107V/A112I 54-3 mutations SEQ ID NO:64 S67G/S107V/A120V 55-3 mutations SEQ ID NO:65 S67G/A112I/A120V 56-3 mutations SEQ ID NO:66 P69L/G104S/S107V 57-3 mutations SEQ ID NO:67 P69L/G104S/A112I 58-3 mutations SEQ ID NO:68 P69L/G104S/120V 59-3 mutations SEQ ID NO:69 P69L/S107V/A112I 60-3 mutations SEQ ID NO:70 P69L/S107V/120V 61-3 mutations SEQ ID NO:71 P69L/A112I/A120V 62-3 mutations
(clone #5) SEQ ID NO:72 G104S/S107V/A112I 63-3 mutations SEQ ID NO:73 G104S/S107V/A120V 64-3 mutations SEQ ID NO:74 G104S/A112I/A120V 65-3 mutations SEQ ID NO:75 S107V/A112I/A120V 66-4 mutations SEQ ID NO:76 S42G/S67G/P69L/G104S 67-4 mutations SEQ ID NO:77 S42G/S67G/P69L/S107V 68-4 mutations SEQ ID NO:78 S42G/S67G/P69L/A112I 69-4 mutations SEQ ID NO:79 S42G/S67G/P69L/A120V 70-4 mutations SEQ ID NO:80 S42G/S67G/G104S/S107V 71-4 mutations SEQ ID NO:81 S42G/S67G/G104S/A112I 72-4 mutations SEQ ID NO:82 S42G/S67G/G104S/A120V 73-4 mutations SEQ ID NO:83 S42G/S67G/S107V/A112I 74-4 mutations SEQ ID NO:84 S42G/S67G/S107V/A120V 75-4 mutations SEQ ID NO:85 S42G/S67G/A112I/A120V 76-4 mutations SEQ ID NO:86 S42G/P69L/G104S/S107V 77-4 mutations SEQ ID NO:87 S42G/P69L/G104S/A112I 78-4 mutations SEQ ID NO:88 S42G/P69L/G104S/A120V 79-4 mutations SEQ ID NO:89 S42G/P69L/S107V/A112I 80-4 mutations SEQ ID NO:90 S42G/P69L/S107V/A120V 81-4 mutations SEQ ID NO:91 S42G/P69L/A112I/A120V 82-4 mutations SEQ ID NO:92 S42G/G104S/S107V/A112I 83-4 mutations SEQ ID NO:93 S42G/G104S/S107V/A120V 84-4 mutations SEQ ID NO:94 S42G/G104S/A112I/A120V 85-4 mutations SEQ ID NO:95 S42G/S107V/A112I/A120V 86-4 mutations SEQ ID NO:96 S67G/P69L/G104S/S107V 87-4 mutations SEQ ID NO:97 S67G/P69L/G104S/A112I 88-4 mutations SEQ ID NO:98 S67G/P69L/G104S/A120V 89-4 mutations SEQ ID NO:99 S67G/P69L/S107V/A112I 90-4 mutations SEQ ID NO:100 S67G/P69L/S107V/A120V 91-4 mutations SEQ ID NO:101 S67G/P69L/A112I/A120V 92-4 mutations SEQ ID NO:102 S67G/G104S/S107V/A112I 93-4 mutations SEQ ID NO:103 S67G/G104S/S107V/A120V 94-4 mutations SEQ ID NO:104 S67G/G104S/A112I/A120V 95-4 mutations SEQ ID NO:105 S67G/S107V/A112I/A120V 96-4 mutations SEQ ID NO:106 P69L/G104S/S107V/A112I 97-4 mutations SEQ ID NO:107 P69L/G104S/S107V/A120V 98-4 mutations SEQ ID NO:108 P69L/G104S/A112I/A120V 99-4 mutations SEQ ID NO:109 P69L/S107V/A112I/A120V 100-4 mutations
(clone #4) SEQ ID NO:110 G104S/S107V/A112I/A120V 101-5 mutations SEQ ID NO:111 S42G/S67G/P69L/G104S/S107V 102-5 mutations SEQ ID NO:112 S42G/S67G/P69L/G104S/A112I 103-5 mutations SEQ ID NO:113 S42G/S67G/P69L/G104S/A120V 104-5 mutations SEQ ID NO:114 S42G/S67G/P69L/S107V/A112I 105-5 mutations SEQ ID NO:115 S42G/S67G/P69L/S107V/A120V 106-5 mutations SEQ ID NO:116 S42G/S67G/P69L/A112I/A120V 107-5 mutations SEQ ID NO:117 S42G/S67G/G104S/S107V/A112I 108-5 mutations SEQ ID NO:118 S42G/S67G/G104S/S107V/A120V 109-5 mutations SEQ ID NO:119 S42G/S67G/G104S/A112I/A120V 110-5 mutations SEQ ID NO:120 S42G/S67G/S107V/A112I/A120V 111-5 mutations SEQ ID NO:121 S42G/P69L/G104S/S107V/A112I 112-5 mutations SEQ ID NO:122 S42G/P69L/G104S/S107V/A120V 113-5 mutations SEQ ID NO:123 S42G/P69L/G104S/A112I/A120V 114-5 mutations SEQ ID NO:124 S42G/P69L/S107V/A112I/A120V 115-5 mutations SEQ ID NO:125 S42G/G104S/S107V/A112I/A120V 116-5 mutations SEQ ID NO:126 S67G/P69L/G104S/S107V/A112I 117-5 mutations SEQ ID NO:127 S67G/P69L/G104S/S107V/A120V 118-5 mutations SEQ ID NO:128 S67G/P69L/G104S/A112I/A120V 119-5 mutations SEQ ID NO:129 S67G/P69L/S107V/A112I/A120V 120-5 mutations
(clone #2) SEQ ID NO:130 S67G/G104S/S107V/A112I/A120V 121-5 mutations
(clone #1) SEQ ID NO:131 P69L/G104S/S107V/A112I/A120V 122-6 mutations SEQ ID NO:132 S42G/S67G/P69L/G104S/S107V/A112I 123-6 mutations SEQ ID NO:133 S42G/S67G/P69L/G104S/S107V/A120V 124-6 mutations SEQ ID NO:134 S42G/S67G/P69L/G104S/A112I/A120V 125-6 mutations SEQ ID NO:135 S42G/S67G/P69L/S107V/A112I/A120V 126-6 mutations SEQ ID NO:136 S42G/S67G/G104S/S107V/A112I/A120V 127-6 mutations SEQ ID NO:137 S42G/P69L/G104S/S107V/A112I/A120V 128-6 mutations
(clone #3) SEQ ID NO:138 S67G/P69L/G104S/S107V/A112I/A120V 129-8 mutations SEQ ID NO:139 S42G/S67G/P69L/N96S/G104S/S107V/A112I/A120V

Example 2 Binding Affinity of sPD-1 Variant for PD-L1 and PD-L2

Binding affinity assays were performed according to the manufacturer's assay protocol. Machine model - Biacore T200, GE Healthcare Life Sciences. The experiments were carried out at 25°C. The analytes were human PD-L1 and human PD-L2. The working buffer was HBS-EP+. Briefly, anti-human Fc IgG was immobilized on a CM5 sensor chip. The sPD-1 variants were attached to an immobilized sensor chip. Multichannel kinetics was performed with the introduction of a serially diluted analyte.

The sPD-1-Fc variant fusion protein showed about a 10,000-fold improvement in PD-L1 binding compared to the WT PD-1-Fc fusion protein ("original Fc fusion protein") and about a 200-fold improvement in PD-L2 binding compared to original hybrid protein Fc (Fig. 8).

Example 3 Binding Activity of the sPD-1-Fc Variant Fusion Protein on MC38 Cells

A FACS binding assay was performed to evaluate the binding activity of the sPD-1-Fc variant fusion protein to mouse colorectal cancer cell line MC38 and human PD-L1 knockout MC38 cells.

Briefly, cells were cultured at 37°C with 5% CO ₂ . Cells were harvested according to standard procedures and the supernatant was discarded. Cells were distributed into staining plates at 0.3 million cells per well. The plates were centrifuged at 300 g at 4°C for 5 minutes. The serial concentration of the fusion protein of the sPD-1-Fc variant and the negative control in FACS buffer containing 2% FBS was titrated 1:5 from 500 µg/mL. 100 μl was added to each well and the cells were incubated for 1 hour at 4°C. Cells were washed twice with 200 μl of FACS buffer, centrifuged at 300 g for 5 minutes and the supernatant was removed before and after each wash. The cells were then resuspended with anti-human IgG-Alexa 488. The plates were incubated for 1 hour at 4°C. Cells were then washed twice for FACS analysis. FACS analyzes were performed using FACS Canto II, BD Biosciences according to the manufacturer's instructions.

sPD-1-Fc variant fusion proteins (e.g., sPD-1-Fc variant version 1 fusion protein and sPD-1-Fc variant version 2 fusion protein) showed higher binding affinity to knockout MC38-PD-L1 cells than the PD group. -1 wild-type (i.e. wild type-Fc PD-1 fusion protein as indicated in SEQ ID NO: 4) (Fig. 9A), although the sPD-1-Fc variant fusion proteins also bind to the original MC38, but to a lower degree (Fig. 9B).

Example 4 T Cell Activation Assay

Isolation of PBMC: A blood sample from an individual donor was diluted with the same volume of sterile PBS, eg 25 ml of sterile PBS was added to 25 ml of fresh whole blood and mixed thoroughly by gentle shaking. 15 ml of Ficoll-Pague medium was transferred to a new 50 ml centrifuge tube, making sure that the volume ratio of Ficoll and blood is 3:4, then the diluted blood sample was carefully added to the surface of Ficoll medium, avoiding mixing, adding as carefully as possible to clearly a layer of two liquids was visible. The tube was carefully transferred to a centrifuge at 400g, 30 min, 20°C, with maximum acceleration and minimum deceleration settings during centrifugation. The mononuclear cell layer was carefully absorbed and transferred to another new sterile centrifuge tube. Sterile PBS buffer was added to the collected PBMC for washing in a 3:1 volume ratio and centrifuged at 300×g, 10 min, 20°C. The cells were then resuspended in 10% FBS + RPMI 1640 for analysis.

Preparation of human PD-L1 knockout Hep3B cells, mycoplasma-free Hep3b-hPDL1-OS8 cells, were prepared in a 15 cm dish and maintained at 60-80% confluence before use. The cells were trypsinized with the TrypLETM Express enzyme, then the trypsin was neutralized and the cells were collected in a 50 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended with mitomycin, 10 μg/ml (in 5-10 ml of 10% FBS + RPMI1640 medium). Cells were incubated at 37°C for 1 hour. Cells were washed with 15-20 ml PBS 4 times before cell counting with a cytometer and then centrifuged at 1000 rpm, 5 min, at 20°C. Cells were resuspended in 10% FBS + RPMI 1640 for analysis.

PBMC stimulation: PBMC was added at 50,000 cells/well, 100 μl/well, and then a series of 50 μl/well protein solutions was added 5X sPD-1 version 2 (PD1 V2), 5X PD1 WT or hIgG4 cells, followed by the addition of Hep3b- hPDL1 at 5000 cells/well in 50 µl/well. The mixed solution was incubated in an incubator at 37°C for 72 hours. The supernatant was collected and frozen at a temperature below -20°C before the ELISA test. IFN-gamma concentration was checked using an ELISA kit. IFN-γ was quantified using Quantifkine ELISA kits purchased from R&D. Procedures were performed according to the Human IFN-γ ELISA Kit Assay Procedure, R&D, Cat. No. DY285.

In FIG. 10 shows that sPD-1-Fc variant 2 fusion proteins are able to activate T cells isolated from human PBMCs.

Example 5 In Vivo Tumor Study Using the MC38-hPD-L1 Colorectal Tumor Cell Line

All animals were purchased and housed in an AAALC accredited, temperature controlled, pathogen free environment. Animals were removed from the study once tumor size reached the ethical limit. C57/B6 mice were inoculated with MC38-hPD-L1 tumor cells and observed for 5 days to confirm tumor growth, and then assigned to each of three treatment groups of 10 animals per group: vehicle control, anti-PD-L1 antibody, 10 mg/kg (positive control group) and sPD-1-Fc variant 2 fusion protein 10 mg/kg (PD1-ECD-Fc group 10 mg/kg). Treatment was started on day 6 after tumor inoculation and animals were removed from the study when tumor size exceeded 2000 mm ³ . Tumor growth over time and survival were recorded as the end point of the experiment.

The sPD-1-Fc variant 2 fusion protein showed superior antitumor activity compared to the positive control group (i.e., anti-PD-L1 antibody) as evidenced by reduction in tumor volume, enhanced tumor growth inhibition, increased survival, and reduced body weight in the sPD-1-Fc variant 2 fusion protein group (i.e., the PD-1-ECD-Fc group) compared to the anti-PD-L1 antibody group.

Example 6 Expression of PD-L2 in Lentivirus Transduced Hep3B-OS8 Cells and MC38 Cells

Hep3B-OS8 cells and MC38 cells were transduced with a lentiviral plasmid expressing human PD-L2.

PDL2 expression in individual clones of Hep3B-OS8 cells and MC38 cells was analyzed by FACS. Briefly, cells were collected according to standard procedures and the supernatant was discarded. The cells were placed in a staining plate at 0.3 million cells per well, and the plate was centrifuged at 300 g at 4° C. for 5 minutes. Added 100 μl/well of rabbit anti-hPD-L2 (1 μg/test) antibodies in FACS buffer, titrated, and incubated for 1 hour at 4°C. Cells were washed twice with 200 μl of FACS buffer, centrifuged at 300 g for 5 min, and the supernatant was removed before and after each wash. Cells were resuspended at 100 μl/well with a 1:1000 dilution of secondary antibody. The plates were incubated for 30 min at 4°C. Cells were washed 2 times with 200 μl of FACS buffer, centrifuged at 300 g for 5 min, and the supernatant was discarded before and after each wash. Cells were resuspended in 100 µl PBS. Cells were stored in the dark and subjected to FACS analysis. FACS analyzes were performed using FACS Canto II, BD Biosciences according to the manufacturer's instructions.

Fig. 13 shows that human PD-L2 is overexpressed in Hep3B and MC38 cell lines.

Example 7 Binding Activity of the sPD-1-Fc Variant Fusion Protein on Hep3b Cells Overexpressing Human PD-L2 (Hep3B-hPDL2)

A FACS binding assay was performed to evaluate the binding activity of the sPD-1-Fc variant fusion protein to human hepatocellular cancer cell line Hep3b cells overexpressing human PD-L2 (Hep3B-hPDL2).

Briefly, cells were cultured at 37°C with 5% CO2. Cells were harvested according to standard procedures and the supernatant was discarded. Cells were distributed into staining plates at 0.3 million cells per well. The plates were centrifuged at 300 g at 4°C for 5 minutes. Serial concentrations of the sPD-1-Fc variant fusion protein, wild-type PD-1, PDL2 tab1, and human IgG4 negative control in FACS buffer containing 2% FBS were titrated 1:5 starting at 500 μg/mL. 100 μl was added to each well and the cells were incubated for 1 hour at 4°C. Cells were washed twice with 200 μl of FACS buffer, centrifuged at 300 g for 5 minutes and the supernatant was removed before and after each wash. The cells were then resuspended with anti-human IgG-Alexa 488. The plates were incubated for 1 hour at 4°C. Cells were then washed twice for FACS analysis. FACS analyzes were performed using FACS Canto II, BD Biosciences according to the manufacturer's instructions.

Fig. 14 shows that the sPD-1-Fc variant fusion protein (eg, sPD-1-Fc variant 2 fusion protein) showed higher binding affinity to Hep3B-hPDL2 cells than the wild-type PD-1 group.

Example 8 T Cell Activation Assay in PBMC and Hep3B-OS8-PDL2 4B9 Co-Cultivation

Isolation of PBMC: A blood sample from an individual donor was diluted with the same volume of sterile PBS, eg 25 ml of sterile PBS was added to 25 ml of fresh whole blood and mixed thoroughly by gentle shaking. 15 ml of Ficoll-Pague medium was transferred to a new 50 ml centrifuge tube, making sure that the volume ratio of ficoll and blood is 3:4, then the diluted blood sample was carefully added to the surface of Ficoll medium, avoiding mixing, adding as carefully as possible so that there are layers of two liquids are clearly visible. The tube was carefully transferred to a centrifuge at 400g, 30 min, 20°C, with maximum acceleration and minimum deceleration settings during centrifugation. The mononuclear cell layer was carefully absorbed and transferred to another new sterile centrifuge tube. Sterile PBS buffer was added to the collected PBMCs for washing in a 3:1 volume ratio and centrifuged at 300×g, 10 min, 20°C. The cells were then resuspended in 10% FBS + RPMI 1640 for analysis.

Preparation of Hep3B hPD-L2 cells: Mycoplasma-free Hep3b-hPDL2-OS8 cells were prepared in a 15 cm dish and maintained at 60-80% confluence before use. The cells were trypsinized with the TrypLETM express enzyme, then the trypsin was neutralized and the cells were collected in a 50 ml centrifuge tube by centrifugation at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended with mitomycin, 10 μg/ml (in 5-10 ml of 10% FBS + RPMI1640 medium). Cells were incubated at 37°C for 1 hour. Cells were washed with 15-20 ml PBS 4 times before cell counting with a cytometer and then centrifuged at 1000 rpm, 5 min, at 20°C. Cells were resuspended in 10% FBS + RPMI 1640 for analysis.

PBMC stimulation: Add PBMC at 50,000 cells/well, 100 µl/well, and then add a series of protein solutions 50 µl/well 5X sPD-1 version 2 (PD1 V2), 5X PD1 WT or hIgG4 at 50 µl/well with followed by the addition of Hep3b-hPDL2. cells, 5000 cells/well, 50 µl/well. The mixed solution was incubated in an incubator at 37°C for 72 hours. The supernatant was collected and frozen at a temperature below -20°C before the ELISA test. IFN-gamma concentration was checked using an ELISA kit. IFN-γ was quantified using Quantifkine ELISA kits purchased from R&D. Procedures were performed according to the Human IFN-γ ELISA Kit Assay Procedure, R&D, Cat. No. DY285.

In FIG. 15 shows that sPD-1-Fc variant 2 fusion proteins are able to activate T cells in the PBMC and Hep3B-OS8-PDL2 4B9 co-culture T cell activation assay.

Example 9 In Vivo Tumor Study Using MC38-hPDL2 Colorectal Tumor Cell Line

All animals were purchased and housed in an AAALC accredited, temperature controlled, pathogen free environment. Animals were removed from the study once tumor size reached the ethical limit. C57/B6 mice were inoculated with MC38-hPD-L2 tumor cells and observed for 5 days to confirm tumor growth, and then allocated to each of three treatment groups of 10 animals per group: vehicle control, anti-PD-L1 antibody, 10 mg/kg (positive control group) and sPD-1-Fc variant 2 fusion protein 10 mg/kg (PD1-ECD-Fc group 10 mg/kg). Treatment was started on day 6 after tumor inoculation and animals were removed from the study when tumor size exceeded 2000 mm3. Tumor growth over time and survival were recorded as the end point of the experiment.

Fig. 16 shows that the variant sPD-1-Fc fusion protein version 2 showed equivalent antitumor activity compared to the positive control group (i.e., anti-PD-1 antibody, pembrolizumab), as evidenced by reduced tumor volume.

Example 10: Neutralization of PD-L1 and PD-L2 to enhance the effectiveness of immune checkpoint inhibitors

abstract

Immune checkpoint inhibitors targeting PD-1 and PD-L1 have made significant contributions to cancer treatment, becoming one of the most widely used immunotherapeutic agents. Unfortunately, low patient response rates are a major clinical problem, and in order to address this shortcoming, much of the research effort has been focused on improving the efficacy and response rate for patients receiving immune checkpoint therapy. In this study, we presented both clinical and biological evidence that PD-L2 is highly expressed in several types of cancers such as ovarian, gastric, and esophageal cancers, all of which respond poorly to PD-1 and PD-L1 inhibitors. We hypothesize that this lack of efficacy is partly due to the overwhelming level of PD-L2 present in tumors. Consistent with the previous discovery that PD-L2 has a 10-fold higher binding affinity for PD-1 compared to PD-L1, high levels of PD-L2 result in insufficient blockade of the PD-1 signaling pathway. To overcome this clinical disadvantage, we engineered a mutant soluble PD-1 bait molecule that binds and neutralizes both PD-L1 and PD-L2 with improved binding affinities of 10,000 and 200 times, respectively, compared to their wild-type interaction. This increase in binding affinity is due in part to conservative amino acid mutations both within and outside the binding boundary. In addition, we have demonstrated that sPD-1 mutant molecules are able to promote T cell-mediated killing without affecting T cell viability, subsequently resulting in superior in vivo efficacy in multiple syngeneic cancer models driven by both PD-L2 and PD-L1.

Introduction

Therapeutic inhibition of the PD-1 signaling pathway has proven to be a successful strategy in the treatment of malignant diseases (Berger and Pu 2018, Gene 638: 20-25, incorporated herein by reference in its entirety), demonstrating an unprecedented sustained clinical response (Abdel-Rahman 2016, Immunotherapy 8 ( 12): 1383-1391, incorporated herein by reference in its entirety). Therapeutic antibodies against PD-1 and PD-L1 are approved for the treatment of various types of cancers as monotherapy or in combination with current standards of care (Abdel-Rahman 2016, Immunotherapy 8 (12): 1383-1391, incorporated herein by reference in its entirety) . Unfortunately, the clinical response to PD-1/PD-L1 inhibitors can be as varied as the heterogeneous nature of the cancers themselves, further revealing the complexity of immune regulation and dysregulation associated with cancer progression (Ruiz de Galarreta, Bresnahan et al. 2019, Cancer Discov 9 (8): 1124-1141, incorporated herein by reference in its entirety). It has been clearly demonstrated that activation of the PD-1 signaling pathway requires binding and activation of PD-L1 and PD-L2 (Latchman, Wood et al. 2001, Nat Immunol 2(3): 261-268; Ishida, Iwai et al. 2002, Immunol Lett 84 (1): 57-62, incorporated herein by reference in its entirety). PD-L1 is the predominant ligand for PD-1 and is a well studied and recognized biomarker of therapeutic response to PD-1 (Freeman, Long et al. 2000, J Exp Med 192 (7): 1027-1034, incorporated herein in its entirety link). However, the role of PD-L2 in tumor immunobiology remains unclear (Bardhan, Anagnostou et al. 2016, Front Immunol 7:550, incorporated herein by reference in its entirety).

There has been ongoing clinical effort to increase the response rate of PD-1/PD-L1 inhibitors by testing these inhibitors in combination with a cocktail of cytotoxic drugs, targeted therapy, and radiotherapy to improve therapeutic efficacy while maintaining a manageable toxicity profile (Sullivan, Hamid et al. 2019 , Nat Med 25 (6): 929-935, incorporated herein by reference in its entirety). However, despite best efforts, it is still puzzling that certain types of cancers, such as ovarian cancer, esophageal cancer, and gastric cancer, still experience a suboptimal clinical response, demonstrating minimal infiltration of CD8+ cytotoxic T cells and limited T cell activity (Meza-Perez and Randall 2017, Trends Immunol 38 (7): 526-536; Sato, Olson et al. 2005, Proc Natl Acad Sci U S. A 102 (51): 18538-18543, in full included in this document by reference). Interestingly, immunohistochemical analysis of patients' tumors revealed additional information regarding the expression of PD-L1 and PD-L2 in tumors. Although PD-L1 expression remains high in many malignancies. PD-L2, a largely understudied second PD-1 ligand, is also reported to be highly expressed in these cancers, which are known to respond poorly to PD-1/PD-L1 inhibitors (Mak, Tong et al. 2016, Clin Cancer Res 22 (3): 609-620, hereby incorporated by reference in its entirety). Although it is widely believed that treatment with anti-αPD-1 antibodies is sufficient to block PD-L1 and PD-L2 mediated PD-1 activation (Garon, Rizvi et al. 2015, N Engl J Med 372 (21): 2018-2028, fully inclusive referenced in this document). However, binding studies by ourselves and others have shown a significantly higher binding affinity between PD-1 and PD-L2 compared to PD-1 and PD-L1 (Lazar-Molnar, Yan et al. 2008, Proc Natl Acad Sci U S A 105 (30): 10483-10488, incorporated herein by reference in its entirety), giving PD-L2 a significant advantage in competing with PD-1 antibodies for receptor binding.

In this study, we demonstrate that PD-L2 overexpression is a clinically significant finding in ovarian, esophageal, and gastric cancer and correlates with high PD-L1 expression, as does the exclusion of CD8+ cytotoxic T cells. This observation prompted us to develop a soluble PD-1 ligand trap with increased binding affinity for PD-L1 and PD-L2. The soluble PD-1 mutant showed a 10,000 and 200 fold improvement in binding affinity for the PD-1 receptor compared to wild-type binding between PD-L1 and PD-L2, respectively. Structural modeling of the receptor-ligand complex revealed unique mutations at both binding interfaces and non-binding sites contributing to this increase in affinity. This increase in binding affinity translated into improved inhibition of PD-1 receptor activity both in vitro and in vivo, superior to antibodies targeting PD-L1 and PD-1, in several types of cancers. More importantly, using both genetic and pharmacological approaches, we have strengthened the biological need for PD-L2 as an independent participant in PD-1 signaling and demonstrated that the use of a high-affinity soluble PD-1 mutant is a clinically viable strategy for targeting cancers. expressing both PD-L1 and PD-L2.

results

PD-L2 is overexpressed in human ovarian, esophageal, gastric, and brain tumors.

Although PD-L1-mediated PD-1 signaling has been the subject of extensive research in immuno-oncology, less is known about the status of PD-L2, especially in cancers that do not respond well to anti-PD-1/PD-L1 therapies (Derks, Nason et al 2015, Cancer Immunol Res 3(10): 1123-1129; Lingohr, Dohmen et al 2019, Epigenomics 11(6): 639-653; hereby incorporated by reference in its entirety). In the current study, we wanted to examine the clinical relevance of PD-L2 during tumor progression and, in particular, the types of cancers that showed suboptimal antitumor effects in response to checkpoint inhibitors. We selected human cancer TMAs from ovarian cancer (n=156), esophageal cancer (n=72), gastric cancer (n=76), and glioblastoma (n=152). Each sample was co-stained with PD-L1 (Latchman, Wood et al.), PD-L2 (grey), and pan-cytokeratin (red) as an epithelial cell marker. The specificity of anti-PD-L1 and anti-PD-L2 antibodies was confirmed in human tonsil tissue to avoid possible cross-reactivity between the two antibodies (FIG. 24E). The expression level of PD-L1 and PD-L2 was assessed according to the staining intensity and further stratified according to the tumor grade. In ovarian, esophageal, and gastric cancers, PD-L1 expression is elevated in malignant tissue, but significantly lower in benign samples (FIGS. 18A-18C). Interestingly, PD-L2 expression levels were also significantly increased in cancers, and its expression pattern is traced with PD-L1 in ovarian, gastric, and esophageal cancers (Fig. 18A-18C), except for glioblastoma (Fig. 18D). In glioblastoma, only PD-L2 expression significantly increased in tumors, regardless of its degree. No change in PD-L1 expression was found between normal and different classes of glioblastoma specimens (FIG. 18D with quantification shown on the right). Although there were very few cells in the sample in non-malignant brain tissue, these cells had an intense signal for PD-L1, hence a high PD-L1 score in normal tissue. Pearson's correlations between PD-L1 and PD-L2 were also calculated and plotted for each type of cancer (FIGS. 24A-24D). Consistent with immunostaining results, we found a positive correlation between PD-L1 and PD-L2 for all four cancer types, supporting our hypothesis that PD-L2 is present in abundance in tumors that respond poorly to immune checkpoint therapy (Fig. 24A-24D). Functionally, we investigated whether the presence of PD-L1 and PD-L2 expression leads to changes in infiltrating CD8+ T cells. We stained ovarian cancer (FIG. 18E) and esophageal cancer (FIG. 18F) samples from the same TMAs and again quantified CD8+ cells stratified according to tumor grade. In ovarian cancer, non-cancerous ovarian tissue with blood vessels was included as a positive control showing that the majority of CD8+ T cells are restricted to blood flow (FIG. 18E upper panel). Representative images of ovarian cancer positive and negative for CD8+ T cell infiltration are shown in FIG. 18E and again quantified according to tumor grade (FIG. 25A). In TMA of esophageal cancer, most of the esophageal cancer samples had low CD8+ T cell staining, with the exception of a few showing higher CD8 cell counts (Fig. 18F and Fig. 25B). Inflammatory esophagitis tissues were included in TMA as a positive control. The absence of CD8+ T cells in the tumor suggests that the presence of both PD-L1 and PD-L2 may contribute to the exclusion of T cells. In summary, our results demonstrated high expression of both PD-L1 and PD-L2 in several cancer types with a known suboptimal clinical response to PD-1/PD-L1 therapy, and this may also play a role in immune exclusion of cytotoxic CD8+ T- lymphocytes in cancerous tissues.

Construction of sPD-1 mutants with excellent binding affinity for PD-L1 and PD-L2

A sophisticated way to counteract both PD-L1 and PD-L2 in a biological system is to use a soluble component of the PD-1 receptor (FIG. 19A). Unfortunately, the natural affinity of wild-type soluble PD-1 and both of its ligands is low in the micromolar range, making wild-type sPD-1 an ineffective antagonist for blocking the PD-1/PD-L1/PD-L2 signaling axis (Lazar-Molnar, Yan et al 2008, Proc Natl Acad Sci U S A 105(30): 10483-10488, incorporated herein by reference in its entirety). In an attempt to develop high binding affinity antagonists to neutralize both PD-L1 and PD-L2, we generated a mutant library by introducing random mutations into the extracellular component of the PD-1 molecule (soluble PD-1). Using a similar strategy that we have previously published (Kariolis, et al. 2014, Nat Chem Biol 10 (11): 977-983, incorporated herein in its entirety by reference), a mutant library was presented as fusion proteins on the surface of yeast cells, further enriched clones with improved PD-L1 binding (FIG. 19B and FIG. 26). After six rounds of sorting, clones with excellent binding affinity were sequenced for further characterization and analysis (FIG. 37). Eight residues were mutated in the sPD-1 domain corresponding to Ser62, Ser87, Pro89, Asn116, Gly124, Ser127, Ala132 and Ala140, with position numbering starting from the signal region (Table 4). An updated version of the sPD-1 mutant (version 2) was created by deleting the mutation in Asn116 to avoid mutation of the N-glycosylation site. Both mutants were fused to the Fc domain of human IgG4 to improve stability and apparent affinities. The apparent affinities of the IgG4 V1 and V2 mutants for human PD-L1 and PD-L2 were quantified using BIAcore® Surface Plasmon Resonance technology and compared with wild-type sPD-1-IgG4. A significant improvement in binding affinities for human PD-L1 and PD-L2 was found in mutant V1 and V2 compared to wild-type sPD-1, demonstrating an approximately 10,000-fold and 200-fold improvement in binding affinity for PD-L1 and PD-L2. , respectively (Fig. 8). In both cases, this improvement in affinity was primarily due to the lower dissociation rate (FIGS. 19C and 19D). Because we did not see significant differences in KD between the sPD-1 V1 mutant and the sPD-1 V2 mutant. All cell and in vivo studies were performed using the sPD-1 V2 mutant. Together, using yeast display technology, we identified sPD-1 mutant clones showing high binding affinity for PD-L1 and PD-L2. These clones can be further characterized to evaluate both structural and biological improvement resulting from site-specific mutations.

Computational modeling to determine the structural basis of high affinity binding

To determine the changes in structural confirmation that lead to this increase in affinity between PD-1/PD-L1 and PD-L2, we performed computer simulations comparing human PD-L1 and PD-L2 in a co-complex with the sPD-1 mutant. The structure of the wild-type human PD-1/PD-L1 complex is available from the PDB (4ZQK and 5IUS). However, since residues in the PD-1 loop region are missing and mutated, we first fixed the missing loop and mutated the residue back into wild-type PD-1 based on 4ZQK. The alignment of the 4ZQK and 5IUS sequences is shown in FIG. 2. The structure of the sPD-1 mutant was modeled using the Schrodinger Suite residue scan module. The crystal structure of PD-1 and PD-L1 is shown in blue and green, respectively, with 8 mutations (Ser62, Ser87, Pro89, Asn116, Gly124, Ser127, Ala132 and Ala140 with position numbering starting from the signal region) shown as orange bars (Fig. .3). Combining knowledge from previously published literature and our structural modeling data, we identified residues G124S, S127V and A132I as mutations within the binding interaction region, S62G, S87G, P89L and A140V lie outside the binding interaction region. G124S and A132I each form one additional hydrogen bond with Tyr 123 and Gln 66 PD-L1, respectively (FIG. 20A, left and right panels). The A132I mutation also resulted in an increase in the PD-L1 complementary surface from 0.72 to 0.85 (Fig. 20B, Fig. 29). To further explore how these mutations affect overall binding between PD-1 and PD-L1, the binding affinity and complex stability variances were calculated before and after a single mutation, with the results shown in Table 4. Calculation of the variance in binding affinity and complex stability was also performed for clustered mutations within and outside the binding boundary (Table 5). A higher negative value indicates increased binding affinity and stability. In general, mutations in the interaction region contributed to both binding affinity and protein stability, as expected, and mutations outside the binding interaction region lead to increased stability. Interestingly, the A140V mutation outside the binding boundary significantly improves the stability of the complex by 25.89 kcal/mol. This observation further reinforced the importance of an objective screening approach to identify mutations outside the binding boundary to improve the overall stability of the co-complex.

Because the structure of the human PD-L2 protein is not available in the PDB, a human PD-L2 model was built using available mouse PD-L2 structures as templates (Fig. 5A). The sequence identity of human PD-L2 and mouse PD-L2 is about 72% (FIG. 6, FIG. 5A). An overlay of the proposed human PD-1/PD-L2 model and mouse PD-1/PD-L2 (PDBID: 3BP5) crystal structure is shown in FIG. 6 and FIG. 5b. The crystal structure of the co-complex of mutant sPD-1 with proposed human PD-L2 is shown in FIG. 7. Compared to the proposed wild-type human PD-1/PD-L2, similar to PD-1/PD-L1 binding, the A132I mutation again retained the same additional hydrogen bond to PD-L2, resulting in increased surface complementarity (Fig. 20C and Fig. 30). Calculation of binding affinity variation and protein stability was also performed for single mutation and clustered mutations within and outside the binding interaction region (Table 8 and Table 9). Based on structural modeling analysis, we have shown that mutations both within and outside the binding interaction region contribute to improved overall binding affinity and stability.

sPD-1 Mutants Block PD-L1 and PD-L2 Mediated PD-1 Activation and Induce T Cell Activation

To demonstrate that sPD-1 mutants can effectively disrupt interactions between PD-L1/PD-L2 and PD-1, we first evaluated the binding activity of sPD-1 mutants to PD-L1 using human PD-L1 knockout MC38 cells (provided by ChemPartner, Shanghai). Both versions of the sPD-1 mutants, wild-type sPD-1 and control hIgG4 were tested using a FACS-based binding assay. Significant improvement in binding signal was observed with both versions of the sPD-1 mutants compared to wild-type sPD-1 and the hIgG4 control, where no fluorescent signal could be detected in the range down to μM for wild-type sPD-1 (Fig. 9A). To validate the binding of sPD-1 to mouse PD-L1 for further in vivo testing, we also analyzed the binding of the sPD-1 mutant to the original wild-type MC38 expressing mouse PD-L1 and PD-L2. An increase in binding signal was also found for both version 1 and version 2 compared to wild-type sPD-1 with an overall lower intensity (Fig. 9B). The binding assay was also repeated in the Hep3B-OS8-hPD-L1 hepatocellular carcinoma cell line (ChemPartner, Shanghai) with similar results (FIG. 31). To test whether sPD-1 mutants are able to block the receptor-mediated interaction between PD-L1 and PD-1. We performed a receptor binding assay (RBA) using wild-type sPD-1, sPD-1 mutants and hIgG4 control. Both sPD-1 mutants showed greater inhibition of PD-L1-mediated PD-1 receptor binding compared to wild-type sPD-1 and hIgG4 (FIG. 21A). To demonstrate that the sPD-1 mutant is able to bind to PD-L2 and block PD-L2 mediated PD-1 activation, we overexpressed human PD-L2 in both MC38 and Hep3B cells (Figures 32 and 33). For binding analysis, we tested the V2 sPD-1 mutant along with wild-type sPD-1, αPD-L2 antibody as a positive control, and hIgG4 as a negative control. The sPD-1 mutant showed better binding to MC38-PD-L2, wild-type sPD-1, and the αPD-L2 antibody binds to MC38-PD-L2 with less efficiency (FIG. 14). Similarly, the sPD-1 mutant also showed an increased ability to block PD-L2 mediated PD-1 activation in the receptor binding assay (FIG. 21B). To test whether the sPD-1 mutant is able to activate human cytotoxic T cells by blocking PD-L1 or PD-L2 mediated signaling, human PBMCs were harvested from healthy donors and incubated with Hep3B-OS8-hPDL1 and Hep3B-OS8-hPDL2 cells. . Added mutant sPD-1, PD-1 wild type and IgG4 and measured the activity of cytotoxic T cells by the level of interferon-γ. The sPD-1 mutant has a high ability to activate T cells in the presence of PD-L1 and PD-L2, respectively, compared to wild-type sPD-1 and IgG4 in a dose dependent manner (FIGS. 10 and 15), supporting the argument that the sPD mutant -1 can functionally stimulate T cell activity by blocking the interaction between PD-L1/PD-L2 and PD-1. Because long-term stimulation of T cell activity via direct binding using αPD-1 antibodies and PD-1 receptors on T cells can potentially affect T cell functionality and viability over time, our sPD-1 mutant was only designed to target cancer cells expressing PD-L1 and PD-L2 in an effort to maintain T cell longevity and functionality. To test this hypothesis, we first made both sPD-1 (V1 and V2) and wild-type sPD-1 mutants, all fused with high purity, low endotoxin human Fc from IgG4 (Fig. 28). Activated human T cells were then treated (via CD3/CD28 cocktail stimulation) with either an αPD-1 antibody or an sPD-1 mutant in the presence or absence of PD-L1 for 12 days. Continuous treatment with αPD-1 slowed T cell proliferation over time, while treatment with the sPD-1 mutant had no effect on T cell growth, showing a growth rate similar to that of CD3/CD28 stimulated T cells. As expected, native T cells did not proliferate in the absence of CD3/CD28 stimulation (FIG. 21C). These observations support the idea that the sPD-1 mutant is able to bind and block the PD-1/PD-L1 and PD-L2 signaling cascade and stimulate T cell activity while maintaining T cell viability and functionality.

sPD-1 Mutant Demonstrates Superior Antitumor Efficacy in Syngeneic Mouse Models of Malignancy

The therapeutic efficacy of the sPD-1 mutant has been evaluated in several syngeneic mouse tumor models. An sPD-1 mutant without a mutation at the N-glycosylation site (Asn116) was used for all in vivo studies. To determine the dosing regimen and pharmacokinetics of the sPD-1 mutant in vivo. The molecule was labeled with a fluorescent dye and mice were dosed with a single dose of 10 mg/kg and images were taken at 30 minutes, 8 hours, 24 hours and 72 hours after treatment (FIG. 22A). In addition, an ELISA detecting a portion of human IgG4 from the mutant sPD-1 molecule was also performed on sera taken over time from an animal receiving the same dose (FIG. 22B). Both fluorescence and ELISA analyzes led to a similar conclusion that the half-life of the mutant sPD-1 molecule is approximately 24 hours post-dose, which is the basis for an in vivo dose of 10 mg/kg every 48 hours. Mice subcutaneously inoculated with MC38-hPDL1 colorectal cancer were treated with vehicle control, sPD-1 mutant, or atezolizumab. A significant reduction in tumor growth was observed in both tumors treated with the sPD-1 mutant and those treated with atezolizumab (Fig. 11A). Compared to atezolizumab treatment, tumors treated with the sPD-1 mutant showed superior efficacy in suppressing tumor growth and increased survival without serious toxicity as measured by body weight change (Figures 11C, 35, 11B), further providing rationale for targeting both PD -L1 and PD-L2 to achieve optimal antitumor activity (Fig. 11B). The ability of the sPD-1 mutant to counteract PD-L2 mediated tumor growth was demonstrated in in vivo models inoculated with MC38 tumors overexpressing human PD-L2 (FIG. 22C). Significant delay in tumor growth was observed in tumor-bearing mice treated with the sPD-1 mutant or anti-αPD-1 antibody, pembrolizumab. Although not statistically significant, there was a trend towards better tumor growth suppression in the sPD-1 mutant group compared to the pembrolizumab group, which may require further investigation (Fig. 22C). The antitumor efficacy of the sPD-1 mutant was also tested in the B16/OVA melanoma mouse model and the ID8 ovarian cancer model. Melanoma tumors treated with the sPD-1 mutant cannot support tumor growth compared to vehicle treated groups (FIG. 22D). It is clinically known that patients with ovarian cancer respond poorly to PD-1 inhibition. In this study, we wanted to test the efficacy of the sPD-1 mutant in a syngeneic model of ovarian cancer. Animals with orthotopically inoculated ID8 ovarian tumor cells were treated with vehicle control; The sPD-1 mutant or mouse αPD-1 antibody and animals were terminated when ascites developed. Kaplan-Meier survival analysis showed an improvement in overall survival for both the mouse αPD-1 antibody treated groups and the sPD-1 mutant treated groups. However, the most significant survival benefits were observed in animals treated with the sPD-1 mutant group, suggesting that the sPD-1 mutant is more effective when directed at ovarian cancer rather than blocking the PD-1 receptor (Fig. 22E). Tumor infiltrating T cells (TIL) were analyzed in B16/OVA (FIG. 22F) and MC38 (FIGS. 22G and 22H) tumor-bearing mice treated with αPD-1 antibody or sPD-1 mutant. Treatment with both inhibitors again resulted in increased infiltration of CD4+, CD8+ cytotoxic T cells, with the most significant changes observed in the sPD-1 mutant treatment groups. Other tumor-associated immune cells were also profiled, but no significant differences were found between treatment groups (FIG. 36). Collectively, we demonstrated superior antitumor efficacy of the sPD-1 mutant tested in multiple syngeneic mouse tumor models, due in part to better TIL infiltration and having the potential to replace both αPD-L1 and αPD-1 antibodies as a new form of checkpoint inhibitor.

The sPD-1 mutant is effective in animal models of PD-L2-driven human cancer (FIG. 23).

To demonstrate that PD-L2 is a functionally important PD-1 ligand, and PD-L2 is able to promote PD-1 mediated tumor growth, especially in cancer types that do not respond well to PD-L1 inhibition. We genetically deleted PD-L1 in ID8 ovarian tumor cells using CRISPR-CAS9 technology and inoculated tumor cells into PD-L1 knockout mice, abolishing PD-L1 expression in both tumor and host (FIGS. 23A and 37A). Interestingly, complete loss of PD-L1 did result in slower tumor growth rates and smaller tumors compared to wild-type ID8 tumor grown in normal C57/B6 mice. However, satisfactory tumor growth did occur when mice were inoculated with large numbers of ID8 cells, possibly due to increased expression of PD-L2 (FIG. 2). Tumors treated with the sPD-1 mutant fail to grow and have shown significant tumor regression in some treated animals, as expected tumors treated with αPD-L1 antibody remain largely insensitive (FIGS. 23B and 23C). In a separate study where ID8 PD-L1 CRISPR cells were orthotopically inoculated intraperitoneally, only three of eight mice in the sPD-1 mutant treated group developed ascites compared with seven of eight mice in the αPD-L1 treated group. Significantly improved survival in mice with ovarian cancer that was exclusively driven by PD-L2 (Fig. 23D). Finally, ID8 tumors were stained for PD-L2 and CD8 expression. As expected, all tumors regardless of treatment groups showed positive expression of PD-L2, however, only tumors treated with the sPD-1 mutant saw infiltration of CD8+ T cells into the tumor due to loss of both PD-L1 and PD-L2 ( Fig. 23E).

In this study, we demonstrated the clinical relevance of PD-L2 in cancer, which is considered a weak immune checkpoint inhibitor responsive response, and presented a therapeutic strategy that preferentially targets both PD-L1 and ultra-high affinity PD-L2. Structural and biological analysis of the sPD-1 mutant strongly supported our hypothesis that improved binding affinity leads to stronger inhibition of the PD-1 pathway by blocking both of its ligands. Together with in vivo data demonstrating enhanced antitumor activity, the sPD-1 mutant represents a viable clinical approach to target cancer types that lack a satisfactory response to PD-1/PD-L1 inhibitors as a result of high expression of PD-L2.

Discussion

Immunotherapy, such as inhibitors of the PD-1 signaling pathway, has revolutionized the field of cancer treatment and significantly improved the survival of cancer patients who had a sustained response to treatment (Topalian, Hodi et al. 2012, N Engl. J. Med. 366 ( 26): 2443-2454, incorporated herein by reference in its entirety). However, while immunotherapy does deserve to be called a "breakthrough" in the fight against cancer, there is definitely no one-size-fits-all approach that will promote an adequate immune response in all treated patients. The response to PD-1/PD-L1 inhibitors varies by type of cancer, and also in an individual patient in the same treatment group, depending on the immunogenic nature of the cancers (Pitt, Vetizou et al. 2016, Immunity 44 (6 ): 1255-1269, incorporated herein by reference in its entirety). The heterogeneity observed with checkpoint inhibitors may be due to several factors including 1), genetic mutations in certain cancer cells causing microinstability (MSI-H) are more immunogenic and therefore show a better response to PD-1 inhibitors (Diaz and Le 2015, N Engl J. Med. 373 (20): 1979, incorporated herein by reference in its entirety). 2). Factors secreted by both cancer cells and cancer-associated stromal cells in the tumor microenvironment, such as PD-L1 and PD-L2, protect cancer cells from immune surveillance by preventing cytotoxic T cell recruitment and activation (Tumeh, Harview et al. 2014, Nature 515 (7528): 568-571, incorporated herein by reference in its entirety). Taken together, strategies that can stimulate the host's immune response to target cancer cells are vital to the success of immune control therapy.

In this study, we are focused on understanding both the level of expression and the correlation of PD-L1 and PD-L2 in cancer types that clinically respond poorly to PD-1 checkpoint therapy (Imai, Hasegawa et al. 2018, Oncol Lett 15 (5 ): 6457-6468, incorporated herein by reference in its entirety). Unlike PD-L1, which is the better known PD-1 ligand, the role of PD-L2 in the development of cancer remains unclear (Yearley, Gibson et al. 2017, Clin Cancer Res 23 (12): 3158-3167, included in full in this document by reference). Interestingly, this is contrary to the popular belief that PD-L1 is the predominant PD-1 ligand that is activated by cancer cells. We observed a significant increase in PD-L2 expression in all patient samples we studied, and its expression level strongly correlates with PD-L1 expression. Interestingly, we also observed PD-L1 expression in both cancerous and non-cancerous tissues, while overexpression of PD-L2 appears to be limited to cancerous tissues, suggesting that PD-L2 expression may be induced during oncological diseases. . Based on these observations, we believe that PD-L2 expression is highly expressed in cancers that do not respond well to PD-1 inhibitors, and that adequate inhibition of both PD-L1 and PD-L2 is required to optimally counteract PD-1 signaling.

Optimal downregulation of the PD-1 signaling pathway can be achieved by disrupting the receptor ligand interaction between PD-1 and PD-L1/L2. Although blocking PD-1 receptors on T cells with anti-PD-1 antibodies resulted in a robust clinical response in a subset of patients, our patient data showed high levels of PD-L2 expression in cancers with a low response rate to PD-1/PD inhibition. -L1, suggesting that the presence of PD-L2 could potentially undermine the inhibitory effort of PD-1. Indeed, our data showed a 10-fold increase in native binding affinity between PD-L2 and PD-1 than binding affinity between PD-L1 and PD-1. Although most anti-αPD-1 antibodies undergo affinity maturation, increasing their ability to bind PD-1 in the presence of its ligands, high levels of PD-L2 expressed by cancer cells can significantly interfere with the ability of αPD-1 antibodies to compete for binding to PD-1. Instead of blocking the PD-1 receptor on T cells, we generated sPD-1 mutants with a significant increase in binding affinity for both PD-L1 and PD-L2. Using a yeast surface display platform with directed evolution approaches (Kariolis, et al. 2014, Nat Chem Biol 10 (11): 977-983, hereby incorporated by reference in its entirety), mutant clones were selected showing a significant increase in binding affinity to both PD- L1 and PD-L2, and the structural framework that provides such high binding affinity was analyzed using computer structural modeling. Comparison of the protein interaction between wild-type sPD-1 and mutant sPD-1 with PD-L1 showed that changes in binding affinity were the result of both obtaining an additional hydrogen bond at Ala 132 and increased stability of the co-complex as a result of mutation in Ser127 and Ala140 with position numbering starting from the signal area. Similar results were observed when comparing the protein interaction between the wild-type PD1/PD-L2 complex and the mutated PD1/PD-L2 complex, again supporting the idea that while mutations occurring in the binding interaction region may improve receptor-ligand binding affinity , mutations that are outside the binding interaction region are equally important for ensuring the stability of the entire construct. Interestingly, two additional studies have been reported that generated high binding affinity sPD-1 clones (Maute, Gordon et al. 2015, Proc Natl Acad Sci U S A 112(47): E6506-6514; Li, Liang et al. 2018, Cancer Sci 109(8): 2435-2445, incorporated herein by reference in its entirety). Both studies reported unique mutation sites contributing to their increased binding affinity, which does not overlap with our current study, again in support of the unbiased approach needed to design increased affinity therapeutics with desired biological properties.

The concept of increased affinity translates into superior biological activity when the in vitro FACS binding assay and RBA assay clearly demonstrated that sPD-1 mutants resulted in increased binding affinity and their ability to block interactions between PD-1 and PD-L1/L2. Functionally, the sPD-1 mutant also resulted in increased T cell activity, which is a sign of suppression of immune checkpoints. In all scenarios, it was clear that both PD-L1 and PD-L2 were sufficient to activate the PD-1 pathway, and the addition of the sPD-1 mutant effectively disrupted signaling mediated by both ligands. In our study, we observed a decrease in T cell viability after continuous treatment with αPD-1 antibodies, whereas T cells treated with sPD-1 treatment had no effect on cell growth. However, it is difficult to confirm whether the decrease in T cell viability after αPD-1 treatment was the result of T cell depletion. Given that the αPD-1 antibody directly binds and stimulates T cells, while the sPD-1 mutant only targets PD-L1/L2 on cancer cells, avoiding direct interaction with the T cell may be beneficial to maintain viability and functionality. T cells.

The current study evaluated the antitumor activity of the sPD-1 mutant against both αPD-L1 and αPD-1 antibodies to answer two questions. 1) Can the sPD-1 mutant outperform both αPD-L1 and αPD-1 antibodies due to its increased ability to block PD-L1 and PD-L2? 2) Can the sPD-1 mutant affect tumor growth in tumor models driven solely by PD-L2? Indeed, the sPD-1 mutant outperformed both the αPD-L1 antibody and the αPD-1 antibody in multiple syngeneic tumor models in mice, supporting the argument that increased receptor-ligand binding affinity improves the therapeutic outcome of PD-1 inhibition. More importantly, since the contribution of PD-L2 to tumor growth remains uncertain, we evaluated the functional significance of PD-L2 using PD-L1 knockout mice inoculated with PD-L1 CRISPR tumors, demonstrating that PD-L2, together with the tumor, is able to stimulate tumor growth in the absence of PD-L1 and can be successfully targeted using sPD-1 mutants but not αPD-L1 antibodies. However, it is worth noting that both higher tumor cell numbers and slower tumor growth have been observed in tumor models lacking PD-L1, but ultimately the presence of PD-L2 is able to support tumor growth, which can be effectively targeted by the sPD-mutant. one.

In summary, this study identified PD-L2 as a potential risk factor associated with poor clinical response to PD-1 inhibitors in several types of cancer. This observation prompted us to develop sPD-1 mutants with superior binding affinity to both PD-L1 and PD-L2 as a strategy to eliminate the interaction between PD-1 and PD-L1/L2. Mutations both within and outside the binding interaction region enhanced binding affinity and resulted in higher antitumor activity compared to both αPD-L1 and αPD-1 antibodies. In addition, the observation that PD-L2-expressing tumors are able to grow in a PD-L1-independent manner in vivo and that their growth is inhibited by the sPD-1 mutant but not by the anti-aPD-L1 antibody further strengthened the antitumor role of PD-L2 in cancerous diseases. These data collectively provided the rationale for the evaluation of the sPD-1 mutant as an alternative to PD-1 inhibitors in the treatment of cancers expressing PD-L1 and/or PD-L2.

Materials and methods

Study design

This study was designed to evaluate the involvement of PD-L2 in both in vivo models and human clinical specimens, and to characterize the structural, biochemical, functional, and therapeutic efficacy of ultra-high binding affinity PD-1 soluble mutants to both PD-L1, so is PD-L2. All tumor microarray samples were commercially purchased from US Biomax (Derwood, MD). A certified veterinary pathologist performed an immunohistochemical evaluation of stained tumor microarray samples. In vivo studies in mice were performed with the approval of AAAPLAC at Stanford University and ChemPartner, Shanghai. The sample size for animal studies was based on previous experience with similar in vivo studies. All animals were randomly divided into treatment groups. Samples were not withdrawn from studies, except in animals requiring early termination due to illness unrelated to the study. The endpoints of the experiments were determined in advance for each experiment. Tumor growth curves were presented for studies in which tumor growth could be measured, and Kaplan-Meier curves were used for the ID8 orthotropic ovarian cancer model. Appropriate statistical analysis was used for each pilot study.

Cell lines

MC38, ID8, ID8-PDL1 CRISPR, B16/OVA, MC38-hPD-L1, MC38-hPD-L2, MC38-hPD-L2-PD-L1 CRISPR, Hep3B-OS8-hPD-L1, Hep3B-OS8-hPD- L2 was maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in a humidified incubator at 37°C and 5% CO2. Cells were trypsinized and passaged at 80% confluence.

Synthesis of the yeast sPD-1 library

DNA encoding the extracellular domain of human PD-1, the amino acids Leu25-Val170, was cloned into the pCT yeast display plasmid using restriction sites NheI and BamHI. Sequence numbering was as used in Mihalis S. Kariolis et al. (Kariolis, et al. 2014, Nat Chem Biol 10 (11): 977-983, incorporated herein by reference in its entirety) to facilitate comparison of their performance with wild-type proteins. An error polymerase derived library was generated using PD-1 extracellular domain DNA as a template and mutations were introduced using Taq imprecise polymerase (Invitrogen) and 8-oxo-dGTP and dPTP nucleotide analogues (TriLink Biotech). Six separate PCR reactions were carried out, in which the concentration of analogs and the number of cycles were varied to obtain a range of mutation frequencies; five cycles (200 µM), ten cycles (2, 20 or 200 µM) and 20 cycles (2 or 20 µM). The products of these reactions were amplified using forward and reverse primers, each of which had a homology of 50 bp. with pCT plasmid in the absence of nucleotide analogs. The amplified DNA was purified by gel electrophoresis and the pCT plasmid was digested with NheI and BamHI. Purified mutant cDNA and linearized plasmid were electroporated at a ratio of 5:1 by weight into EBY100 yeast, where they were assembled in vivo by homologous recombination (Kariolis, et al. 2014, Nat Chem Biol 10 (11): 977-983, hereby incorporated by reference in full ). The library size was estimated to be 7.4 x 10 ⁷ by seeding by dilution.

Library screening

Yeasts presenting high affinity PD-1 mutants were isolated from the library using fluorescence activated cell sorting (FACS). For FACS rounds 1-3, equilibrium binding sorts were performed in which yeast were incubated at room temperature in phosphate-buffered saline with 1 mg/ml BSA (PBSA) with the following nominal concentrations of Gas6 (R&D Systems): sort 1) 10 nM PD- L1 for 3 h; sorting 2) 2 nM PD-L1 for 3 h; sorting 3) 0.2 nM PD-L1 for 24 hours. After incubation with PD-L1, the yeast was precipitated, washed and resuspended in PBSA with 1:250 chicken anti-c-Myc (Invitrogen) for 1 h at 4°C. The yeast was then washed, pelleted, and relabeled on ice for 30 min using PBSA at a 1:100 dilution of goat anti-chicken antibodies labeled with Alexa Fluor 555 (Invitrogen) and mouse anti-HIS antibodies Hilyte Fluor 488 (Anaspec).

For FACS rounds 4-6, dissociation rate kinetic sorts were performed in which yeast were incubated with 2 nM PD-L1 for 3 hours at room temperature, after which the cells were washed twice to remove excess unbound Gas6 and resuspended in PBSA containing ~50- multiple molar excess of PD-1 (R&D Systems) to make debinding events irreversible. The duration of the debinding step was as follows: sorting 4) 4 hours; sorting 5) 4 h; sorting 6) 24 hours, with all debinding reactions carried out at room temperature. During the last hour of the dissociation reaction, chicken anti-c-Myc was added to a final dilution of 1:250. The yeast was precipitated, washed, and relabeled as previously described. Labeled yeasts were sorted by FACS using a Vantage SE flow cytometer (Stanford FACS Core Facility) and CellQuest software (Becton Dickinson). Sorting was performed in such a way that 1-3% of clones with the highest ratio of Gas6 binding/c-Myc expression were selected, which enriched the library with clones with the highest binding affinity for Gas6. In sort 1, 10 ⁸ cells were screened and at least ten times the number of clones collected in the previous sort round were analyzed in subsequent rounds to ensure adequate sampling of library diversity. Selected clones were expanded and subjected to further FACS cycles. After sorting 5 and 6, plasmid DNA was isolated using a Zymoprep kit (Zymo Research Corp.), transformed into XL-1 blue supercompetent cells, and isolated using a miniprep plasmid kit (Qiagen). Sequencing was performed at Sequetech Corp.

Analysis of yeast display sort products was performed using the same reagents and protocols as described for library sorts. Samples were analyzed on FACSCalibur (BD Biosciences) and data was analyzed using FlowJo software (Treestar Inc.).

Obtaining a recombinant protein

Soluble wild-type PD-1 and hIgG4Fc-related mutants were cloned into pCPC signal peptide plasmid (ChemPartner, Shanghai) and transiently transfected into human embryonic kidney HEK293 cells and cultured in 4 L of OPM-293 CD03. The products were then purified with MabSelect SuRe protein A resin (GE Healthcare) and further isolated by superdex 200 size exclusion chromatography. Each purification step was followed by SDS-PAGE confirmation and SEC-HPLC analysis. End products were tested for endotoxin and verified at 0.09 IU/mg.

Analysis of computer simulation

The structure of the human PD1/PDL1 complex is available from the Protein Data Bank, PDBID - 4ZQK and 5IUS. However, in both PDB structures, some PD1 residues are missing from the loop region and some of the residues were mutated compared to wild-type PD1. The missing loop and mutated residue were rebuilt back into wild-type PD1 based on the 4ZQK crystal structure. The mutated PD1 structure was modeled using the Schrodinger Suite Residue Scan module. Residues within 5.0 Å of the mutant residue were refined by side chain prediction and backbone sampling. Calculation of binding affinity variation and complex stability was also performed for multiple mutations. A more negative binding affinity or stability value indicates a greater increase. A PDL2 homology model was built to investigate the interaction between human PD1 and PDL2. After looking for homology in the PDB data set without redundancy, three mouse PDL2 PDB structures were selected as templates. Comparison of PD1 binding interaction region between human PDL2 model and mouse PDL2 crystal structure (PDBID: 3BP5).

Immunoassays

The slides were deparaffinized and antigen retrieval was performed using 10 mM citric acid buffer, 0.05% Tween 20, pH 6. The slides were removed from the buffer and cooled at room temperature for 15 minutes. Endogenous peroxidase was quenched by a 1:10 dilution of 34% hydrogen peroxide in water for 15 minutes. Avidin blocker and biotin were added for 15 minutes each. Added protein block using 2% fetal calf serum for 20 minutes. Serum and antibodies were diluted in PBT (1XPBS, 0.1% BSA, 0.2%, 0.01% Tween 20). Antibodies αHuman PD-L1 1:500 (#AF154, R&D systems, Minneapolis, MN), αΗuman PD-L2 1:500 (#MABC1120, EMD Millipore, MA), αΗuman CD8 1:500 (#Ab4005, Abcam, Cambridge, MA), αMouse PD-L1 1:750 (#17952-1-AP, Proteintech, IL), αMouse PD-L2 1:750 (#Ab21107, Abcam, Cambridge, MA) and αMouse CD8 1:500 (#Ab203035, Abcam, Cambridge, MA) were incubated overnight at °C. For immunohistochemistry, anti-rabbit and rat antibodies were added to each slide and incubated at 37°C for 30 minutes, then incubated with STREP-HRP for 30 minutes at 37°C, and signals were developed using the DAB substrate kit (#34002, ThermoFisher Scientific., Waltham, Massachusetts). To determine immunofluorescence, all staining was performed in the dark. Secondary antibodies 1:400 diluted in 2% BSA and 0.1% tween 20 in PBS were incubated at room temperature for 1 hour. A 0.2% solution of Sudan Black B in 70% ethanol through a syringe filter prewarmed to room temperature was added to glass slides for 10 minutes at room temperature. DAPI (#F6057, Sigma-Aldrich, St. Louis, MO), 0.1 μg/ml was used for counterstaining and then covered with a coverslip. Human IgG4 (# BMS2059, ThermoFisher Scientific, Waltham, MA) was quantitated by ELISA according to the manufacturer's instructions.

Surface plasmon resonance

Human PD-L1 (#10084-H08H, SinoBiological, Wayne, PA) and human PD-L2 (#10292-H08H, SinoBiological, Wayne, PA) were used as analyte. Prepared working buffer HBS-EP+ (10 mm HEPES, 150 mm NaCl, 3 mm EDTA and 0.05% P20, pH 7.4). The flow rate was 30 μl/min. Antibodies against Fc from human IgG were immobilized on the CM5 sensor chip. PD1-Fc was captured by the immobilized sensor chip. An injection of a serially diluted analyte was used. Glycine pH 1.5 was used for surface regeneration for 30 s. Multichannel kinetics were measured on a Biacore T200. Kd analysis was calculated using Biacore T200 evaluation software (GE Healthcare Life Sciences).

Binding analysis based on flow cytometry

MC38-hPD-L1, MC38-hPD-L2, Hep3B-OS8-hPD-L1, Hep3B-OS8-hPD-L2 cells and stock MC38 cells were cultured under standard tissue culture conditions. The cells were harvested and the supernatant was discarded, then applied to the staining plate at 3 x 10 ⁵ cells per well. The tablet was centrifuged at 300 g at 4°C for 5 minutes. Various concentrations of sPD-1 mutants and a negative control were diluted in FACS buffer containing 2% FBS, 100 μl/well was added. Cells were incubated for 1 hour at 4°C, washed twice with 200 μl of FACS buffer and centrifuged at 300 g for 5 minutes. The supernatant was removed before and after each wash. Cells were resuspended at 100 μl/well in 1:1000 diluent with anti-human IgG antibody conjugated to Alexa 488 (# A28175, ThermoFisher, Waltham, MA). The plates were incubated for 1 hour at 4°C. Cells were washed twice with FACS buffer and centrifuged at 300 g for 5 minutes. The supernatant was removed and the cells were resuspended in 100 μl of cold PBS. Cells were stored in the dark and FACS analysis was performed on FACS CantoII (BD Biosciences, San Jose, CA).

Receptor Blocking Assay

Hep3B-OS8-hPD-L1, Hep3B-OS8-hPD-L2, MC38-hPD-L1 and MC38-hPD-L2 cells were cultured in PRMI 1640 medium, 10% FBS, G418 and hygro, in a T175 flask to a confluence of 60-80 %. Cells were harvested and resuspended in dilution buffer. Cells were plated in a 96-well Corning #3799 round bottom plate at a density of 2 x 10 ⁵ cells/well. The sPD-1 mutant at several concentrations: 600, 120, 24, 4.8, 0.96, 0.192, 0.00384 nM and 0 nM were resuspended in dilution buffer. Wild-type sPD-1 was also obtained in dilution buffer. The plate was centrifuged at 500 g for 3 minutes and the supernatants were discarded. Cells were resuspended with 50 µl of antibody followed by 50 µl of ligand. The plates were incubated at 4°C for 2 hours, then centrifuged and washed. Streptavidin-Alexa Flour 488 (# S11223, Thermo Fisher, Waltham, MA) diluted 1:1000 in dilution buffer was prepared and incubated with cells at 4° C. for 1 hour. The plate was centrifuged and washed, then resuspended in 200 μl of FACS buffer. Cells were run on FACS Canto II (BD Biosciences, San Jose, CA). Curves were plotted with MFI values and IC50 and high points were calculated.

T cell stimulation and proliferation assay

Blood samples from healthy donors were diluted with an equal volume of sterile PBS and mixed with gentle shaking. 15 ml of Ficoll-Paque PLUS medium (-1440-02, GE Healthcare, Pittsburgh, PA) was transferred to a fresh 50 ml centrifuge tube with a 3:4 volume ratio of ficoll to blood. The diluted blood sample was then carefully layered onto the surface of the Ficoll medium to avoid mixing. The tube was centrifuged at 400 g for 30 minutes at 20° C. with maximum acceleration and minimum deceleration settings during centrifugation. After centrifugation, 4 interfaces were observed with layers of plasma, mononuclear cells, ficoll medium, and erythrocytes seen from top to bottom. Carefully removing the layer of mononuclear cells, carefully transferred to a new sterile centrifuge tube. Sterile PBS buffer was added to the collected PBMC for washing. The tube was centrifuged at 300×g for 10 minutes at 20° C. with maximum acceleration and minimum deceleration settings during centrifugation. Cells were resuspended in 10% FBS + RPMI 1640 for analysis. Mycoplasma-free Hep3b-hPDL1-OS8 cells were prepared in a 15 cm dish maintaining 60-80% confluence prior to use. Cells were trypsinized with TrypLETM Express (# 12605-036, Thermo Fisher, Waltham, MA), collected in a 50 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and cells were resuspended with mitomycin (# H33020786, Zhejiang, China) 10 μg/ml in 5-10 ml of 10% FBS + RPMI 1640 medium. Cells were incubated at 37°C for one hour. After incubation, the cells were washed in PBS, counting the cells with a cytometer. Cells were centrifuged at 1000 rpm for 5 minutes at 20°C. The cells were then resuspended in 10% FBS + RPMI 1640 for analysis. Stimulation of PBMCs was performed by adding PBMCs at 5E4 cells/well at 100 μl/well to a 96-well microplate. 50 µl/well of a series of 5X OD1 v2, 5X PD1 WT or hIgG4 protein solutions was added, followed by APC Hep3b-hPDL1-OS8 at 5000 cells/well at 50 µl/well. The mixed solution was incubated at 37°C for 72 hours. The supernatant was collected and placed at -20°C before the ELISA test. IFN-γ concentration was tested using an ELISA kit. IFN-γ was quantified using ELISA quantitation kits purchased from R&D. The assay procedure described in the human IFN-γ ELISA kit, R&D, cat. No. DY285. The plate was read using a plate reader for ELISA at a wavelength of 450 nm.

PD-L2 expression in lentivirus-transduced Hep3B-OS8 cells and MC38 cells

The Hep3B-OS8 (4E8) cell line was transduced with pLVX-IRES-hygro-hPDL2: homemade CP grown in 1640 medium. MC38 cells were transduced with pLVX-IRES-hygro-hPDL2. Cells were harvested according to standard procedures and the supernatant was discarded. Cells were dispensed into a 96-well round bottom staining plate: Corning, REF # 3799, at 3e5 cells per well. The tablet was placed in a centrifuge at 300 g at 4°C for 5 minutes. 100 μl of rabbit anti-hPD-L2 in 1xPBS + 2% FBS FACS titration buffer was added to each well and incubated for one hour at 4°C. Cells were washed twice with 200 μl of FACS buffer and centrifuged at 300 g for 5 minutes. The supernatant was removed before and after each wash. Cells were resuspended at 100 µl/well with a 1:1000 dilution of secondary donkey anti-rabbit IgG (H + L) conjugated to Alexa488 (life technology) and incubated for 1 hour at 4°C. Cells were again washed twice with 200 μl of FACS buffer and centrifuged at 300 g for 5 minutes. The supernatant was removed before and after each wash. Cells were resuspended in 100 µl PBS. Cells were stored in the dark and subjected to FACS analysis.

In vivo tumor studies

All animal experiments were reviewed and approved by the Stanford University Institutional Animal Care and Use Committee (IACUC) and the Animal Ethics Committee, ChemPartner, Shanghai. hPD-L1 knockout C57B/6 mice were purchased from Biocytogen (Beijing, China) and wild-type C57/B6 mice were purchased from Biocytogen (Beijing, China) or The Jackson Laboratory (Bar Harbor, Maine). PD-L1 knockout mice against a background of C57B/6 were kindly donated (by Dr. Dean W. Felsher of Stanford University). Female mice aged 6-8 weeks were used for ID8 ovarian tumor studies and male mice aged 6-8 weeks were used for MC38 colorectal studies and B16/OVA melanoma studies. Mice were maintained in pathogen-free animal housing at constant temperature and humidity and controlled 12-hour light-dark cycles. For the MC38, MC38-hPD-L1, MC38-hPD-L2 colorectal study and the B16/OVA melanoma study, 1 x 10 ⁶ cells were injected subcutaneously and randomly assigned to the control group or the corresponding treatment group after confirmation of tumor growth approximately 7 days after tumor inoculation. Tumor growth was measured throughout the study and body weight was recorded. Animals were sacrificed at the same time that most tumors had reached the ethical point. For the ID8 ovary study, 5 x 10 ⁶ cells were injected intraperitoneally and each animal was sacrificed when developing peritoneal ascites for survival analysis. For tumor studies conducted in PD-L1 knockout mice, 10 x 10 ⁶ ID8 cells were injected subcutaneously or intraperitoneally for two separate studies.

Statistical analysis

Pearson's correlation was used for all correlation analyzes of tumor samples. IHC H values were determined by a certified veterinary pathologist. All of tumor volume, survival, in vivo quantitative immunohistochemical evaluation were performed using Prism software (GraphPad Software Inc). Tukey-Kramer ANOVA was used to compare multiple treatment groups with each other. P<0.05 was considered significant. Repeated measures ANOVA was used to compare multiple treatment groups measured over time. Statistical analysis of survival curves was performed in the ID8 survival study. To compare mean survival among groups, a log-rank test (Mantel-Cox) was performed; P ≤ 0.05 was considered statistically significant.

Example 11 Neutralization of PD-L1 and PD-L2 to enhance the efficacy of immune checkpoint inhibitors

Screening of mutant clones against human PD-L1 and PD-L2

Yeast display libraries with 128 sPD-1 mutants, consisting of all possible combinations of permutations, originated from a high binding affinity sPD-1 mutant with 7 mutations (62-87-89-124-127-132-140 with position numbering starting from signal region) were isolated from the library using fluorescence-activated cell sorting (FACS). Equilibrium binding sorts were performed in which the yeast was incubated at room temperature in 1 mg/ml BSA phosphate buffered saline (PBSA) with the following nominal concentrations of PDL1-Fc and PD-L2-Fc. Clones with the highest binding were collected and sequenced for further analysis.

Characterization of selected clones

Fig. 38A illustrates the amino acid mutations present in the original PD-1 mutant clone, and all mutations were used to create a library of 128 sPD-1 mutant clones that includes all possible permutations of the 7 mutations. In FIG. 38B shows a list of the top 5 mutant clones selected from the 128 sPD-1 mutant clone library for further binding analysis. FIG. 39 shows dose-dependent binding curves of the original sPD-1 mutant and clones #1-5 to PD-L1, and FIG. 40 summarizes the Kd values of the original sPD-1 mutant and clones #1-5 binding to PD-L1. In FIG. 41 shows dose-dependent binding curves of the original sPD-1 mutant and clones #1-5 to PD-L2, and FIG. 42 summarizes the Kd values of the original sPD-1 mutant and clones #1-5 binding to PD-L2.

The above examples are provided to give those of ordinary skill in the art a full disclosure and description of how to make and use embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors consider their invention. Modifications of the methods of carrying out the invention described above that are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the description indicate the level of skill of specialists in the field to which the invention relates. All sources cited in this disclosure are incorporated by reference to the same extent as if each source were incorporated by reference entirely individually.

All section headings and designations are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from different headings and sections in accordance with the spirit and scope of the invention described herein.

All sources cited in this document are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application were specifically and individually designated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this application may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are provided by way of example only.

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SEQUENCE LIST

<110> THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR

UNIVERSITY

AKSO BIOPHARMACEUTICAL, INC.

<120> SPD-1 VARIANT - FC FUSION PROTEINS

<130> 1151248

<140> PCT/US2019/050742

<141> 2019-09-12

<150> 62/888.320

<151> 2019-08-16

<150> 62/731.488

<151> 2018-09-14

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 4

<211> 384

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 4

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly

145 150 155 160

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

165 170 175

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

180 185 190

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

195 200 205

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

210 215 220

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

225 230 235 240

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

245 250 255

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

260 265 270

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

275 280 285

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

290 295 300

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

305 310 315 320

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

325 330 335

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

340 345 350

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

355 360 365

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

370 375 380

<210> 5

<211> 384

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 5

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Ser

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly

145 150 155 160

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

165 170 175

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

180 185 190

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

195 200 205

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

210 215 220

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

225 230 235 240

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

245 250 255

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

260 265 270

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

275 280 285

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

290 295 300

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

305 310 315 320

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

325 330 335

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

340 345 350

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

355 360 365

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

370 375 380

<210> 6

<211> 384

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 6

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly

145 150 155 160

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

165 170 175

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

180 185 190

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

195 200 205

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

210 215 220

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

225 230 235 240

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

245 250 255

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

260 265 270

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

275 280 285

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

290 295 300

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

305 310 315 320

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

325 330 335

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

340 345 350

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

355 360 365

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

370 375 380

<210> 7

<211> 20

<212> PRT

<213> Homo sapiens

<400> 7

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg

twenty

<210> 8

<211> 404

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 8

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

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

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

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

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Gly Gly Gly Gly Ser Glu

165 170 175

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

180 185 190

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

195 200 205

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

210 215 220

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

225 230 235 240

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

245 250 255

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

260 265 270

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

275 280 285

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

290 295 300

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

305 310 315 320

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

325 330 335

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

340 345 350

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

355 360 365

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

370 375 380

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

385 390 395 400

Ser Leu Gly Lys

<210> 9

<211> 404

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 9

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

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

115 120 125

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

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Gly Gly Gly Gly Ser Glu

165 170 175

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

180 185 190

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

195 200 205

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

210 215 220

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

225 230 235 240

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

245 250 255

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

260 265 270

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

275 280 285

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

290 295 300

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

305 310 315 320

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

325 330 335

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

340 345 350

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

355 360 365

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

370 375 380

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

385 390 395 400

Ser Leu Gly Lys

<210> 10

<211> 404

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 10

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Val Leu

115 120 125

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

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Gly Gly Gly Gly Ser Glu

165 170 175

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

180 185 190

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

195 200 205

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

210 215 220

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

225 230 235 240

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

245 250 255

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

260 265 270

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

275 280 285

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

290 295 300

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

305 310 315 320

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

325 330 335

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

340 345 350

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

355 360 365

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

370 375 380

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

385 390 395 400

Ser Leu Gly Lys

<210> 11

<211> 150

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

PD1 sequence"

<400> 11

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 12

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 12

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 13

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 13

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 14

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 14

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 15

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 15

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 16

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 16

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 17

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 17

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 18

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 18

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 19

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 19

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 20

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 20

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 21

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 21

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 22

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 22

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 23

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 23

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 24

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 24

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 25

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 25

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 26

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 26

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 27

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 27

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 28

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 28

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 29

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 29

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 30

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 30

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 31

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 31

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 32

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 32

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 33

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 33

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 34

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 34

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 35

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 35

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 36

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 36

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 37

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 37

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 38

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 38

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 39

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 39

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 40

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 40

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 41

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 41

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 42

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 42

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 43

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 43

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 44

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 44

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 45

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 45

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 46

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 46

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 47

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 47

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 48

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 48

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 49

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 49

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 50

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 50

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 51

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 51

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 52

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 52

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 53

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 53

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 54

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 54

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 55

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 55

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 56

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 56

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 57

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 57

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 58

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 58

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 59

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 59

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 60

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 60

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 61

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 61

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 62

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 62

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 63

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 63

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 64

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 64

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 65

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 65

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 66

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 66

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 67

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 67

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 68

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 68

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 69

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 69

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 70

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 70

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 71

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 71

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 72

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 72

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 73

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 73

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 74

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 74

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 75

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 75

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 76

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 76

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 77

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 77

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 78

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 78

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 79

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 79

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 80

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 80

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 81

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 81

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 82

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 82

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 83

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 83

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 84

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 84

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 85

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 85

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 86

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 86

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 87

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 87

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 88

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 88

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 89

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 89

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 90

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 90

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 91

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 91

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 92

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 92

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 93

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 93

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 94

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 94

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 95

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 95

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 96

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 96

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 97

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 97

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 98

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 98

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 99

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 99

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 100

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 100

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 101

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 101

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 102

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 102

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 103

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 103

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 104

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 104

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 105

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 105

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 106

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 106

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 107

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 107

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 108

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 108

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 109

<211> 149

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 109

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Phe Val Leu Asn Trp Tyr Arg

35 40 45

Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp

50 55 60

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

65 70 75 80

Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp

85 90 95

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

100 105 110

Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg Ala

115 120 125

Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln

130 135 140

Phe Gln Thr Leu Val

145

<210> 110

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 110

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 111

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 111

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 112

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 112

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 113

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 113

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 114

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 114

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 115

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 115

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 116

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 116

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 117

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 117

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 118

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 118

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 119

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 119

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 120

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 120

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 121

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 121

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 122

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 122

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 123

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 123

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 124

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 124

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 125

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 125

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

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

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 126

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 126

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 127

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 127

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 128

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 128

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 129

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 129

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 130

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 130

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 131

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 131

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 132

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 132

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 133

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 133

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 134

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 134

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala Pro Lys Ile

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 135

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 135

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 136

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 136

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 137

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 137

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 138

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 138

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 139

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 139

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

1 5 10 15

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

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Ser

85 90 95

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

100 105 110

Gln Ile Lys Glu Ser Leu Arg Val Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 140

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> SITE

<222> (1)..(10)

<223> /note="This sequence may encompass 1-5 'Gly Ser'

repeating units"

<400> 140

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 141

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> SITE

<222> (1)..(25)

<223> /note="This sequence may encompass 1-5 'Gly Ser Gly Gly Ser'

repeating units"

<400> 141

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

1 5 10 15

Ser Gly Gly Ser Gly Ser Gly Gly Ser

20 25

<210> 142

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> SITE

<222> (1)..(25)

<223> /note="This sequence may encompass 1-5 'Gly Gly Gly Gly Ser'

repeating units"

<400> 142

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

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 143

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> SITE

<222> (1)..(20)

<223> /note="This sequence may encompass 1-5 'Gly Gly Gly Ser'

repeating units"

<400> 143

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

1 5 10 15

Gly Gly Gly Ser

twenty

<210> 144

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 144

Gly Gly Gly Gly Ser

fifteen

<210> 145

<211> 20

<212> PRT

<213> Homo sapiens

<400> 145

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro

twenty

<210> 146

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> source

<223> /note="See specification as filed for detailed description of

substitutions and preferred embodiments"

<400> 146

Gly Ser Gly Gly Ser

fifteen

<210> 147

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> source

<223> /note="See specification as filed for detailed description of

substitutions and preferred embodiments"

<400> 147

Gly Gly Gly Gly Ser

fifteen

<210> 148

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> source

<223> /note="See specification as filed for detailed description of

substitutions and preferred embodiments"

<400> 148

Gly Gly Gly Ser

one

<210> 149

<211> 175

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 149

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

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

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

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

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

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

165 170 175

<210> 150

<211> 175

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 150

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

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

115 120 125

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

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

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

165 170 175

<210> 151

<211> 175

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 151

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Gly Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Gly Gln Leu Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Val Leu

115 120 125

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

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

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

165 170 175

<210> 152

<211> 229

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 152

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 153

<211> 119

<212> PRT

<213> Homo sapiens

<400> 153

Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val

1 5 10 15

Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser

20 25 30

Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr

35 40 45

Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp

50 55 60

Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met

65 70 75 80

Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly

85 90 95

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

100 105 110

Glu Leu Arg Val Thr Glu Arg

115

<210> 154

<211> 118

<212> PRT

<213> Homo sapiens

<400> 154

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

1 5 10 15

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

20 25 30

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

35 40 45

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

50 55 60

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

65 70 75 80

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

85 90 95

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

100 105 110

Thr Glu Arg Arg Ala Glu

115

<210> 155

<211> 129

<212> PRT

<213> Homo sapiens

<400> 155

Gly Pro Gly Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe

1 5 10 15

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

20 25 30

Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe His Val Val Trp His Arg

35 40 45

Glu Ser Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala Phe Pro Glu Asp

50 55 60

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

65 70 75 80

Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp

85 90 95

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

100 105 110

Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Ala Ala

115 120 125

Ala

<210> 156

<211> 202

<212> PRT

<213> Homo sapiens

<400> 156

Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His

1 5 10 15

Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val

20 25 30

Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr

35 40 45

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

50 55 60

Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly

65 70 75 80

Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr

85 90 95

Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile

100 105 110

Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr

115 120 125

Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala

130 135 140

Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser

145 150 155 160

Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe

165 170 175

Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln

180 185 190

Ser Gln Met Glu Pro Arg Thr His Pro Thr

195 200

<210> 157

<211> 201

<212> PRT

<213> Mus sp.

<400> 157

Leu Phe Thr Val Thr Ala Pro Lys Glu Val Tyr Thr Val Asp Val Gly

1 5 10 15

Ser Ser Val Ser Leu Glu Cys Asp Phe Asp Phe Phe Glu Cys Thr Glu

20 25 30

Leu Glu Gly Ile Arg Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser

35 40 45

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

50 55 60

Lys Ala Leu Phe His Ile Pro Ser Val Gln Val Arg Asp Ser Gly Gln

65 70 75 80

Tyr Arg Cys Leu Val Ile Cys Gly Ala Ala Trp Asp Tyr Lys Tyr Leu

85 90 95

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

100 105 110

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

115 120 125

Tyr Pro Leu Ala Glu Val Ser Trp Gln Asn Val Ser Val Pro Ala Asn

130 135 140

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

145 150 155 160

Leu Arg Leu Lys Pro Gln Pro Ser Arg Asn Phe Ser Cys Met Phe Trp

165 170 175

Asn Ala His Met Lys Glu Leu Thr Ser Ala Ile Ile Asp Pro Leu Ser

180 185 190

Arg Met Glu Pro Lys Val Pro Arg Thr

195 200

<210> 158

<211> 202

<212> PRT

<213> Mus sp.

<400> 158

Met Leu Phe Thr Val Thr Ala Pro Lys Glu Val Tyr Thr Val Asp Val

1 5 10 15

Gly Ser Ser Val Ser Leu Glu Cys Asp Phe Asp Phe Phe Glu Cys Thr

20 25 30

Glu Leu Glu Gly Ile Arg Ala Ser Leu Gln Lys Val Glu Asn Asp Thr

35 40 45

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

50 55 60

Gly Lys Ala Leu Phe His Ile Pro Ser Val Gln Val Arg Asp Ser Gly

65 70 75 80

Gln Tyr Arg Cys Leu Val Ile Cys Gly Ala Ala Trp Asp Tyr Lys Tyr

85 90 95

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

100 105 110

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

115 120 125

Gly Tyr Pro Leu Ala Glu Val Ser Trp Gln Asn Val Ser Val Pro Ala

130 135 140

Asn Thr Ser His Ile Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser

145 150 155 160

Val Leu Arg Leu Lys Pro Gln Pro Ser Arg Asn Phe Ser Cys Met Phe

165 170 175

Trp Asn Ala His Met Lys Glu Leu Thr Ser Ala Ile Ile Asp Pro Leu

180 185 190

Ser Arg Met Glu Pro Lys Val Pro Arg Thr

195 200

<210> 159

<211> 29

<212> PRT

<213> Homo sapiens

<400> 159

Ala Leu Phe Thr Val Glu Leu Thr Ser Gln Thr Ser Pro His Gln Gln

1 5 10 15

Cys Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu

20 25

<210> 160

<211> 29

<212> PRT

<213> Mus sp.

<400> 160

Met Leu Phe Thr Ala Glu Val Arg Ser Gln Thr Ser Leu Gln Gln Arg

1 5 10 15

Cys Leu Ile Cys Gly Ala Ala Trp Asp Tyr Lys Tyr Leu

20 25

<210> 161

<211> 23

<212> PRT

<213> Homo sapiens

<400> 161

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro

twenty

<210> 162

<211> 19

<212> PRT

<213> Homo sapiens

<400> 162

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

Ala Gly Pro

<210> 163

<211> 70

<212> PRT

<213> Homo sapiens

<400> 163

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

35 40 45

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro

50 55 60

Glu Phe Leu Gly Gly Pro

65 70

<---

Claims (53)

1. An sPD-1-Fc variant fusion protein that binds to and antagonizes PD-1 ligands, containing: a) a soluble PD-1 variant (sPD-1) domain comprising an amino acid substitution compared to SEQ ID NO: 1, wherein said amino acid substitution is at a position selected from the group consisting of 120, 112, 107, 104, 67, 69, 96 and 42; and where the specified domain variant sPD-1 includes a set of amino acids G104S/S107V/A112I/A120V compared with SEQ ID NO: 1; b) an optional linker; and c) Fc domain. 2. An Fc fusion protein according to claim 1, wherein said Fc fusion protein comprises, from the N-terminus to the C-terminus: a) the specified sPD-1 variant domain; b) said optional linker; and c) the specified Fc domain. 3. The Fc fusion protein according to claim 1, wherein said Fc fusion protein comprises, from the N-terminus to the C-terminus: a) the specified Fc domain; b) said optional linker; and c) the specified sPD-1 variant domain. 4. Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain shows at least 95% identity with SEQ ID NO: 1. 5. An Fc fusion protein according to claim 1, wherein said sPD-1 variant domain has amino acid substitutions at five of said positions, six of said positions, seven of said positions, or eight of said positions. 6. An Fc fusion protein according to claim 1, wherein said sPD-1 variant domain further comprises one or more amino acid substitutions selected from the group consisting of: S67G, P69L, N96S, and S42G. 7. An Fc fusion protein according to claim 1, wherein said sPD-1 variant domain includes a set of amino acid substitutions selected from the group consisting of S42G/S67G/P69L/G104S/S107V/A112I/A120V, S42G/S67G/P69L/N96S/G104S/S107V/A112I/A120V, P69L/G104S/S107V/A112I/A120V, S67G/G104S/S107V/A112I/A120V, S67G/P69L/G104S/S107V/A112I/A120V, and G104S/S107V/A112I/A120V. 8. Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain includes a set of amino acid substitutions S42G/S67G/P69L/G104S/S107V/A112I/A120V. 9. Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain includes a set of S42G/S67G/P69L/N96S/G104S/S107V/A112I/A120V amino acid substitutions. 10. An Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain includes a set of amino acid substitutions P69L/G104S/S107V/A112I/A120V. 11. Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain includes a set of amino acid substitutions S67G/G104S/S107V/A112I/A120V. 12. An Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain includes a set of amino acid substitutions S67G/P69L/G104S/S107V/A112I/A120V. 13. Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain includes a set of G104S/S107V/A112I/A120V amino acid substitutions. 14. Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain has SEQ ID NO: 2. 15. Fc fusion protein according to claim 1, characterized in that said sPD-1 variant domain has SEQ ID NO: 3. 16. An Fc fusion protein according to claim 1, wherein said sPD-1 variant domain has SEQ ID NO: 110. 17. An Fc fusion protein according to claim 1, wherein said sPD-1 variant domain has SEQ ID NO: 130. 18. An Fc fusion protein according to claim 1, wherein said sPD-1 variant domain has SEQ ID NO: 131. 19. An Fc fusion protein according to claim 1, wherein said sPD-1 variant domain has SEQ ID NO: 138. 20. An Fc fusion protein according to claim 1, wherein said Fc domain is a human IgG Fc domain or a human IgG Fc variant domain. 21. An Fc fusion protein according to claim 20, wherein said human IgG Fc domain includes a human IgG4-CH2-CH3 hinge. 22. An Fc fusion protein according to claim 20, wherein said Fc domain is a human IgG Fc variant domain. 23. An Fc fusion protein according to claim 22, wherein said human IgG Fc variant domain comprises a human IgG4-CH2-CH3 hinge with the S228P amino acid substitution. 24. An Fc fusion protein according to any one of the preceding claims, wherein said linker is selected from the group consisting of (GS)n, SEQ ID NO: 146, SEQ ID NO: 147 and SEQ ID NO: 148, where n is selected from group consisting of 2, 3, 4 and 5. 25. An Fc fusion protein according to claim 24, wherein said linker comprises SEQ ID NO: 147. 26. An Fc fusion protein according to claim 1, wherein said Fc fusion protein comprises SEQ ID NO: 5. 27. An Fc fusion protein according to claim 1, wherein said Fc fusion protein comprises SEQ ID NO: 6. 28. A nucleic acid encoding said Fc fusion protein according to claim 1, containing a nucleic acid encoding an sPD-1-Fc variant fusion protein according to claim 1. 29. An expression vector comprising the specified nucleic acid according to claim 28. 30. Host cell for expression of the fusion protein of the sPD-1-Fc variant according to claim 1, containing the specified nucleic acid according to claim 28. 31. A method for producing a sPD-1-Fc variant fusion protein, comprising: a) culturing said host cell according to claim 30 under conditions under which said Fc fusion protein is expressed; and b) isolating said Fc fusion protein. 32. A method for treating, reducing or preventing metastasis or tumor invasion in a cancer subject, comprising administering to the subject a therapeutically effective dose of one or more of said Fc fusion proteins according to claim 1. 33. The method according to p. 32, characterized in that the cancer is selected from the group consisting of melanoma, glioma, lymphoma, myeloma, head and neck cancer, esophageal cancer, kidney cancer, lung cancer, breast cancer, liver cancer, colorectal cancer , gallbladder cancer, stomach cancer, pancreatic cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, and any other solid malignant tumor. 34. A method of treating an object with an infection, comprising administering to the object a therapeutically effective dose of one or more of the indicated Fc fusion proteins according to claim 1. 35. The method of claim 34, wherein the infection is selected from the group consisting of a fungal infection, a bacterial infection, and a viral infection. 36. The method according to claim 35, characterized in that the viral infection is selected from the group consisting of hepatitis B virus infection, hepatitis C virus infection, human papillomavirus infection, human immunodeficiency virus (HIV) infection, human T-lymphotrophic virus infection ( HTLV), Epstein-Barr virus infection, herpes virus infection, cytomegalovirus infection and any other chronic viral infection. 37. The method of claim 32, wherein an effective dose of one or more Fc fusion proteins inhibits, reduces, or modulates signaling mediated by wild-type PD-1 in the subject. 38. The method of claim 32 wherein an effective dose of one or more Fc fusion proteins increases the T cell response in the subject.

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