Multispecific ligand binding molecules and their applications

Abstract

The present invention relates to ligand-binding molecules that can bind to one or more growth factors including VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC, their glycosylation-engineered forms, fusion proteins and compositions containing such ligand-binding molecules, as well as methods for producing and using the same.

Description

Multispecific ligand binding molecules and their applications Technical Field

[0001] The present invention relates to the field of proteins, and in particular, to ligand-binding molecules, as well as glycosylation-engineered forms, fusion proteins and conjugates of such ligand-binding molecules, nucleic acid sequences encoding such molecules, and methods for producing and using the same. Background Art

[0002] The vascular endothelial growth factor (also known as VEGF)-related gene family includes multiple secreted glycoproteins, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. VEGF-A is a 45,000 Da glycoprotein with broad angiogenic activity. Its gene consists of 8 exons and 7 introns. It is divided into five monomers based on different splicing patterns: VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. VEGF165 is one of the most important homologous monomers of VEGF-A.

[0003] VEGF-A plays an important role in the early formation of blood vessels and is expressed by all vascularized tissues. In addition, many other cells can also express VEGF-A under conditions of hypoxic stress, such as macrophages, platelets, dendritic cells (DCs), activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells, and tumor cells.

[0004] VEGF-C and VEGF-D play a key role in lymphangiogenesis during the embryonic and postnatal stages. Homozygous VEGF-C deficiency in mice is lethal during the embryonic stage, and heterozygous deficiency results in defects in lymphatic vessel development in postnatal mice. VEGF-C and VEGF-D may also play an important role in angiogenesis, particularly during pathological stages, such as tumor growth. VEGF-E was isolated from a group infected with the scutellaria virus and stimulates endothelial cell proliferation and migration, as well as enhancing vascular permeability (Vladimir Joukov et al., The EMBO Journal 15(2):290-98, February 1996).

[0005] VEGF ligands mediate angiogenesis through a variety of different receptors (i.e., through ligand-receptor interactions). Two receptors were initially identified in endothelial cells: specific tyrosine kinase receptors (RTKs), VEGFR-1 (Fit-1) and VEGFR-2 (Fik-1). They were later confirmed to be expressed on a variety of adult hematopoietic cell lines. These two receptors consist of an extracellular domain with seven immunoglobulin-like structures, a membrane domain, and a tyrosine kinase domain. They are both transmembrane receptors and belong to the RTK type III class. Their common feature is the presence of a tyrosine kinase insert within the catalytic domain. The activity of this tyrosine kinase is activated by receptor-ligand binding. Receptor phosphorylation triggers numerous enzymatic and other reactions within the cell, playing a crucial role in cell growth and differentiation. Recently, another RTK, VEGFR3 (Fit-4), was identified and found to be closely associated with lymphangiogenesis. Different VEGF family members possess distinct receptor binding sites.

[0006] Inhibiting angiogenesis is an effective treatment for angiogenesis-related diseases, such as tumor growth and ophthalmic vascular proliferation. Over the past few decades, VEGF-related signaling has been extensively studied, and many drugs are used for treatment, including Lucentis (ranibizumab), Eylea (aflibercept), Avastin (bevacizumab), Cyramza (ramucirumab), Inlyta (axitinib), Stivarga (regorafenib), Cometriq (cabozantinib), OFEV (nintedanib), and Lenvim (envatinib).

[0007] Wet age-related macular degeneration (wAMD) and diabetic macular edema (DME) affect a wide range of patients, progress rapidly, and have severe consequences (visual loss), making them the leading causes of blindness. Currently, the main treatment option is to target VEGF-A, which offers significantly improved efficacy compared to traditional treatments (such as laser photocoagulation). Consequently, targeted drugs (such as ranibizumab and aflibercept) have also achieved significant market success. However, single-target VEGF-A therapy still cannot completely overcome these diseases. For example, after AMD treatment, only one-third of patients can fully benefit, the remaining patients have no significant improvement in vision, and one-third of patients are still close to blindness (Rofagha S, Bhisitkul RB, Boyer DS, Sadda SR, Zhang K. Seven-Year Outcomes in Ranibizumab-Treated Patients in ANCHOR, MARINA, and HORIZON: A multicenter cohort study (SEVEN UP) [J]. Ophthalmology, 2013, 120(11): 2292-2299). Currently, there are many drugs targeting VEGF-A that are on the market and in clinical use, with affinities (K D ) range from picomolar to nanomolar levels and have shown similar effects in clinical treatment, suggesting that single-target VEGF-A therapy may have reached its upper limit of efficacy.

[0008] In summary, while extensive research on VEGF-related signaling has been conducted over the past few decades, significant unmet clinical needs remain in diseases related to angiogenesis and tumors. The present invention addresses this need by providing novel molecules that target the VEGF, PlGF, and PDGF families, which have important clinical implications for the treatment of angiogenesis-related diseases. Summary of the Invention

[0009] The present invention relates to ligand-binding molecules (i.e., multispecific ligand-binding molecules) that can bind to one or more growth factors, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A, PDGF-B, and PDGF-C, as well as glycosylated versions thereof, compositions comprising such ligand-binding molecules, and methods for their production and uses. The present invention further demonstrates the in vivo and in vitro efficacy of such ligand-binding molecules, particularly their anti-angiogenic effects.

[0010] In some aspects, the present disclosure provides an isolated ligand-binding molecule comprising a VEGFR2-derived sequence that has enhanced binding to at least one, more preferably two, more preferably three, more preferably four, more preferably five, more preferably six, and most preferably seven of VEGF-A, VEGF-C, VEGF-D, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC compared to native VEGFR2 (e.g., native full VEGFR2 extracellular domain, e.g., extracellular sequence Met 1-Glu 764). The VEGFR2-derived sequence is an amino acid sequence having at least 95% identity to any of the following amino acid sequences:

[0011] (a) the amino acid sequence defined by positions 117-218 of SEQ ID NO: 2 (e.g., LFV-A);

[0012] (b) the amino acid sequence defined by positions 117-327 of SEQ ID NO: 2 (e.g., LFV-B);

[0013] (c) the amino acid sequence defined by positions 123-327 of SEQ ID NO: 2 (e.g., LFV-C, LFV-G, LFV-H, LFV-I, LFV-J, LFV-K, LFV-L);

[0014] (d) the amino acid sequence defined by positions 117-421 of SEQ ID NO: 2 (e.g., LFV-D);

[0015] (e) the amino acid sequence defined by positions 23-327 of SEQ ID NO: 2 (e.g., LFV-E); and

[0016] (f) the amino acid sequence defined by positions 23-421 of SEQ ID NO: 2 (eg, LFV-F).

[0017] In some embodiments, the VEGFR2-derived sequence is an N-terminal and / or C-terminal truncated fragment of any one of (a)-(f), for example, truncated by 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids or consisting thereof.

[0018] In some embodiments, the VEGFR2-derived sequence comprises one or more amino acid insertions, substitutions, or deletions compared to the sequence at corresponding positions in the native VEGFR2 extracellular domain, such as insertions, deletions, or substitutions at glycosylation sites.

[0019] In some embodiments, various modifications can be made at glycosylation sites to remove glycosylation, particularly eliminating known glycosylation sites such as the "NXT" and / or "NXS" triaminase, for example, by mutating N to Q or A. Specific glycosylation sites can be selected, for example, from amino acid positions 46, 66, 96, 143, 158, 245, and 318 corresponding to SEQ ID NO: 2. In some embodiments, the Asn at the above sites is substituted with Gln or Ala or other suitable amino acids. Specifically, substitutions at the glycosylation sites can be selected from the following: N143A, N143Q, N158A, N158Q, N245Q, and N318Q.

[0020] In some embodiments, the VEGFR2-derived sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID Nos: 4-15 and 18.

[0021] In some embodiments, the ligand binding molecule comprises a VEGFR1-derived sequence in addition to a VEGFR2-derived sequence. The VEGFR1-derived sequence may be selected from the group consisting of:

[0022] (i) an amino acid sequence that is at least 95% identical to the amino acid sequence shown at positions 132-230 of SEQ ID NO: 1; and

[0023] (ii) an amino acid sequence of (i) with the N-terminus and / or C-terminus truncated, for example, truncated by 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid.

[0024] In some embodiments, the ligand binding molecule comprises an amino acid sequence having at least 95% identity to the amino acid sequence shown at positions 117-327 or 123-327 of SEQ ID NO: 2, operably linked to the VEGFR1 derived sequence. For example, the ligand binding molecule comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID Nos: 16-17 and 19-21.

[0025] In some further embodiments, the ligand binding molecule comprises a VEGFR3-derived sequence in addition to a VEGFR2-derived sequence. The VEGFR3-derived sequence may be selected from the group consisting of:

[0026] (i) an amino acid sequence that is at least 95% identical to the amino acid sequence shown at positions 25-216, 25-115, 47-115, 154-210, 248-314, or 25-329 of SEQ ID NO: 3; and

[0027] (ii) an amino acid sequence of (i) with the N-terminus and / or C-terminus truncated, for example, truncated by 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid.

[0028] In some additional embodiments, the ligand binding molecule comprises an amino acid sequence having at least 95% identity to the amino acid sequence shown at positions 117-327, 123-327, or 221-312 of SEQ ID NO: 2, operably linked to the VEGFR3-derived sequence. For example, the ligand binding molecule comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID Nos: 22-30.

[0029] In some embodiments, the ligand binding molecule further comprises an Fc region of an immunoglobulin selected from IgA, IgM, IgE, IgD, and IgG, including IgG1, IgG2, IgG3, IgG4, preferably an Fc region of human IgG1 or a variant thereof, optionally further comprising a hinge region. The hinge region and Fc region of the immunoglobulin can be operably connected to the C-terminus of the VEGFR2-derived sequence, VEGFR1-derived sequence, or VEGFR3-derived sequence via a linker (e.g., GPG) or without a linker. In some embodiments, the Fc region or its variant is a polymeric form comprising macromolecular compounds such as phospholipids.

[0030] In some embodiments, the Fc region is as shown in the amino acid sequence of positions 104-330 of SEQ ID No:31.

[0031] In some specific embodiments, the ligand binding molecules disclosed herein comprise or consist of any one of SEQ ID Nos: 32-59.

[0032] In some embodiments, the ligand binding molecule further comprises a signal peptide.

[0033] In some embodiments, the ligand-binding molecule is capable of binding to at least one of the VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC proteins, preferably at least two, more preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six, more preferably at least seven, more preferably at least eight, and more preferably all nine. Preferably, the ligand-binding molecule is capable of binding to multiple targets, i.e., it is a multispecific ligand-binding molecule. The VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC proteins can be derived from or derived from humans, mice, rats, or cynomolgus monkeys. For example, the proteins can be human, including processed or unprocessed forms, and phosphorylated or non-phosphorylated forms.

[0034] In some embodiments, the ligand binding molecule can bind to a target protein with a K of 5 nM or less. D Binds to at least one of VEGF-A, VEGF-C, VEGF-D, PDGF-AB, preferably at least two, more preferably at least three, and most preferably all four, as determined by SPR.

[0035] In some embodiments, the ligand binding molecule can bind to the ligand at a K of 50 pM or less, preferably 40 pM or less, more preferably 30 pM or less, more preferably 20 pM or less, more preferably 10 pM, more preferably 5 pM, more preferably 4 pM, more preferably 3 pM, more preferably 1 pM, or less. D Binds to at least one of VEGF-A and VEGF-C as determined by SPR.

[0036] In some embodiments, the ligand binding molecule can bind to the ligand with a K of 20 nM or less, preferably 15 nM or less, more preferably 10 nM or less, more preferably 5 nM or less, more preferably 3 nM or less. D Binding to VEGF-D as determined by SPR.

[0037] In some embodiments, the ligand binding molecule can bind to the ligand with a K of 50 nM or less, preferably 40 nM or less, more preferably 30 nM or less, more preferably 28 nM or less, more preferably 26 nM or less, more preferably 24 nM or less, more preferably 22 nM or less, more preferably 20 nM or less, more preferably 18 nM or less, more preferably 15 nM or less, more preferably 10 nM or less. DBinding to at least one, preferably at least two, preferably at least three, more preferably all four of PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC as determined by SPR.

[0038] In some embodiments, the ligand binding molecule can inhibit or block the binding of at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC proteins to the corresponding receptor of the protein.

[0039] In some embodiments, the receptor is selected from VEGFR-1 receptor, VEGFR-2 receptor, VEGFR-3 receptor, PDGFR-α receptor and PDGFR-β receptor. The receptor can be in monomeric form, homodimeric form or any heterodimer form depending on its binding form when playing a role in physiology or pathology.

[0040] In some aspects, the present invention further provides a fusion protein comprising the ligand binding molecule disclosed above operably linked to a heterologous peptide. The heterologous peptide can be selected from the following: another ligand binding molecule, such as a common human receptor protein extracellular domain (ECD) or a truncated or recombinant polypeptide thereof; an antibody variable region fragment and a combination thereof, such as an antibody Fab, a single chain antibody (scFv), a nanobody, a human heavy chain variable region single domain antibody (V H ); active peptides and protein aptamers.

[0041] In some embodiments, the heterologous peptide is a heterologous peptide that targets VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD, Ang-2, TGF-β, CTGF (connective tissue growth factor), EGF (epidermal growth factor), S1P, Galectin, Decorin, or a receptor for the aforementioned protein.

[0042] In some embodiments, the operably linked is directly linked or linked via a linker.

[0043] In some embodiments, the ligand-binding molecule is directly linked to the N-terminus or C-terminus of the heterologous peptide or is linked via a linker. For example, the C-terminus is directly linked to the N-terminus of the heterologous peptide via a peptide bond, or the N-terminus is directly linked to the C-terminus of the heterologous peptide via a peptide bond. In some embodiments, the linker is a polypeptide or a chemical group.

[0044] In some aspects, the present invention also provides a conjugate comprising a ligand binding molecule or fusion protein as described above conjugated to at least one module selected from the following: a modifying module, a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), or a purification module. For example, the modifying module is a polyethylene glycol module, optionally attached to the amino terminus of the ligand binding molecule.

[0045] In some aspects, the present invention also provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding the ligand binding molecule or fusion protein. The nucleic acid molecule may further comprise a promoter sequence or other regulatory sequence connected to the polynucleotide sequence.

[0046] In some aspects, the present invention further provides a vector comprising the nucleic acid molecule. The vector may include, but is not limited to, a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, a liposome vector, and any combination thereof. In some embodiments, the vector is a replication-defective adenoviral vector, wherein the nucleic acid molecule is operably linked to a promoter and is flanked by adenoviral polynucleotide sequences.

[0047] In some aspects, the present invention further provides a host cell transformed or transfected with the polynucleotide or vector described above. The host cell is preferably a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a human cell.

[0048] In some aspects, the present invention also provides a method for preparing a ligand binding molecule, comprising the steps of:

[0049] The host cells described above are cultured under appropriate conditions to express the ligand-binding molecule; and the ligand-binding molecule is isolated from the culture supernatant of the host cells.

[0050] In some aspects, the present invention also provides a pharmaceutical composition comprising the ligand binding molecule or fusion protein as disclosed above or a nucleic acid molecule encoding the ligand binding molecule, and a pharmaceutically acceptable adjuvant or carrier, such as a diluent or excipient. The composition can be formulated for topical administration or intravitreal injection or implantation. The composition can be in the form of a solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion, or suspension.

[0051] In some aspects, the present invention also provides a method of inhibiting neovascularization in a subject and a disease or condition caused by neovascularization, the method comprising administering to the subject a ligand binding molecule or pharmaceutical composition as disclosed herein in an amount effective to inhibit neovascularization in the subject.

[0052] In some embodiments, the neovascularization is retinal neovascularization and / or choroidal neovascularization.

[0053] In some embodiments, the disease or condition is selected from choroidal vasculopathy or neovascularization, retinal angiogenesis, macular angiogenesis, and an ocular disorder associated with fundus leakage.

[0054] In some embodiments, the composition is administered topically to the subject's eye, or the composition is administered by intravitreal injection, by intravitreal implant, or by topical administration.

[0055] In some aspects, the present invention also provides uses of the ligand binding molecules or fusion proteins in the preparation of a medicament for treating neovascularization and diseases or conditions caused by neovascularization in a subject.

[0056] In some aspects, the present invention also provides the ligand binding molecules or fusion proteins for use in treating angiogenesis and diseases or conditions caused by angiogenesis in a subject.

[0057] In some aspects, the invention provides a kit comprising a ligand binding molecule or fusion protein or pharmaceutical composition as described herein.

[0058] The above is an overview and therefore contains simplifications, generalizations, and omissions of details as necessary; therefore, those skilled in the art will recognize that the overview is illustrative only and is not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and uses and / or other subject matter described herein will become apparent from the teachings presented herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG1 shows the ELISA results of various fusion protein ligand binding molecules derived from VEGFR2 and VEGFR3 binding to VEGF-A165 ( FIG1a ), VEGF-C ( FIG1b ), or VEGF-D ( FIG1c ).

[0060] FIG2 shows the ELISA results of binding of various ligand-binding molecules derived from VEGFR2 to VEGF-C.

[0061] Figures 3-4 show the blocking ELISA results of various ligand-binding molecules on the binding between VEGFR2-VEGF C (Figure 3a), VEGFR2-VEGF D (Figure 3b), VEGFR3-VEGF C (Figure 4a) and VEGFR3-VEGF D (Figure 4b), respectively.

[0062] FIG5 shows the results of ELISA of the binding of various ligand-binding molecules derived from VEGFR2 to VEGF-A165 ( FIG5a ) and VEGF-A121 ( FIG5b ).

[0063] FIG6 shows the blocking ELISA results of various ligand-binding molecules on the binding between VEGFR2-VEGF A165 ( FIG6 a ) and VEGFR2-VEGF-A121 ( FIG6 b ), respectively.

[0064] FIG7 shows the pharmacokinetic results of the ligand-binding molecule in mice.

[0065] Figure 8 shows the therapeutic effect of ligand-binding molecules in a rat CNV model.

[0066] FIG9 shows the blocking ELISA results of the ligand-binding molecules with glycosylation removed against VEGF-A 165-VEGFR2 ( FIG9a ), VEGFC-VEGFR2 ( FIG9b ), VEGF D-VEGFR2 ( FIG9c ), and VEGF D-VEGFR3 ( FIG9d ).

[0067] Figure 10 shows the blocking ELISA results of ligand binding molecules derived from VEGFR1 and VEGFR2 against VEGF-A165-VEGFR2 (Figure 10a), VEGF-A165-VEGFR1 (Figure 10b), VEGF C-VEGFR2 (Figure 10c), VEGF D-VEGFR2 (Figure 10d), and VEGF D-VEGFR3 (Figure 10e).

[0068] FIG11 shows the results of ELISA of the binding of ligand-binding molecules derived from VEGFR1 and VEGFR2 to VEGF-B ( FIG11 a ) and PlGF ( FIG11 b ).

[0069] FIG12 shows the blocking ELISA results of the ligand-binding molecules after fusion and glycosylation engineering on VEGF-A165-VEGFR2 ( FIG12a ), VEGF C-VEGFR2 ( FIG12b ), and VEGF D-VEGFR2 ( FIG12c ).

[0070] FIG13 shows the results of ELISA for the binding of LFV-Q to VEGF-B ( FIG13 a ) and PlGF ( FIG13 b ).

[0071] FIG14 shows the SEC single peak purity detection results after one-step purification of LFV-M before and after removal of glycosylation sites.

[0072] Figure 15 shows the blocking ELISA results of LFV-Q, LFV-S, LFV-M, and LFV-R molecules on VEGF-A165-VEGFR2 (Figure 15a), VEGF-A165-VEGFR1 (Figure 15b), VEGF C-VEGFR2 (Figure 15c), and VEGF D-VEGFR2 (Figure 15d).

[0073] FIG16 shows the results of ELISA for the binding of LFV-Q, LFV-S, LFV-M, and LFV-R to VEGF-B ( FIG16 a ) and PlGF ( FIG16 b ).

[0074] Figure 17 shows the ELISA binding efficacy of LFV-Q, LFV-B, LFV-R, and LFV-C to VEGF-A (Figure 17a), VEGF-C (Figure 17b), and VEGF-D (Figure 17c) compared to VEGFR2-Fc.

[0075] Figures 18a-18b show the experimental results of LFV-R and LFV-Q inhibiting the proliferation of HUVEC cells.

[0076] Figures 19a-19b show the experimental results of LFV-R and LFV-Q treatment of laser-induced choroidal neovascularization in rats. DETAILED DESCRIPTION

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, patent applications, and other publications cited herein are incorporated by reference in their entirety. In the event of a conflict between a definition set forth herein and a definition set forth in a patent, patent application, or other publication incorporated by reference herein, the definition set forth herein shall prevail.

[0078] As used herein, the term "VEGF" refers to the VEGF family, which consists of VEGF glycoproteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E). VEGF-A encompasses any form of VEGF-A, including the natural protein of VEGF-A (processed or unprocessed form), full-length VEGF-A, its fragments (e.g., truncated forms, extracellular / transmembrane domains) or variants (e.g., splice variants, allelic variants or artificially engineered variants) and modified forms thereof (e.g., glycosylation). There are five VEGF-A isoforms in the human body according to different shearing modes, namely VEGF121, VEGF145, VEGF165, VEGF189, VEGF206, of which VEGF165 is the most important homologous monomer of VEGF-A. Similarly, VEGF-B, VEGF-C, and VEGF-D also encompass their native proteins (including processed or unprocessed forms), full-length or fragments (e.g., truncated forms, extracellular / transmembrane domains), or variants (e.g., splice variants, allelic variants, or artificially engineered variants), as well as modified forms (e.g., glycosylated).

[0079] As used herein, the term "vascular endothelial growth factor receptor" or "VEGF receptor" includes VEGFR1, VEGFR2 and VEGFR3, which are members of the receptor tyrosine kinase family. The proteins of these three receptors contain a large extracellular region consisting of 6 or 7 immunoglobulin (Ig)-like domains (respectively referred to as D1-D7), a single transmembrane (TM) helix and a cytoplasmic region with tyrosine kinase activity and additional regulatory sequences. When referring to "VEGFR2", "VEGFR1", "VEGFR3", depending on the context, this article may refer to the full length or all or part of its extracellular domain. Examples of the full-length amino acid sequences of the natural proteins of human VEGFR2 and VEGFR3 are SEQ ID No: 2 and SEQ ID No: 3, respectively. All VEGF-A subtypes can bind to VEGFR1 and VEGFR2 simultaneously, VEGF-B specifically binds to and activates VEGFR-1, and the specificity of VEGF-E is related to VEGFR-2. VEGF-C and VEGF-D primarily exert their effects by binding to VEGFR3, but they can also bind to VEGFR2, though this binding is weaker than their binding to VEGFR3. VEGF-A can also bind to VEGFR2, but again, this binding is weaker than their binding to VEGFR1. Previously, because VEGFR2 can bind to multiple VEGF molecules, its specificity was considered inferior to that of VEGFR1. Therefore, the development of aflibercept was primarily based on VEGFR1, while VEGFR2, particularly its binding properties to VEGF-C and VEGF-D, has not received sufficient attention or research.

[0080] As used herein, the term "platelet-derived growth factor" or "PDGF" belongs to the vascular endothelial growth factor family and is a polypeptide growth factor containing sugar chains with a relative molecular weight of approximately 30 kD. PDGF is essentially a homodimeric molecule composed of four polypeptide chains, A, B, C, and D, formed by disulfide bonds. There are five isoforms: PDGF-AA, BB, AB, CC, and DD. PDGF was first discovered in the α-granules of platelets. It has now been demonstrated that diploid cells, such as monocytes, vascular smooth muscle cells, endothelial cells, embryonic cells, mesangial cells (MCs), and collecting duct cells, can produce PDGF. PDGF promotes pericyte migration, recruitment, and distribution. PDGF-B is an extremely important regulatory gene for vascular maturation and stabilization during development. PDGF-A, B, and C have certain effects on promoting lymphangiogenesis, with PDGF-B, in particular, being considered to have a strong effect on promoting lymphangiogenesis and having a certain relationship with angiogenesis. In addition, PDGF is the earliest discovered connective tissue growth factor, which can promote the growth and differentiation of endothelial cells, glial cells, fibroblasts and vascular smooth muscle cells, and has been clinically used in the repair treatment of trauma.

[0081] As used herein, the terms "PDGF-AA," "PDGF-AB," "PDGF-BB," and "PDGF-CC" refer to the dimeric forms AA, AB, BB, CC of PDGF, as well as fragments or variants thereof. These generally bind to and exert their effects on PDGF receptors as homodimers or heterodimers fixed by disulfide bonds. PDGF A and B chains can form both homodimers and heterodimers. PDGF C chain primarily exerts its effects as a homodimer. Experiments have shown that PDGF is an important mitogenic factor that has the ability to stimulate the division and proliferation of specific cell populations and plays an important role in pathological fibrosis of tissues.

[0082] The PDGF receptor is composed of two subunits, α and β, and exists in three dimer forms: αα, αβ, and ββ, all of which can bind to PDGF molecules. The α unit primarily recognizes the PDGF A, B, and C chains, allowing the αα receptor to bind to PDGF-AA, AB, BB, and CC. The β unit primarily recognizes the PDGF B chain, allowing the ββ receptor to primarily bind to PDGF-BB. The αβ receptor can bind to PDGF-AB and BB. Binding of PDGF to the PDGF receptor promotes receptor dimerization, which in turn transmits signals downstream, promoting cell proliferation and stimulating cell chemotaxis in autocrine and paracrine ways. Its primary effects are on cells such as vascular smooth muscle cells, fibroblasts, and glial cells.

[0083] As used herein, the term "operably linked" refers to the juxtaposition of two or more biological sequences of interest (with or without spacers, linkers, or intervening sequences) such that they are in a relationship that permits them to function in the intended manner. When used with respect to polypeptides, it refers to the connection of polypeptide sequences in a manner that permits the connected product to have the intended biological function. For example, a ligand-binding molecule can be operably linked to an immunoglobulin constant region to provide a stable product with ligand-binding activity. For another example, a ligand-binding molecule can be operably linked to an immunoglobulin constant region via an intervening sequence therebetween, and such an intervening sequence can be a spacer or can comprise a longer sequence.

[0084] As used herein, when used with respect to amino acid sequences (e.g., peptides, polypeptides, or proteins), the term "fusion" or "fused" refers to the combination of two or more amino acid sequences, for example, by chemical bonding or recombination, to form a single amino acid sequence that does not exist in nature. A fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct comprising the recombinant polynucleotide into a host cell.

[0085] As used herein, the term "isolated" refers to a state obtained from a natural state by artificial means. If a substance or component is "isolated" in nature, it may be because its natural environment has changed, or the substance has been separated from the natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide naturally exists in a living organism, and the same highly pure polynucleotide or polypeptide separated from the natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impure substances that do not affect the activity of the isolated substance.

[0086] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptides or polypeptide molecules or two or more nucleic acid molecules as determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0087] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as a ligand and a ligand-binding molecule. In certain embodiments, the ligand-binding molecules provided herein bind to a molecule with a specific binding affinity of ≤10 -6 M (e.g., ≤5x10 -7 M, ≤2x10 -7 M, ≤10 -7 M, ≤5x10 -8 M, ≤2x10 -8 M, ≤10 -8 M, ≤5x10 -9 M, ≤4x10 -9 M, ≤3x10 -9 M, ≤2x10 -9 M, or ≤10 -9The binding affinity (K D (or KD) specifically binds to human VEGF-A and / or human PDGF. As provided in the SPR experiments herein, K d is the dissociation constant, K a is the association constant, and K D is the equilibrium dissociation constant (K d / K a ). K D It can be determined by using any conventional method known in the art, including but not limited to surface plasmon resonance methods, microthermophoresis methods, HPLC-MS methods, and flow cytometry (eg, FACS) methods.

[0088] As used herein, the ability to "block binding" refers to the ability of a ligand binding molecule of the invention to inhibit the binding interaction between two molecules (e.g., VEGF-A, C, D and VEGFR2, VEGF-C, D and VEGFR3) to any detectable extent. In certain embodiments, the ligand binding molecule that blocks the binding between two molecules inhibits the binding interaction between the two molecules by at least 85% or at least 90%. In certain embodiments, the inhibition may be greater than 85%, or greater than 90%.

[0089] As used herein, the term "effective amount" refers to an amount of an active compound, or an amount of a material, composition, or dosage comprising an active compound, which, when administered according to a desired treatment regimen, is effective for producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, an effective amount herein may refer to an amount effective to inhibit neovascularization in a subject, an amount effective to inhibit at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC in a subject (e.g., in the eye), or an amount to stimulate at least one of the VEGFR-1, VEGFR-2, VEGFR-3, and PDGF receptor families expressed in ocular cells or blood vessels of the eye.

[0090] I. Ligand-binding molecules comprising an amino acid sequence derived from VEGFR2

[0091] In some aspects, the present invention provides an isolated ligand binding molecule that specifically binds to one or more VEGF / PDGF molecules. The VEGF / PDGF molecules can be selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC.

[0092] Preferably, the ligand binding molecule is capable of specifically binding to at least two of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC, more preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six, more preferably at least seven, and more preferably at least eight. Most preferably, the ligand binding molecule has specific binding affinity for VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC.

[0093] As used herein, the expression "specifically binds to" or "has specific binding affinity for" when referring to a growth factor encompasses specific binding not only to the free, active form of the growth factor, but also to other forms of the growth factor. For example, VEGF-A has multiple isoforms, some of which circulate and others are associated with heparin sulfate proteoglycans on the cell surface. A ligand binding molecule that specifically binds to VEGF-A binds to at least one circulating isoform, preferably all circulating isoforms, and more preferably to other isoforms. Thus, reference to a ligand binding molecule that specifically binds to VEGF-A encompasses its ability to specifically bind to VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A189, and VEGF-A206. As another example, VEGF-C is translated into a precursor molecule having extended amino-terminal and carboxyl-terminal propeptides, which are cleaved to produce the "fully processed" form of VEGF-C. A ligand binding molecule that specifically binds to VEGF-C encompasses binding to the fully processed form of VEGF-C, and preferably also encompasses binding to partially processed and unprocessed forms thereof.

[0094] In some embodiments, the ligand binding molecule comprises one or more ligands derived from native VEGFR2, and has a significantly improved binding affinity for one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC compared to the entire extracellular domain of native VEGFR2 or a control ligand binding molecule. For example, the ligand binding molecule has a significantly improved binding affinity for one or more of VEGF-A, VEGF-C, and VEGF-D compared to the entire extracellular domain of native VEGFR2 or a control ligand binding molecule.

[0095] Preferably, the VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC are from or derived from mammals such as humans, mice, monkeys, more preferably, they are natural VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC of humans, including various isoforms, partially processed or unprocessed precursor forms thereof.

[0096] In some embodiments, one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC are present as free soluble proteins or in an anchored form expressed on the cell surface.

[0097] The present invention also encompasses ligand-binding molecules capable of binding to abnormally overexpressed forms of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC, thereby promoting the restoration of normal function in the body through binding to these abnormally overexpressed growth factors. As is known in the art, in some pathological conditions, VEGF / PDGF / PlGF growth factors are overexpressed, causing these growth factors to exhibit abnormal functions in body tissues.

[0098] In some embodiments, the ligand binding molecules preferably have a K of about 1 nM or less (e.g., 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM, 5 pM, 1 pM or less). D Binds to human VEGF-A, and / or human VEGF-C. The ligand binding molecule preferably binds to human VEGF-A with a K of about 10 nM or less (e.g., 5 nM, 1 nM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM or less). D Binds to at least one of human VEGF-D, human PDGF-AB, and human PDGF-BB. The ligand binding molecule preferably has a K of about 50 nM or less (e.g., 30 nM, 10 nM, 5 nM, 1 nM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM or less). D Binds to human PDGF-AA and / or human PDGF-CC.

[0099] In some embodiments, the ligand binding molecule can bind to the ligand with a K of 50 pM or less, preferably 40 pM or less, more preferably 30 pM or less, more preferably 20 pM or less, more preferably 10 pM or less. D Binds to at least one of VEGF-A and VEGF-C as determined by SPR.

[0100] In some embodiments, the ligand binding molecule can bind to the ligand with a K of 20 nM or less, preferably 15 nM or less, more preferably 10 nM or less, more preferably 5 nM or less, more preferably 3 nM or less. D Binding to VEGF-D as determined by SPR.

[0101] In some embodiments, the ligand binding molecule can be used with an EC of 1 nM or less, preferably 0.5 nM or less, more preferably 0.4 nM or less, more preferably 0.3 nM or less, more preferably 0.2 nM or less. 50 Binds to VEGF-C; can bind to VEGF-C with an EC of 1 nM or less, preferably 0.5 nM or less, more preferably 0.4 nM or less, more preferably 0.3 nM or less, more preferably 0.2 nM or less, more preferably 0.1 nM or less 50 Binding to VEGF-A (eg, VEGF-A165) as determined by ELISA.

[0102] In some embodiments, the ligand binding molecule can bind to the ligand with a K of 50 nM or less, preferably 40 nM or less, more preferably 30 nM or less, more preferably 28 nM or less, more preferably 26 nM or less, more preferably 24 nM or less, more preferably 22 nM or less, more preferably 20 nM or less, more preferably 18 nM or less, more preferably 15 nM or less, more preferably 10 nM or less. D Binding to at least one, preferably at least two, preferably at least three, more preferably all four of PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC as determined by SPR.

[0103] In some embodiments, the ligand binding molecules disclosed herein are capable of blocking the binding between one or more growth factors selected from the group consisting of:

[0104] (a) VEGF-A and VEGFR-1, VEGFR-2 or PDGFR-α;

[0105] (b) VEGF-B, PlGF, and VEGFR-1

[0106] (c) VEGF-C and VEGFR-2 or VEGFR-3;

[0107] (d) VEGF-D and VEGFR-2 or VEGFR-3;

[0108] (e) PDGF-A and PDGFR-α;

[0109] (f) PDGF-B and PDGFR-α or PDGFR-β;

[0110] (g) PDGF-C and PDGFR-α;

[0111] (h) PDGF-D and PDGFR-β.

[0112] Optionally, the ligand binding molecule is capable of blocking the binding between one or more growth factors selected from the group consisting of:

[0113] (a) any of VEGF-A, B, C, and D and PDGFR-α or human PDGFR-β;

[0114] (b) Any one of PDGF-A, B, C, D and any one of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α and PDGFR-β.

[0115] As demonstrated in the Examples, the ligand-binding molecules disclosed herein can bind to VEGFR2 at lower EC values ​​than native VEGFR2 molecules or control ligand-binding molecules. 50 (eg, less than 1 nM, less than 0.5 nM, less than 0.4 nM) to block the binding of VEGF-C or VEGF-D to VEGFR-2 or VEGFR-3.

[0116] As known in the art, any one or more of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α and PDGFR-β can be activated by binding to VEGF / PDGF growth factors in the form of homodimerization, or can be activated by binding to VEGF / PDGF growth factors in the form of any heterodimerization. Taking human VEGFR-2 as an example, the dimerization forms formed accordingly include, but are not limited to, human VEGFR2-human VEGFR2, human VEGFR2-human VEGFR1, human VEGFR2-human VEGFR3, human VEGFR2-human PDGFRα, and human VEGFR2-human PDGFRβ dimers. Therefore, when referring to blocking the binding between a growth factor and a receptor in this article, it is intended to encompass blocking the binding between a growth factor and a receptor in various forms (e.g., monomers, homodimers, heterodimers, etc.).

[0117] In some embodiments, the ligand binding molecule comprises one or more amino acid sequences derived from VEGFR2 (particularly the extracellular region of VEGFR2), wherein the amino acid sequence derived from VEGFR2 is a fragment of VEGFR2 or an amino acid sequence having at least 80% identity thereto. Specifically, the fragment of VEGFR2 can be selected from the following:

[0118] (a) the amino acid sequence defined by positions 20 to 116 of SEQ ID NO: 2, or the amino acid sequence defined by positions 46 to 110 of SEQ ID NO: 2;

[0119] (b) the amino acid sequence defined by positions 117 to 218 of SEQ ID NO: 2, or the amino acid sequence defined by positions 141 to 207 of SEQ ID NO: 2;

[0120] (c) the amino acid sequence defined by positions 219 to 327 of SEQ ID NO: 2, or the amino acid sequence defined by positions 224 to 320 of SEQ ID NO: 2;

[0121] (d) the amino acid sequence defined by positions 328-421 of SEQ ID NO: 2, or the amino acid sequence defined by positions 328-414 of SEQ ID NO: 2; and

[0122] (e) an N-terminal or C-terminal truncated or extended fragment of (a), (b), (c) or (d) (e.g., truncated or extended by no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids).

[0123] The ligand binding molecule may comprise two or more amino acid sequences derived from the extracellular region of VEGFR2. In some embodiments, the amino acid sequence derived from the extracellular region of VEGFR2 is a fragment of the extracellular region of VEGFR2 (preferably human VEGFR2). Two or more amino acid sequences derived from the extracellular region of VEGFR2 may be directly linked to each other or linked via a spacer, or, in the case where the ligand binding molecule is a double-chain dimer, may be on two separate chains.

[0124] In some embodiments, the ligand binding molecule comprises an N-terminal truncated fragment of (b), such as the amino acid sequence defined at positions 123-218 of SEQ ID NO: 2. In some embodiments, the ligand molecule comprises both the amino acid sequences of (b) and (c), such as the amino acid sequence of (b) is directly linked to the amino acid sequence of (c), i.e., the amino acid sequence defined at positions 117-327 of SEQ ID NO: 2. In some embodiments, the ligand molecule comprises both an N-terminal truncated fragment of (b) and the amino acid sequence of (c), such as the N-terminal truncated fragment of (b) is directly linked to the amino acid sequence of (c), i.e., the amino acid sequence defined at positions 123-327 of SEQ ID NO: 2.

[0125] In some embodiments, the ligand binding molecules described herein comprise the amino acid sequence as set forth at positions 20-116 of SEQ ID NO:2, the amino acid sequence as set forth at positions 117-218 of SEQ ID NO:2, the amino acid sequence as set forth at positions 219-327 of SEQ ID NO:2, the amino acid sequence as set forth at positions 328-421 of SEQ ID NO:2, or an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the foregoing sequences.

[0126] In some embodiments, the ligand binding molecules described herein comprise the amino acid sequence as set forth at positions 117-327 of SEQ ID NO:2, the amino acid sequence as set forth at positions 23-327 of SEQ ID NO:2, the amino acid sequence as set forth at positions 117-421 of SEQ ID NO:2, the amino acid sequence as set forth at positions 23-421 of SEQ ID NO:2, or an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the foregoing sequences.

[0127] In some embodiments, the amino acid sequence with identity is the amino acid sequence as set forth at positions 20-116 of SEQ ID NO:2, the amino acid sequence as set forth at positions 117-218 of SEQ ID NO:2, the amino acid sequence as set forth at positions 219-327 of SEQ ID NO:2, the amino acid sequence as set forth at positions 328-421 of SEQ ID NO:2, the amino acid sequence as set forth at positions 117-327 of SEQ ID NO:2, the amino acid sequence as set forth at positions 23-327 of SEQ ID NO:2, the amino acid sequence as set forth at positions 117-421 of SEQ ID NO:2, or an N-terminal and / or C-terminal truncated and / or extended fragment of the amino acid sequence as set forth at positions 23-421 of SEQ ID NO:2. Preferably, the truncation or extension is no more than 40 amino acids, for example no more than 30 amino acids, no more than 20 amino acids, no more than 15 amino acids, no more than 10 amino acids, no more than 8 amino acids, no more than 6 amino acids, no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids or no more than 1 amino acid. In some specific embodiments, the ligand binding molecule described herein comprises the amino acid sequence as shown at positions 123-218 of SEQ ID NO: 2 or the amino acid sequence as shown at positions 123-327 of SEQ ID NO: 2.

[0128] In some embodiments, the identical amino acid sequence comprises one or more amino acid modifications, such as amino acid substitutions, insertions, deletions, etc., compared to the amino acid sequence set forth at positions 20-116 of SEQ ID NO: 2, the amino acid sequence set forth at positions 117-218 of SEQ ID NO: 2, the amino acid sequence set forth at positions 219-327 of SEQ ID NO: 2, the amino acid sequence set forth at positions 328-421 of SEQ ID NO: 2, the amino acid sequence set forth at positions 117-327 of SEQ ID NO: 2, the amino acid sequence set forth at positions 23-327 of SEQ ID NO: 2, the amino acid sequence set forth at positions 117-421 of SEQ ID NO: 2, or the amino acid sequence set forth at positions 23-421 of SEQ ID NO: 2. The amino acid modification can be a mutation at a glycosylation site to eliminate the glycosylation site, such as eliminating an N-glycosylation site or an O-glycosylation site. In some embodiments, the modification is a mutation at one or more of positions Asn46, Asn66, Asn96, Asn143, Asn158, Asn245, and Asn318 of SEQ ID NO:2.

[0129] II. Ligand-binding molecules comprising amino acid sequences derived from VEGFR2 and other receptors

[0130] In some aspects, the ligand binding molecules of the present invention comprise, in addition to the amino acid sequence derived from VEGFR2, amino acid sequences derived from other receptors for improved binding or blocking of at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC. The other receptors may be other receptor tyrosine kinases, such as VEGFR-1, VEGFR-3, PDGFR-α, and / or human PDGFR-β.

[0131] In some embodiments, the ligand binding molecules of the present disclosure comprise one or more amino acid sequences derived from VEGFR2 and one or more amino acid sequences derived from VEGFR3 (e.g., human VEGFR3, particularly the extracellular region of VEGFR3). Specifically, the amino acid sequence derived from VEGFR2 is operably linked to the amino acid sequence derived from VEGFR3. The operably linked sequences can be directly linked to each other or linked via a spacer.

[0132] In some embodiments, the amino acid sequence derived from VEGFR3 is a fragment of VEGFR3 or an amino acid sequence having at least 80% identity thereto. Specifically, the fragment of VEGFR3 can be selected from the following: the amino acid sequence defined by positions 25-115 of SEQ ID NO:3; the amino acid sequence defined by positions 154-210 of SEQ ID NO:3; the amino acid sequence defined by positions 248-314 of SEQ ID NO:3; the amino acid sequence defined by positions 25-210 of SEQ ID NO:3; the amino acid sequence defined by positions 154-314 of SEQ ID NO:3; the amino acid sequence defined by positions 25-314 of SEQ ID NO:3; and N-terminal or C-terminal truncations and / or extensions of any of the above (e.g., truncations or extensions of no more than 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids).

[0133] The ligand binding molecule may comprise two or more amino acid sequences derived from the extracellular region of VEGFR3 (preferably human VEGFR3). In some embodiments, the amino acid sequence derived from the extracellular region of VEGFR3 is a fragment of the extracellular region of human VEGFR3. In the case of comprising two or more amino acid sequences derived from the extracellular region of VEGFR3, the ligand binding molecule may be directly connected to each other or connected via a spacer, or, in the case where the ligand binding molecule is a double-chain dimer, may be on two separate chains.

[0134] In some embodiments, the ligand binding molecules described herein comprise the amino acid sequence as shown at positions 25-210 of SEQ ID NO:3, the amino acid sequence as shown at positions 25-314 of SEQ ID NO:3, or an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the above sequence.

[0135] In some embodiments, the amino acid sequence having identity is an N-terminal or C-terminal truncated fragment of the amino acid sequence as shown in positions 25-210 of SEQ ID NO:3 or the amino acid sequence as shown in positions 25-314 of SEQ ID NO:3. For example, the truncation is no more than 40 amino acids, such as no more than 30 amino acids, no more than 20 amino acids, no more than 15 amino acids, no more than 10 amino acids, no more than 8 amino acids, no more than 6 amino acids, no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid. In some specific embodiments, the ligand binding molecules described herein comprise the amino acid sequence as shown in the amino acid sequence as shown in positions 47-210 of SEQ ID NO:3.

[0136] In some embodiments, the identical amino acid sequence comprises one or more amino acid modifications, such as amino acid substitutions, insertions, deletions, etc., compared to the amino acid sequence shown at positions 25-210 of SEQ ID NO:3 or the amino acid sequence shown at positions 25-314 of SEQ ID NO:3.

[0137] As known in the art, the spacer can take various forms. In some embodiments, the spacer is a peptide sequence having a length between 1-100 amino acids, preferably 1-50 amino acids. In some embodiments, the spacer is derived from the connecting sequence at the corresponding position in natural VEGFR-2 or VEGFR-3 (i.e., the connecting sequence at the two ends of the sequence of the derived fragment). In some embodiments, the spacer comprises an amino acid sequence at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the natural peptide sequence or its portion connecting the fragment in natural human VEGFR-2 or human VEGFR-3.

[0138] Alternatively, the spacer may or may not be used and may be a commonly used linker sequence in the art, such as (G4S)n, AAA or other natural short peptide sequences. As will be appreciated by those skilled in the art, the spacer may have a wide range of amino acid sequences.

[0139] In some specific embodiments, the amino acid sequence derived from human VEGFR3 is linked to the amino acid sequence derived from human VEGFR2 via a spacer, wherein the amino acid sequence derived from human VEGFR3 is the amino acid sequence defined by positions 25-210 of SEQ ID NO: 3, and the amino acid sequence derived from human VEGFR2 is the amino acid sequence defined by positions 219-327 of SEQ ID NO: 2, and the spacer comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence defined by positions 211-231 of SEQ ID NO: 3 or positions 211-220 of SEQ ID NO: 2. The amino acid sequence derived from human VEGFR3 can be at the N-terminus or the C-terminus of the amino acid sequence derived from human VEGFR2.

[0140] In some embodiments, the ligand binding molecules of the present disclosure comprise one or more amino acid sequences derived from VEGFR2 and one or more amino acid sequences derived from VEGFR1 (e.g., human VEGFR1, particularly the extracellular region of VEGFR1). Specifically, the amino acid sequence derived from VEGFR2 is operably linked to an amino acid sequence derived from VEGFR3. The operably linked sequences may be directly linked to each other or linked via a spacer.

[0141] In some embodiments, the amino acid sequence derived from VEGFR1 is a fragment of VEGFR1 or an amino acid sequence having at least 80% identity thereto. Specifically, the fragment of VEGFR1 can be selected from the group consisting of the amino acid sequence defined by positions 132-230 of SEQ ID NO:1 and N-terminal and / or C-terminal truncated and / or extended fragments thereof (e.g., truncated or extended by no more than 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids).

[0142] In some embodiments, the identical amino acid sequence comprises one or more amino acid modifications, such as amino acid substitutions, insertions, deletions, etc., compared to the amino acid sequence shown as positions 132-230 of SEQ ID NO: 1.

[0143] In some aspects, the ligand binding molecule comprises one or more amino acid sequences derived from VEGFR2 and one or more amino acid sequences derived from VEGFR1. Specifically, the amino acid sequence derived from VEGFR2 is operably linked to the amino acid sequence derived from VEGFR1. The operably linked sequence can be directly connected to each other or connected via a spacer. In some embodiments, the amino acid sequence derived from VEGFR1 is a fragment of VEGFR1 or an amino acid sequence with at least 80% identity thereto. In some embodiments, the fragment of VEGFR1 is selected from the amino acid sequence shown in positions 132-230 of SEQ ID NO:1, or a truncated and / or extended fragment of its N-terminal and / or C-terminal ends.

[0144] III. Ligand binding molecules comprising an Fc region and optionally a signal peptide

[0145] The ligand binding molecules of the present invention can be monomers (i.e., single chains), dimers (i.e., double chains), or multimers. In dimeric or multimeric structures, the individual chains of the ligand binding molecules are covalently or non-covalently linked to each other. In some embodiments, the linkage occurs between the VEGFR-2 derived sequences of the ligand binding molecules.

[0146] In some embodiments, the ligand binding molecules provided by the present invention further comprise an immunoglobulin constant domain sequence, such as a constant domain sequence of human IgG, more specifically a hinge region and an Fc region, such as an Fc region sequence of IgG1, IgG2, IgG3, or IgG4. The immunoglobulin constant domain sequence can be connected to a sequence derived from VEGFR1 and / or VEGFR2 and / or VEGFR3 via a linker. As known in the art, Fc refers to the portion of an antibody consisting of the second and third constant regions of the first heavy chain of an antibody bound to the second and third constant regions of the second heavy chain via a disulfide bond, and optionally the Fc region further comprises all or part of the hinge region. The Fc region herein includes both wild-type Fc regions and variants thereof, with different mutations for various purposes. The variant may comprise one or more amino acid residue modifications, such as substitutions, in the Fc region.

[0147] In certain embodiments, the Fc region variant comprises one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such a variant can have an extended pharmacokinetic half-life because it binds to FcRn at acidic pH, thereby enabling it to escape degradation in lysosomes and then be transported and released from the cell. Methods of engineering antibody molecules to improve binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0148] In certain embodiments, the Fc region variant comprises one or more amino acid substitutions that alter antibody-dependent cellular cytotoxicity (ADCC), or alter complement-dependent cytotoxicity (CDC) by improving or reducing CIq binding and / or CDC.

[0149] In certain embodiments, the ligand binding molecules provided herein comprise a human IgG4 constant region in which amino acid residue 228 is altered, e.g., Ser228Pro (S228P, which can prevent or reduce chain exchange), and / or amino acid residue 235 is altered, e.g., Leu235Glu (L235E, which can alter Fc receptor interactions.

[0150] In certain embodiments, the ligand-binding molecules provided herein comprise one or more amino acid substitutions within the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include introducing a protrusion into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protrusion can be positioned within the cavity to facilitate interaction between the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating protein molecules with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0151] In certain embodiments, the ligand binding molecules provided herein comprise the native Fc sequence of human IgG1, as shown in Asp104-Lys330 of SEQ ID NO: 31. In some embodiments, the ligand binding molecules provided herein comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical, or 100% identical to the amino acid sequence at positions 104-330 of SEQ ID NO: 31.

[0152] In some embodiments, the ligand binding molecule further comprises a signal peptide at the amino terminus to facilitate secretion and purification in cells.

[0153] IV. Properties of ligand-binding molecules

[0154] The present invention has discovered ligand-binding molecules comprising one or more amino acid sequences derived from VEGFR2 (and / or amino acid sequences derived from other receptors) that have binding and blocking abilities for VEGF-A, VEGF-C, and VEGF-D, particularly VEGF-C and VEGF-D. In some embodiments, the ligand-binding molecules have significantly improved binding or blocking abilities for VEGF-A, VEGF-C, and VEGF-D, with the binding or blocking abilities for VEGF-A being superior to bevacizumab (Avastin) and similar to or superior to aflibercept (Eylea), and the binding or blocking abilities for VEGF-C and VEGF-D being significantly superior to the control substance OPT. The ligand-binding molecules can effectively inhibit angiogenesis by blocking molecules of the VEGF-A, VEGF-C, VEGF-D, and PDGF family, playing a role in the treatment of various diseases, particularly ophthalmic diseases or tumors.

[0155] The present invention found that the ligand binding molecule can block the binding of VEGF-A, VEGF-C, and VEGF-D to the receptor, and can also bind to the PDGF molecule. As verified in the examples:

[0156] 1) K of LFV-B binding to VEGF-C as determined by Biacore D 5.6 times stronger than the control OPT, LFV-E's K D Slightly better than OPT; in the VEGF-C blocking experiment, the EC of LFV-B 50 It is 1.4-2.2 times stronger than OPT (blocking experiments on VEGFR2 and VEGFR3 binding to VEGF-C, respectively). The EC of LFV-C is 50 1.3-2.4 times stronger than OPT, LFV-E's EC 50It is 1.57-2.2 times stronger than OPT, and the control drugs Avastin and Eylea cannot block it.

[0157] 2) K of LFV-B binding to VEGF-D as determined by Biacore D 7.8 times stronger than OPT, LFV-E's K D Slightly better than OPT; in the VEGF-D blocking experiment, the EC of LFV-B 50 It is 4.7-7.4 times stronger than OPT (blocking experiments on VEGFR2 and VEGFR3 binding to VEGF-D, respectively). The EC 50 4.6-5.9 times stronger than OPT, LFV-E's EC 50 It is 4.8-3.8 times stronger than OPT, and the control drugs Avastin and Eylea cannot block it.

[0158] 3) K of LFV-B binding to VEGF-A as determined by Biacore D was 9.3 pM, close to or similar to Eylea; in the VEGF-A 165-VEGFR2 blocking ELISA experiment, the EC 50 The EC of LFV-C is 4.67nM, which is between Avastin (6.68nM) and Eylea (3.2nM). 50 was 4.57 nM, close to that of LFV-B; in the VEGF-A 121-VEGFR2 blocking ELISA experiment, the EC 50 The EC of LFV-C is 7.63nM, which is better than Avastin (22.37nM) and Eylea (9.55nM). 50 It is 9.35nM, close to Eylea and better than Avastin.

[0159] 4) In in vivo ophthalmic experiments on AMD in mice, LFV-B and LFV-C also showed better improvement trends compared to the control drug Eylea.

[0160] Furthermore, in the present invention, novel ligand-binding molecules (e.g., LFV-M, LFV-N, LFV-Q, LFV-R, LFV-S) comprising one or more amino acid sequences derived from VEGFR2 operably linked to one or more amino acid sequences derived from VEGFR1 were discovered. In some embodiments, the ligand-binding molecules have further improved binding or blocking ability to VEGF-A, while retaining good binding or blocking ability to VEGF-C and VEGF-D, and having binding ability to VEGF-B and PlGF, as demonstrated in the Examples:

[0161] 1) In the VEGF-A 165-VEGFR2 blocking ELISA experiment, the EC values ​​of LFV-M and LFV-N were 50 The EC values ​​of LFV-M and LFV-N were 1.60 and 1.87 nM, respectively, which were better than the original molecule LFV-C (3.18 nM) that was not fused with VEGFR1, and also better than the control drugs Eylea (2.23 nM) and Avastin (4.58 nM). In the VEGF-A 165-VEGFR1 blocking ELISA experiment, the EC values ​​of LFV-M and LFV-N were 50 They are 3.14 and 3.30 nM respectively, which are better than Eylea (4.64 nM) and Avastin (7.31 nM).

[0162] 2) In the VEGF-C-VEGFR2 blocking ELISA experiment, the EC of LFV-M and LFV-N 50 They were 27.6 and 27.3 nM respectively, close to LFV-C (26.2 nM) and slightly better than the control OPT (32.1 nM).

[0163] 3) In the VEGF-D-VEGFR2 blocking ELISA experiment, the EC 50 The EC values ​​of LFV-M and LFV-N were 2.17 and 1.79 nM respectively, which were close to LFV-C (2.30 nM) and significantly better than the control OPT (24.59 nM). In the VEGF-D-VEGFR3 blocking ELISA experiment, the EC values ​​of LFV-M and LFV-N were 50 They were 2.74 and 2.31 nM respectively, close to LFV-C (2.62 nM), and significantly better than the control OPT (25.22 nM).

[0164] 4) In the VEGF-B and PlGF binding ELISA experiments, LFV-M and LFV-N also showed good binding ability, while LFV-C showed very weak binding.

[0165] Furthermore, the present invention found that after removing the N-glycosylation of LFV-M, the resulting molecule LFV-R has further optimized blocking efficacy against VEGF family molecules, as well as binding efficacy against VEGF-B and PlGF molecules, as verified in the Examples:

[0166] (1) In the VEGF A 165-VEGFR2 binding blocking ELISA experiment, the EC 50The EC of LFV-R was 1.901nM, which was better than 2.645nM of LFV-M and the control drug Eylea (3.937nM). In the VEGF A 165-VEGFR1 binding blocking ELISA experiment, the EC of LFV-R was 50 It is 6.972nM, which is better than LFV-M's 7.797nM and close to the control drug Eylea (6.959nM).

[0167] (2) In the VEGF D-VEGFR2 binding blocking ELISA experiment, the EC of LFV-R 50 It is 4.933nM, which is better than 5.623nM of LFV-M.

[0168] (3) In the VEGF-B binding experiment, the EC of LFV-R 50 It is 125.6ng / ml, which is better than LFV-M's 181.7ng / ml.

[0169] (4) In the PlGF binding experiment, the EC of LFV-R 50 It is 32.01ng / ml, which is better than 43.85ng / ml of LFV-M.

[0170] V. Polynucleotides, Vectors, and Host Cells

[0171] In some aspects, the present invention also provides polynucleotides encoding such ligand binding molecules. The polynucleotides can be used to express ligand binding molecules. In some embodiments, the polynucleotides can also be used as therapeutic agents in active form for achieving in vivo expression of polypeptide ligand binding molecules.

[0172] In certain embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding at least one of the following amino acid sequences and / or a nucleotide sequence having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and / or variants thereof having only degenerate substitutions:

[0173] (a) the amino acid sequence shown at positions 117-218 of SEQ ID NO: 2;

[0174] (b) the amino acid sequence shown at positions 117-327 or 123-327 of SEQ ID NO: 2;

[0175] (c) the amino acid sequence shown at positions 117-421 of SEQ ID NO: 2;

[0176] (d) the amino acid sequence shown at positions 23 to 327 of SEQ ID NO: 2; and

[0177] (e) The amino acid sequence shown at positions 23 to 421 of SEQ ID NO: 2.

[0178] Optionally, the polynucleotide further comprises a nucleotide sequence encoding at least one of the following amino acid sequences and / or a nucleotide sequence having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto, and / or variants thereof having only degenerate substitutions:

[0179] (i) the amino acid sequence shown at positions 25-115 or 47-115 of SEQ ID NO: 3;

[0180] (ii) the amino acid sequence shown at positions 154 to 210 of SEQ ID NO: 3;

[0181] (iii) the amino acid sequence shown at positions 248 to 314 of SEQ ID NO: 3; and

[0182] (iv) The amino acid sequence shown at positions 132 to 230 of SEQ ID NO: 1.

[0183] The polynucleotides encoding the ligand binding molecules are readily isolated and sequenced using conventional procedures known in the art. The polynucleotides may also be obtained synthetically. Preferably, the polynucleotides are codon-optimized for expression in eukaryotic host cells, particularly mammalian cells.

[0184] Using known recombinant techniques, the polynucleotide encoding the ligand binding molecule can be inserted into a vector for further cloning (amplification of the DNA) or expression. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0185] In some embodiments, the present disclosure provides a vector (e.g., an expression vector) comprising a polynucleotide encoding a ligand binding molecule as provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the polynucleotide, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, liposomes, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, p FUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2. 2. pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0186] The vector that comprises the polynucleotide sequence of coding ligand binding molecule can be imported into host cell for cloning or gene expression.For cloning or suitable host cell of expression DNA in the vector herein is prokaryotic organism, yeast or higher eukaryotic cell.For this purpose suitable prokaryotic organism comprises true bacteria, for example gram-negative or gram-positive bacteria, such as escherichia coli.Except prokaryotic organism, eukaryotic microorganism such as filamentous fungi or yeast is suitable clone or expression host for the vector that provides.Saccharomyces cerevisiae or common baker's yeast are most commonly used in lower eukaryotic host microorganisms.Yet many other genera, species and bacterial strains are generally obtainable and useful in this article.

[0187] Suitable host cells for expressing the ligand binding molecules provided herein can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants from hosts and corresponding permitted insect host cells have been identified. However, vertebrate cells are of greatest interest, and the propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells grown in suspension culture as subclones, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In some preferred embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some other preferred embodiments, the host cell is other mammalian cell lines, such as human cell lines.

[0188] Host cells are transformed with the above-described expression or cloning vectors for production of the ligand binding molecules and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the gene encoding the desired sequence.

[0189] Host cells for producing the ligand binding molecules provided herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), minimal essential medium (MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's modified Eagle's medium (DMEM), Sigma) are suitable for culturing host cells. In addition, Ham et al., Meth.Enz.58:44 (1979), Barnes et al., Anal.Biochem.102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or any culture medium described in US Pat. Re. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds generally present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to those of ordinary skill in the art.

[0190] In some embodiments, the ligand binding molecule is expressed in a culture medium containing 500 ng of ligand binding molecules. The ... When using recombinant technology, the ligand binding molecule can be produced in the cell, in the periplasmic space, or directly secreted into the culture medium. If the ligand binding molecule is produced in the cell, the first step is, for example, by centrifugal or ultrafiltration to remove the particle debris of the host cell or cleavage fragment. Cell debris can be removed by centrifugation. When the ligand binding molecule is secreted into the culture medium, usually at first use a commercially available protein concentration filter, for example Amicon or MilliporePellicon ultrafiltration unit, to concentrate the supernatant from this expression system. Protease inhibitors such as PMSF can be included in any of the above-mentioned steps to inhibit proteolysis, and can include antibiotic to prevent the growth of adventitious pollutants.

[0191] Ligand-binding molecules produced by cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0192] In certain embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of ligand-binding molecules. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the ligand-binding molecule. Protein A can be used for purifying antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J.Immunol.Meth.62:1-13 (1983)). The matrix to which the affinity ligand is attached is most typically agarose, but other matrices can also be used. Mechanically stable matrices (such as controlled pore glass or poly (styrene divinyl) benzene) allow faster flow rates and shorter processing times than achieved by agarose. Other protein purification techniques, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica gel, on heparin SEPHAROSE TM Chromatography on anion or cation exchange resins (eg, polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation may also be used, depending on the antibody to be recovered.

[0193] VI. Conjugates

[0194] In some aspects, the present invention also provides a conjugate comprising a ligand binding molecule and a conjugate module conjugated thereto. A conjugate module is a portion that can be attached to a ligand binding molecule. It is encompassed that a variety of conjugate modules can be connected to the ligand binding molecules provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate modules can be connected to the ligand binding molecules by methods such as covalent binding, affinity binding, insertion (intercalation), coordination binding, complexing, association, blending or addition.

[0195] In certain embodiments, the ligand-binding molecules disclosed herein can be engineered to include specific sites outside of the ligand-binding portion that can be used to bind one or more conjugate moieties. For example, such sites can include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent attachment to the conjugate moiety.

[0196] In certain embodiments, the conjugate includes a conjugate module connected to the ligand binding molecule. In some other embodiments, the ligand binding molecule as described herein is directly attached to the N-terminal amino acid of the conjugate module by a peptide bond at the C-terminus, or is directly attached to the C-terminal amino acid of the conjugate module by a peptide bond at the N-terminus. It is also possible to form a polypeptide chain by chemical bonds, including but not limited to amide bonds and conjugate modules. In certain embodiments, the ligand binding molecule can be connected to the first conjugate module indirectly or by the second conjugate module. For example, the ligand binding molecule can be conjugated to biotin and then indirectly conjugated to the second conjugate module conjugated to avidin. The conjugate module can be a clearance modification module, a toxin (such as a chemotherapeutic agent), a detectable label (such as a radioisotope, a lanthanide, a luminescent label, a fluorescent label or an enzyme-substrate label) or a purification module.

[0197] "Toxin" can be any agent that is harmful to cells or can damage or kill cells. Examples of toxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarboxyzine), alkane alkylating agents (e.g., methylethylamine, thiabendazole chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., actinomycin (formerly dactinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0198] Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioactive isotopes (e.g., 123 I. 124 I. 125 I. 131 I. 35 S. 3 H.111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y. 88 Y. 90 Y. 177 Lu, 211 At 186 Re、 188 Re、 153 Sm, 212 Bihe 32 P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection.

[0199] In certain embodiments, the conjugate module can be a removal modifier, which helps increase the half-life of the ligand binding molecule. Illustrative examples include water-soluble polymers, such as copolymers of PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol, etc. Polymer can have any molecular weight and can be side chain or non-side chain. The quantity of the polymer attached to the ligand binding molecule can vary, and if attached to more than one polymer, they can be identical or different molecules. In some specific embodiments, the ligand binding molecule provided by the invention is conjugated to at least one polyethylene glycol module, and the polyethylene glycol module can be attached to the amino terminal of the ligand binding molecule.

[0200] In certain embodiments, the conjugate moiety can be a purification moiety, such as a magnetic bead.

[0201] VII. Pharmaceutical Compositions

[0202] In some aspects, the present invention provides compositions, such as pharmaceutical compositions, comprising a ligand binding molecule of the present invention formulated with a pharmaceutically acceptable carrier. Such compositions comprise at least one ligand binding molecule, fusion protein, or conjugate of the present invention. In some embodiments, the pharmaceutical composition is a formulation suitable for intravitreal injection.

[0203] As used herein, "pharmaceutically acceptable carrier" includes any and all pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances that are physiologically compatible, other components known in the art, or various combinations thereof, etc. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, transdermal, intravitreal injection or implantation administration, etc. Depending on the route of administration, the active compound, i.e., the ingredient of the present invention, can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0204] The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0205] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants.

[0206] These compositions may also include adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Sterilization methods and the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.) may ensure the presence of microorganisms. Including isotonic agents such as sugars, sodium chloride, etc. into the compositions may also be desirable. In addition, by including substances that delay absorption, such as aluminum monostearate and gelatin, the absorption of the injectable form may be prolonged.

[0207] The pharmaceutical composition may be in the form of a solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion or suspension.

[0208] In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions or solid forms suitable for producing liquid solutions, suspensions or emulsions. The preparation for injection can include sterile and / or non-pyrolysis solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be mixed with solvents before use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle before use, and sterile and / or non-pyrolysis emulsions. The solution can be aqueous or non-aqueous.

[0209] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Such media and agents for pharmaceutically active substances are known in the art. Unless any conventional media or agents are incompatible with the active compound, use of any conventional media or agents in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds may also be added to the compositions.

[0210] Pharmaceutical compositions must be sterile and stable under the conditions of manufacture and storage usually. The composition can be formulated as a solution, microemulsion, liposome or other ordered structures suitable for high drug concentration. The carrier can be a solvent or dispersion medium, which comprises, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by utilizing a coating (such as lecithin), in the case of a dispersant, by maintaining the required particle size and by utilizing a surfactant, suitable fluidity can be maintained. In many cases, it is preferred to include isotonic agents such as sugar, polyols (such as mannitol, sorbitol) or sodium chloride in the composition. By including the material of delayed absorption (such as monostearate and gelatin) in the composition, the absorption of the injectable composition can be extended.

[0211] Sterile injection solutions can be prepared by adding the active compound in the amount required with one of the components listed above as needed, or a combination thereof, to a suitable solvent and then aseptically microfiltering. Typically, dispersions can be prepared by adding the active compound to a sterile carrier comprising a basic dispersion medium and other required components from the material listed above. In the case of sterile powders for the preparation of sterile injections, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional required components from a previously sterile-filtered solution.

[0212] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form depends on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier to produce a single dosage form is generally that amount of the composition that produces a therapeutic effect. Generally, based on 100%, this amount ranges from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0213] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dosage may be adjusted based on the dose.

[0214] The dosage may be proportionally reduced or increased as required by the exigencies of the therapeutic situation. It is particularly advantageous to formulate the pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound appropriate to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are dictated by and directly dependent on: (a) the unique properties of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for treating individual sensitivities.

[0215] For administration of the ligand binding molecule, dosage ranges are about 0.0001 to 100 mg / kg of host body weight, and more typically 0.01 to 5 mg / kg. For example, dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or within the range of 1-10 mg / kg. Exemplary treatment regimens include administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0216] Alternatively, the ligand binding molecule can be used as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary with the half-life of the substance administered in the patient. The dosage and frequency of administration may depend on whether the treatment is preventive or therapeutic. In preventive applications, administration is performed at relatively infrequent intervals for a long time with relatively low dosages. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively short time intervals and relatively high dosages are sometimes required until the progression of the disease is slowed or terminated, and preferably until the patient demonstrates partial or complete improvement of symptoms of the disease. Thereafter, a preventive regimen may be given to the patient.

[0217] Actual dosage levels of the active ingredients and small molecules in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend upon various pharmacokinetic factors, including the activity of the particular combination of the present invention employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in conjunction with the particular combination employed, the age, sex, weight, condition, general health, and prior medical history of the patient to be treated, and similar factors well known in the medical arts.

[0218] VIII. Methods of Using Ligand Binding Molecules

[0219] In some aspects, the present invention provides a method of inhibiting, preventing or treating neovascularization in a subject and a disease or condition caused by neovascularization, the method comprising administering to the subject a ligand binding molecule, a conjugate thereof or a pharmaceutical composition as described herein. The neovascularization includes retinal neovascularization and / or choroidal neovascularization; the disease or condition includes polypoidal choroidal vasculopathy, retinal neovascularization, and an ocular disorder associated with fundus leakage.

[0220] In some embodiments, the eye condition is selected from age-related macular degeneration, diabetic eye conditions, polypoidal choroidal vasculopathy, fundus fibrosis, retinal vein occlusion-related lesions, retinopathy of prematurity, and macular telangiectasia. Preferably, the pharmaceutical composition comprising the ligand binding molecule described herein is topically administered to the subject's eye by eye drops, intravitreal injection, or intravitreal implant.

[0221] In some embodiments, the ligand binding molecules, conjugates thereof, or pharmaceutical compositions disclosed herein can be administered in a single administration or multiple administrations. Preferably, the desired therapeutic effect can be achieved by a single administration without the need for administration with other targeted therapeutic drugs.

[0222] In some embodiments, the ligand-binding molecules, conjugates thereof, or pharmaceutical compositions disclosed herein can be administered alone or in combination with one or more additional therapeutic means or agents. For example, the ligand-binding molecules, conjugates thereof, or pharmaceutical compositions disclosed herein can be administered in combination with additional therapeutic agents, such as chemotherapeutic agents, or other therapeutic methods. For example, in the treatment of ocular diseases, other therapeutic methods include laser photocoagulation, vitrectomy, and the like.

[0223] In some of these embodiments, the ligand binding molecules, conjugates or pharmaceutical compositions disclosed herein that are administered in combination with one or more additional therapeutic agents can be administered simultaneously or sequentially or alternately with one or more additional therapeutic agents. A ligand binding molecule administered "in combination" with another therapeutic agent need not be administered simultaneously with the agent or in the same composition. Even in the case where the ligand binding molecule and the second agent are administered via different routes, the phrases used in this article also consider that the ligand binding molecule administered before or after another agent is administered "in combination" with the agent. Where possible, the additional therapeutic agent administered in combination with the ligand binding molecules disclosed herein is administered according to the schedule listed in the additional therapeutic agent product information sheet or according to Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)) or a regimen well known in the art.

[0224] In some aspects, the present invention also provides a kit comprising the ligand-binding molecule, its conjugate or pharmaceutical composition.

[0225] IX. Advantageous Effects of the Invention

[0226] In angiogenesis-related diseases such as tumor growth and ophthalmic angiogenesis, inhibiting angiogenesis is an effective treatment. In the past few decades, a large number of studies have been conducted on VEGF-related signals, and many drugs have been used for treatment. In order to break through the limitations of single-target therapy, simultaneous targeting of PDGF-BB and VEGF-A has been highly anticipated. Inhibiting PDGF-BB can cause pericyte apoptosis, thereby breaking the resistance to anti-VEGF-A treatment. In addition, PDGF-BB is closely related to tissue fibrosis. Fundus fibrosis is the late development form of AMD and the cause of blindness. It is a major reason why VEGF-A therapy ultimately fails (Rofagha S. et al. (2013), J. Ophthalmology. 120(11): 2292-2299) (Rosenfeld PJ et al. (2011), Ophthalmology. 118(3): 523-530). Among the PDGF-targeted therapies, the more eye-catching ones are the PDGF-BB antagonist E10030 introduced by Novartis and the bispecific antibody ABBV642 developed by AbbVie. The former achieved more effective therapeutic effects than the single use of ranibizumab in phase I and II clinical trials for AMD, and had a significant improvement effect on fundus fibrosis. Although E10030 did not show a comparative advantage over the single targeted VEGF-A therapy in the larger-scale phase III clinical trial, due to its effective intervention effect on fibrosis, its research on the treatment of fibrosis is still ongoing.

[0227] Another company, Opthea, has developed a product, OPT-302, which can simultaneously target VEGF-C and VEGF-D. It is injected in combination with a product targeting VEGF-A. In clinical trials, it has clearly demonstrated significantly better efficacy and a wider response than targeting VEGF-A alone, thus bringing the treatment of diseases such as nAMD into the second generation. Moreover, this therapy also has a good effect on patients with polypoidal choroidal vasculopathy (PCV), which is common in Asians. PCV is a subtype of wet macular degeneration, which is different from wet macular degeneration. It has no effective and extensive response to targeted VEGF-A therapy and is highly prevalent in Asia (Imamura Y et al. (2010), Surv. Ophthalmol. 55(6):501-515). In my country, the proportion of PCV among patients newly diagnosed with AMD is as high as 33% (Lande A. et al. (2010), Prog Retin Eye Res. 29(1):19-29), and in Japan it is 54.7% (Maruko I. et al. (2007), Am JOphthalmol. 144:15-22). Due to the large population base in Asia, there is currently a huge treatment gap for PCV.

[0228] However, this therapy is a combination treatment that requires the simultaneous intravitreal injection of two drugs, namely a VEGF-A-targeting drug and OPT-302, resulting in high treatment costs. Furthermore, this therapy requires monthly transocular injections, which not only increases treatment costs but is also inconvenient, leads to poor patient compliance, and increases the risk of accidents such as injection-induced intraocular infection. Furthermore, this therapy requires simultaneous targeting of VEGF-A, VEGF-C, and VEGF-D, requiring a high affinity for all of them (at or below the 10pM level), which hinders the development of alternatives using the currently popular monoclonal antibody technology (one antibody generally binds to only one target, with an affinity generally around 100pM).

[0229] The inventors unexpectedly discovered that ligand-binding molecules containing specific amino acid sequences derived from VEGFR2 (e.g., positions 123-327 or 117-327 of SEQ ID NO:2) have superior binding to VEGF-C or VEGF-D compared to OPT. To further investigate this, they constructed and expressed various VEGF Trap molecules containing different fragments from VEGFR2, and their binding and blocking abilities to VEGF-A, VEGF-C, VEGF-D, and others were studied and characterized in detail.

[0230] The results showed that ligand-binding molecules containing amino acid sequences derived from VEGFR2, particularly the amino acid sequences shown at positions 123-327 or 117-327 of SEQ ID NO:2, exhibited significantly better binding and blocking abilities to VEGF-C or VEGF-D than OPT or the entire VEGFR2 extracellular domain, and also had significantly better binding and blocking abilities to VEGF-A.

[0231] At the same time, the inventors also unexpectedly characterized that ligand-binding molecules containing amino acid sequences derived from VEGFR2, particularly the amino acid sequences shown at positions 117-327 or 123-327 of SEQ ID NO: 2, also have a certain binding ability to PDGF family growth factors such as PDGF-AA, AB, BB, and CC, and are the main structural domains involved in binding, and are superior to the binding of recombinant molecules containing the entire extracellular domain of natural VEGFR2 to PDGF. Since high expression of PDGF family molecules is believed to be one of the reasons for insufficient response to targeted VEGF-A therapy, resistance, and post-treatment fundus fibrosis, simultaneously targeting the PDGF family may benefit more patients, especially for fundus lesions caused by diabetes. Literature reports that PDGF-AA, PDGF-AB, and PDGF-BB are highly expressed in such fundus lesions, and therefore have the potential to treat fundus lesions caused by diabetes.

[0232] In addition, compared to OPT molecules, ligand-binding molecules comprising an amino acid sequence derived from VEGFR2, particularly the amino acid sequence shown at positions 117-327 or 123-327 of SEQ ID NO:2, have significantly better initial monomer purity and final yield during expression and purification in HEK293 and CHO cells, which means that they can have lower production costs and drug costs.

[0233] In addition, novel ligand-binding molecules comprising one or more amino acid sequences derived from VEGFR2 operably linked to one or more amino acid sequences derived from VEGFR1 and glycosylated versions thereof (e.g., LFV-M, LFV-N, LFV-Q, LFV-R (LFV-R HPLC-SEC purity is higher than LFV-M), LFV-S) have also been invented. While retaining good binding and blocking abilities to VEGF-C and VEGF-D, they have optimized binding and blocking abilities to VEGF-A. In particular, they have been found to have blocking abilities to VEGF A-VEGFR1, and their blocking abilities to VEGF A-VEGFR1 and VEGF A-VEGFR2 are superior to those of the control drugs Eylea and Avastin. In addition, they also have binding abilities to VEGF-B and PlGF.

[0234] Thus, the present invention uses a ligand-binding molecule that can block VEGF-A, VEGF-C, and VEGF-D, while also binding to VEGF-B, PlGF, and PDGF family molecules. This helps meet unmet clinical needs and achieve excellent therapeutic effects, while also solving the high cost problem of using two or more drugs simultaneously. At the same time, it has high affinity and blocking ability for VEGF-A, VEGF-C, and VEGF-D. For example, LFV-B and LFV-C have stronger affinity for VEGF-C and VEGF-D than OPT, and their affinity and blocking ability for VEGF-A are significantly better than Avastin, and close to or better than Aflibercept. This helps to extend the injection interval and reduce the number of injections, thereby further reducing treatment costs, while solving the various disadvantages brought about by frequent injections.

[0235] By using the ligand-binding molecules of the present invention or pharmaceutical compositions comprising the same, a single drug can replace multiple drugs in combination, thereby filling the gap in treatment methods for patients who have no significant response to VEGF-A targeting, and reducing the number of injections by virtue of high affinity, thereby achieving optimal therapeutic effects while significantly reducing the burden on patients, reducing treatment inconveniences, improving compliance, and reducing the risks of intraocular infection caused by injections.

[0236] Sequence summary of the present invention

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250] Example

[0251] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0252] Example 1 - Design and expression of recombinant receptor molecules

[0253] First, various Trap polypeptides containing different VEGFR2-derived sequences were constructed, as shown in Table 1A below. Some of these Trap polypeptides contain fragments derived from VEGFR1 or VEGFR3. These fragments can be operably linked via a linker or connector. The mutation site numbers used in this invention, such as N143A, N158Q, N318Q, etc., are relative to the corresponding positions in SEQ ID NO: 2.

[0254] Table 1A

[0255]

[0256]

[0257] Each of the designed Trap polypeptides can be linked to a human immunoglobulin IgG1 constant domain sequence (fused to the Fc hinge region) via a short, artificial linker sequence, Gly-Pro-Gly (GPG). Following gene synthesis, vector construction, and extraction, each of these sequences was transiently transfected into HEK293 or CHO cells (purchased from Gibco Life) and purified using Protein A affinity chromatography followed by secondary purification using molecular sieves. (LFV-D, LFV-F, and LFV-001 / V0 molecules met purity requirements after a single Protein A purification step and therefore did not undergo secondary purification using molecular sieves.) The resulting truncated VEGFR2-Fc fusion polypeptides are also referred to as recombinant receptor molecules or ligand-binding molecules.

[0258] Eylea, Avastin, R3A and OPT were used as controls. Eylea follows the sequence disclosed in WO0075319. R3A is constructed by connecting the amino acids at positions 25-329 of VEGFR3 to the constant domain sequence of human immunoglobulin IgG1 (fused with the Fc Hinge hinge region). OPT is constructed as described in WO2015 / 123715A1, which uses amino acids at positions 25-329, including amino acids at positions 47-314 of VEGFR3, and performs glycosylation modification at positions 104-106 as described in WO2015 / 123715A1, transforms the NDT sequence into a non-QDT sequence, and connects it to the constant domain sequence of human immunoglobulin IgG1 (fused with the Fc Hinge hinge region). The molecule constructed is referred to herein as OPT.

[0259] The expression and purification status of various recombinant receptor molecules are shown in Table 1B below.

[0260] Table 1B

[0261]

[0262] Example 2 - Construction, production and binding testing of recombinant receptor molecules derived from VEGFR2 and VEGFR3

[0263] 2.1 Construction and expression of recombinant receptor molecules

[0264] In order to construct a novel receptor-associated trap protein with better human binding ability, a fragment from human VEGFR3 (25-216 of SEQ ID NO: 3) and a fragment from human VEGFR2 (221-327 of SEQ ID NO: 2) were used as constituent elements, and a series of linker sequences were designed between them (as shown in Table 2A below).

[0265] Specifically, LFV-001 or V0 used a sequence from native VEGFR3 (positions 217-227 of SEQ ID NO:3); LFV-010 used a sequence that was further extended toward the C-terminus of the LFV-001 sequence in VEGFR3; to introduce flexibility in length, LFV-002 removed Thr from the linker sequence based on LFV-001; and LFV-003 replaced this Thr with Gly to introduce greater flexibility in the torsional torsional behavior of the main chain. On the other hand, in LFV-004 to 008, the VEGFR3-derived linker sequence used in LFV-001 underwent a series of mutations to the linker sequence of the corresponding region of VEGFR2 (as shown in Table 1 below). In LFV-008, an Ile residue derived from VEGFR2 was mutated to an Ala residue with a smaller side chain and the same polarity to reduce steric clash between the side chains. As control trap proteins, LFV-B containing only the VEGFR2 fragment shown as 117-327 of SEQ ID NO: 2 and R3A containing only the VEGFR3 fragment shown as 25-329 of SEQ ID NO: 3 were designed.

[0266] Table 2A

[0267]

[0268]

[0269] Each of the above-mentioned trap polypeptides can be constructed by linking a short artificial linker sequence Gly-Pro-Gly (GPG) with a human IgG1 immunoglobulin constant domain sequence (fused to the Fc Hinge hinge region). After gene synthesis, vector construction and extraction, they are transiently transfected into HEK293 cells (purchased from Gibco Life) and then purified by one-step affinity elution with protein A. They are hereinafter referred to as recombinant receptor molecules, and their expression characteristics are shown in Table 2B below:

[0270] Table 2B.

[0271]

[0272]

[0273] 2.2 Binding test with VEGF-A, VEGF-C, and VEGF-D

[0274] VEGF-A165, VEGF-C, and VEGF-D polypeptide molecules were purchased from ACROBiosystems. 100-300 ng / well of VEGF molecules were prepared in PBS and coated overnight at 4°C. The next day, the plates were washed with PBS and blocked with 1% BSA at room temperature for 2 hours. The plates were then washed with PBS. The recombinant receptor molecules expressed in Example 1 were diluted from high to low concentrations using PBT (0.5% BSA dissolved in PBS containing 0.05% Tween-20) and added to each well. The plates were incubated at room temperature at 300 rpm for 1.5 hours. The plates were washed three times with PBST (PBS containing 0.05% Tween-20). A goat anti-human Fc secondary antibody conjugated to horseradish peroxidase diluted in PBT was added and incubated at room temperature at 300 rpm for 1 hour. The plates were washed three times with PBST, and TMB was added for color development. The color was terminated with 5% hydrochloric acid, and the OD value at 450 nm was read using a microplate reader.

[0275] The results are shown in Figure 1 , where various recombinant ligand-binding molecules exhibited binding ability to VEGF-A (represented by VEGF-A165), VEGF-C, and VEGF-D. It was also unexpectedly discovered that the control LFV-B molecule exhibited stronger binding ability to VEGF-C and VEGF-D than R3A.

[0276] Example 3 - Binding of recombinant receptor molecules to VEGF-C

[0277] 100 ng / well of VEGF-C molecules were prepared in PBS and coated overnight at 4°. The next day, the plates were washed with PBS and blocked with 1% BSA solution at room temperature for 2 hours. The plates were washed with PBS, and the recombinant receptor molecules expressed in Example 1 were diluted from high to low concentrations and added to each well. The plates were incubated at room temperature at 300 rpm for 1.5 hours, washed three times with PBST solution, and a goat anti-human Fc secondary antibody conjugated to horseradish peroxidase was added. The plates were incubated at room temperature at 300 rpm for 1 hour, washed three times with PBST solution, and TMB was added for color development. The color was terminated with 5% hydrochloric acid solution, and the OD value at 450 nm was read using a microplate reader.

[0278] The results are shown in Figure 2. It can be seen that the relevant recombinant receptor molecules have good VEGF-C binding ability, among which LFV-B and its truncated form LFV-C have the best VEGF-C binding ability, which is better than OPT, and LFV-E also has a binding ability close to OPT. 50 The values ​​are shown in Table 3 below:

[0279] Table 3.

[0280] R3ALFV-ELFV-DLFV-BLFV-CLFV-AOPTLFV-FV0EC 50(ng / ml)161.685.19191.716.2424.16 / 54.59916398.5EC 50 (nM)1.350.711.60.160.24 / 0.456.53.3 surface

[0281] Example 4 - Blocking Experiment of VEGFR2 Binding of Recombinant Receptor Molecules to VEGF-C and VEGF-D

[0282] 500 ng / well of VEGFR2-Fc (Met 1-Glu 764) molecules were coated at 4° overnight, and the plates were washed with PBS the next day. BSA solution was added and blocked at room temperature for 2 hours. During the blocking period, each recombinant receptor molecule was diluted from high to low concentration and mixed with 100 ng / well of VEGF-C or VEGF-D (both with His tags). The plates were incubated at room temperature for 30 minutes. The VEGFR2-Fc plate was washed, and the incubation solution of the recombinant receptor molecules and VEGF-C and VEGF-D was added thereto. The plates were incubated at room temperature for 1 hour. The plates were washed with PBST, and anti-polyhistidine secondary antibody coupled to horseradish peroxidase was added. The plates were incubated at room temperature for 1 hour. The plates were washed with PBST, TMB was added for color development, and 5% hydrochloric acid solution was used to terminate the reaction. The OD value at 450 nm was read using a microplate reader.

[0283] The results are shown in Figure 3. The relevant recombinant receptor molecules have a certain blocking ability, among which LFV-B and its truncated forms LFV-C and LFV-E have the best blocking ability, and are better than OPT. V0 molecules also have a good blocking ability for VEGF-C. The blocking EC of each recombinant receptor molecule on the binding of VEGF-C and VEGF-D to VEGFR2 50 The values ​​are shown in Tables 4 and 5 below respectively.

[0284] Table 4. Blocking ECs for VEGFR2-VEGFC Binding 50 value

[0285] LFV-ALFV-DLFV-FLFV-BOPTLFV-CLFV-EV0R3AEyleaAvastinEC 50 (ng / ml) / 5828024008208535672252227535937808 / / EC 50 (nM) / 485.7170.620.8529.722.5218.9629.965.1 / / surface

[0286] Table 5. Blocking ECs for VEGFR2-VEGFD Binding 50 value

[0287] LFV-ALFV-DLFV-FLFV-BOPTLFV-CLFV-EV0R3AEyleaAvastinEC 50 (ng / ml) / 920.4521.626.78241.334.0250.541542532.2 / / EC 50 (nM) / 7.73.70.272.010.340.4212.854.4 / / surface

[0288] Example 5 - VEGFR3 binding blocking experiment of recombinant receptor molecules to VEGF-C and VEGF-D

[0289] 500 ng / well of VEGFR3-Fc (Tyr25-IIe776) molecules were coated at 4° overnight, and the plates were washed with PBS the next day. BSA solution was added and blocked at room temperature for 2 hours. During the blocking period, each recombinant receptor molecule was diluted from high to low concentration, mixed with 10 ng / well VEGF-C or 500 ng / well VEGF-D (both with His tags), and incubated at room temperature for 30 minutes. The VEGFR3-Fc plate was washed, and the incubation solution was added thereto. The plates were incubated at room temperature for 1 hour. The plates were washed with PBST, and anti-polyhistidine secondary antibody coupled to horseradish peroxidase was added. The plates were incubated at room temperature for 1 hour. The plates were washed with PBST, TMB was added for color development, and 5% hydrochloric acid solution was used to terminate the reaction. The OD value at 450 nm was read using a microplate reader.

[0290] The results are shown in Figure 4. It can be found that the blocking results are basically consistent with the blocking experiments on VEGFR2. 50 The values ​​are shown in Tables 6 and 7 below respectively.

[0291] Table 6. Blockade EC of VEGFR3-VEGFC 50 value

[0292] LFV-ALFV-DLFV--FLFV-BOPTLFV-CLFV-EV0R3AEvleaAvastinEC 50 (ng / ml) / 43232915114.6300.4106.1135.8188.6560.7 / / EC 50 (nM) / 36.0320.721.152.51.061.131.574.67 / / surface

[0293] Table 7. Blockade EC of VEGFR3-VEGFD 50 value

[0294]

[0295] Example 6 - Binding of recombinant receptor molecules to VEGF-A

[0296] 50 ng / well of VEGF-A 165 or VEGF-A 121 molecules (purchased from ACROBiosystems) were coated at 4° overnight, and the plates were washed with PBS the next day, blocked at room temperature for 2 hours, washed with PBS, and the recombinant receptor molecules were diluted from high to low concentrations and added to each well, incubated at room temperature for 1.5 hours, washed three times with PBST solution, and a goat anti-human Fc secondary antibody conjugated to horseradish peroxidase was added, incubated at room temperature for 1 hour, washed three times with PBST solution, TMB was added for color development, terminated with 5% hydrochloric acid solution, and the OD value at 450 nm was read using a microplate reader.

[0297] The results are shown in Figure 5. It can be seen that all VEGFR2-related recombinant receptor molecules except LFV-A, LFV-D, and LFV-F have good VEGF-A binding ability. Among them, LFV-B and its truncated form LFV-C have the best VEGF-A binding ability, which is better than Eylea. LFV-E has a VEGF-A binding ability close to that of Eylea. 50 The values ​​are shown in Tables 8 and 9 below, respectively.

[0298] Table 8. Binding EC to VEGF-A165 50 value

[0299] LFV-CLFV-ELFV-BLFV-DLFV-FEyleaEC 50 (ng / ml)12.6121.199.268476.1983.417.39EC 50 (nM)0.1260.1770.0933.976.990.174 surface

[0300] Table 9. Binding EC to VEGF-A 121 50 value

[0301] LFV-CLFV-ELFV-BLFV-DLFV-FEyleaEC 50 (ng / ml)47.5972.131.14 / / 71.89EC 50 (nM)0.4760.6010.311 / / 0.719 surface

[0302] Example 7 - Blocking experiment of VEGF-A binding to VEGFR2 by recombinant receptor molecules

[0303] 500 ng / well of VEGFR2-Fc molecules were coated at 4° overnight, and the plates were washed with PBS the next day. BSA solution was added and blocked at room temperature for 2 hours. During the blocking period, each recombinant receptor molecule was diluted from high to low concentration, and then mixed with 60 ng / well of VEGF-A 165 or 120 ng / well of VEGF-A 121 (both with His tags) and incubated at room temperature for 30 minutes. The VEGFR2-Fc plate was washed, and the incubation solution was added thereto. The plates were incubated for 1 hour at room temperature for binding. The plates were washed with PBST, and anti-polyhistidine secondary antibody coupled to horseradish peroxidase was added. The plates were incubated for 1 hour at room temperature for binding. The plates were washed with PBST, TMB was added for color development, and 5% hydrochloric acid solution was used to terminate the reaction. The OD value at 450 nm was read using a microplate reader.

[0304] The results are shown in Figure 6. LFV-B and its truncated form LFV-C have good blocking ability, reaching a level better than bevacizumab and close to that of Eylea. 50 The values ​​are shown in Tables 10 and 11 below, respectively.

[0305] Table 10. Blocking EC of VEGF-A 165 50 value

[0306]

[0307] Table 11. Blocking EC of VEGF-A 121 50 value

[0308]

[0309] Example 8 - Binding affinity detection experiment for VEGF-A, VEGF-C, and VEGF-D

[0310] The affinity was tested using surface plasmon resonance (SPR) on a Biacore T200 instrument. Protein A chips were used to capture recombinant receptor molecules (including LFV-B and LFV-E, with OPT as a control). The running buffer was 1×HBS-EP, and antigens of various concentrations were flowed in at a flow rate of 30 μl / min.

[0311] The test results are shown in Table 12 below. It can be seen that LFV-B has significantly better affinity for VEGF-C and VEGF-D than OPT, and also has a strong affinity for VEGF-A, reaching 9.3 pM. LFV-E has slightly better affinity for VEGF-C and VEGF-D than OPT. Compared with LFV-B, LFV-E has a significantly weaker apparent affinity due to its relatively low Ka value. However, their Kd values ​​are similar, resulting in similar blocking potency in the blocking experiment. The relatively lower blocking potency of OPT may be explained by its significantly weaker Kd value.

[0312] Table 12A.

[0313]

[0314] Table 12B

[0315]

[0316] Example 9-Binding detection experiment of PDGF family molecules

[0317] In this example, the binding between VEGFR2 and its related truncated forms of trap molecules and PDGF family molecules was also tested. Briefly, surface plasmon resonance was used to detect the binding of LFV-B, LFV-E, and VEGFR2-Fc (using the full extracellular domain sequence of VEGFR2: Met 1-Glu 764) molecules to PDGF family molecules. In initial detection experiments, it was found that PDGF family molecules (purchased from R&D Systems) were prone to nonspecific binding within the chip, resulting in a false increase in the measured value and making the detection impossible. Subsequently, a CM5 chip was used to couple and load PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC molecules, respectively, and then the recombinant receptor molecules were introduced, thereby effectively eliminating the impact of nonspecific binding on affinity determination.

[0318] The relevant test results are shown in Table 13 below. It can be seen that the entire VEGFR2 extracellular domain has a certain binding affinity to PDGF molecules, but LFV-C and LFV-B have significantly superior binding to PDGF family molecules. This unexpectedly indicates that they are the main segment responsible for binding to PDGF, potentially leading to more effective therapeutic effects and benefiting more patients by binding to and blocking PDGF family molecules. LFV-E has a similar binding affinity to PDGF as LFV-B and LFV-C.

[0319] Table 13.

[0320]

[0321] Example 10 - Pharmacokinetics in mice

[0322] C57BL / 6 mice were divided into three groups, each with six mice. Each group was injected via the tail vein with LFV-B, LFV-E, or the control substance, OPT, at 4 mg / kg. Blood was collected at 1, 2, 12, 24, 72 hours (3 days), 168 hours (7 days), 336 hours (14 days), and 504 hours (21 days) after injection. Serum was collected by centrifugation and the target molecule was quantified using an anti-human Fc antibody capture ELISA. Pharmacokinetic calculations were performed using WinNonlin (Phoenix™, version 8.0) and other related software. The resulting PK data are shown in Figure 7 and Table 14 below:

[0323] Table 14.

[0324]

[0325]

[0326] It can be seen that the three molecules have similar in vivo PK characteristics, the serum concentration of LFV-E is relatively the highest, and the half-life of LFV-B is relatively good.

[0327] Example 11 - Laser-induced choroidal neovascularization in rats

[0328] Brown Norway rats were anesthetized and photocoagulated around the optic disc using a laser photocoagulator. The formation of bubbles indicated rupture of Bruch's membrane. Twelve days after photocoagulation, the rats were divided into groups and administered intraocularly on the second day of grouping at 4 μl or 40 μg / eye. Fluorescein angiography and fundus photography were performed on the 10th or 14th day after administration, and the number of grade 4 fluorescent spots was counted in each group.

[0329] The results are shown in Figure 8 , which shows that compared with the PBS and Eylea control groups, LFV-B and LFV-C showed a trend of four-level light spot relief.

[0330] Example 12 - Glycosylation modification design and detection

[0331] The N-glycosylation sites in LFV-E (Asn46, Asn66, Asn96) and the N-glycosylation sites in LFV-B and LFV-C (Asn143, Asn158, Asn245, Asn318) were point mutated to remove N-glycosylation, preferentially mutating Asn to Gln or Ala.

[0332] The sequences with designed point mutations underwent gene synthesis, vector construction, extraction, cell transfection, expression, and purification. ELISAs were used to assess their ability to block VEGFR2 / R1-VEGFA, VEGFR2 / R3-VEGFC, and VEGFR2 / R3-VEGFD binding, compared to the unmutated original molecules. Several variants were selected based on a comprehensive evaluation of expression levels and single-peak purity. HEK 293 cells were used for transfection, expression, and purification, and PK assays were performed in mice, comparing the results to the unmutated original molecules. The glycosylated recombinant proteins were also recombined with heterologous peptides, and ELISA, cell-based, and animal studies were performed to further optimize the selection.

[0333] Point mutations were performed on the four N-glycosylation sites (Asn143, Asn158, Asn245, and Asn318) of LFV-C. Their mutation designs and expression and purification are shown in Table 15 below:

[0334] Table 15.

[0335]

[0336] Then, using the conditions in the above examples, the ELISA method was used to detect the blocking ability of different concentrations of LFV-H, LFV-J, LFV-K, and LFV-L compared to non-mutated LFV-C on the binding of VEGF-A165, VEGF-C, and VEGF-D to the receptors. The experimental conditions used in the VEGF-A 165-VEGFR1 blocking ELISA are as follows: 200 ng / well of VEGFR1-Fc (Met1-Asn756) molecules were coated at 4° overnight, the plates were washed with PBS the next day, and BSA solution was added for blocking at room temperature for 2 hours. During the blocking period, each recombinant receptor molecule was diluted from high to low concentration, and then mixed with 40 ng / well of VEGF-A 165 (with His tag), incubated at room temperature for 30 minutes, the VEGFR1-Fc plate was washed, the incubation solution was added thereto, incubated at room temperature for 1 hour, the plates were washed with PBST, and anti-polyhistidine secondary antibody conjugated to horseradish peroxidase was added, incubated at room temperature for 1 hour, the plates were washed with PBST, TMB was added for color development, 5% hydrochloric acid solution was used to terminate the reaction, and the OD value at 450 nm was read using a microplate reader.

[0337] The test results are shown in Figure 9. It can be seen that compared with the non-glycosylated form, the LFV-K molecule has a better blocking ability on the binding of VEGF-A, VEGF-C, and VEGF-D to the receptor, and has relatively greater development potential.

[0338] Example 13 - Design of a new VEGFR2-VEGFR1 fusion protein

[0339] To further enhance the ability of the recombinant receptor molecule to block VEGF-A binding to the receptor, the region 132-230 of VEGFR1, or a combination of the region 132-230 of VEGFR1 and the region 225-327 of VEGFR2, was introduced into LFV-B or LFV-C molecules or their glycosylated modified molecules, and transient transfection and expression purification were performed in CHO cells. Then, the ELISA method was used to detect its blocking ability against VEGFR2 / R1-VEGFA, VEGFR2 / R3-VEGFC, and VEGFR2 / R3-VEGFD. The system described in Examples 8 and 9 was used to detect its blocking ability against VEGF and PDGF family molecules to explore whether it has enhanced blocking ability against VEGF-A binding to the receptor, as well as binding ability against VEGF-B and PlGF, while retaining good blocking ability against VEGF-C, VEGF-D and PDGF family molecules binding to the receptor. Biacore was used to detect its binding ability against PDGF family molecules, and cell or animal experiments were performed on the preferred fusion protein.

[0340] In one experiment, the 132-230 region of VEGFR1 was linked to the nitrogen and carbon termini of the LFV-C molecule, respectively, and the combination of the 132-230 region of VEGFR1 and the 225-327 region of VEGFR2 was constructed into the LFV-C molecule, and the GS linker was used for connection. The full sequences of the constructed molecules LFV-M, LFV-N, and LFV-O are shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 46. LFV-M is a form in which the 132-230 region of VEGFR1 is constructed at the nitrogen terminus of LFV-C, LFV-N is a form in which the 132-230 region of VEGFR1 is constructed at the carbon terminus of LFV-C, and LFV-O is a recombinant form of VEGFR1 (132-230)-VEGFR2 (225-327) and LFV-C. Their expression and purification in CHO cells are shown in Table 16:

[0341] Table 16

[0342]

[0343] Then, using the conditions in the above example, the ELISA method was used to detect their blocking abilities against VEGF-A, VEGF-C, and VEGF-D. The results are shown in Figure 10. It can be seen that LFV-M and LFV-N have the same or slightly better blocking abilities against VEGF-C and VEGF-D binding to receptors than LFV-C, and have significantly improved blocking abilities against VEGF-A binding to receptors compared to LFV-C. In particular, in the VEGF A-VEGFR1 blocking experiment, LFV-M and LFV-N exhibited blocking abilities superior to those of Eylea.

[0344] In addition, their binding ability to VEGF-B and PlGF was tested: 200 ng / well VEGF-B or PlGF molecules (both purchased from ACROBiosystems) were prepared in PBS and coated overnight at 4°. The plates were washed with PBS the next day, blocked with 1% BSA solution at room temperature for 2 hours, washed with PBS, and the recombinant receptor molecules were diluted from high to low concentrations and added to each well. The plates were incubated at room temperature and 300 rpm for 1.5 hours, washed three times with PBST solution, and goat anti-human Fc secondary antibody conjugated with horseradish peroxidase was added. The plates were incubated at room temperature and 300 rpm for 1 hour. The plates were washed three times with PBST solution, TMB was added for color development, terminated with 5% hydrochloric acid solution, and the OD value at 450 nm was read using a microplate reader.

[0345] The results are shown in FIG11 . Both LFV-M and LFV-N showed a certain degree of binding to VEGF-B and PlGF.

[0346] Example 14 - Design and verification of fusion protein of glycosylated VEGFR2 sequence and VEGFR1 sequence

[0347] The LFV-B molecule or its truncated or extended form is introduced with the glycosylation modification used in the LFV-K and LFV-L molecules, and recombined with the VEGFR1 position 132-230 region to produce a new fusion protein, which is expressed and purified, and then characterized and analyzed at the molecular and cellular levels and animal experiments are carried out in order to obtain a more effective therapeutic molecule.

[0348] In one experiment, an LFV-B molecule with the N245Q mutation, designated LFV-P, was used as a control. LFV-P was combined with the VEGFR1 region 132-230 to produce a new fusion protein, designated LFV-Q. The sequence of LFV-P is shown in SEQ ID NO:18, and the sequence of LFV-Q is shown in SEQ ID NO:19. LFV-P and LFV-Q were expressed and purified, and then assayed by ELISA using the conditions described in the previous examples for their ability to block VEGF-A, VEGF-C, and VEGF-D receptor binding, as well as their ability to bind VEGF-B and PlGF.

[0349] The results of blocking the binding of VEGF-A, VEGF-C, and VEGF-D to VEGFR2 are shown in Figure 12. It can be seen that after glycosylation modification, LFV-P has a better ability to block the binding of VEGF-A, VEGF-C, and VEGF-D to the receptors than LFV-B. After fusion with the 132-230 region of VEGFR1, LFV-Q has the best blocking ability against the binding of VEGF-A to the receptor. The results of binding to VEGF-B and PlGF are shown in Figure 13. It can be seen that after fusion with the 132-230 region of VEGFR1, LFV-Q also has a good binding ability to VEGF-B and PlGF.

[0350] Example 15- Detection of Binding of LFV-Q to PDGF Family Molecules

[0351] The binding of LFV-Q molecules to PDGF family molecules was detected using surface plasmon resonance. The specific implementation steps were consistent with the above Example 9, that is, the method of first coupling and loading PDGF and then flowing the fusion protein molecules to be tested was adopted to eliminate the nonspecific binding of PDGF molecules to the chip.

[0352] The relevant test results are shown in Table 17 below:

[0353] Table 17

[0354]

[0355] It can be seen that LFV-Q has a definite binding ability to PDGF family molecules.

[0356] Example 16 - Further glycosylation variant studies of LFV-M and LFV-Q

[0357] As described above, LFV-Q uses a deglycosylated form of position N245 of sequence 117-327 derived from VEGFR2, i.e., N245 is mutated to Q. To further study glycosylation, position N245 in LFV-M is also mutated to Q to remove glycosylation. The resulting new molecule is named LFV-R, and the sequence of LFV-R is shown in SEQ ID NO:20. At the same time, position Q245 of LFV-Q is restored to N for functional comparison before and after glycosylation, and the resulting molecule is named LFV-S.

[0358] First, LFV-R and LFV-S were transiently expressed and purified. During this process, it was found that after the removal of glycosylation at position N245, the SEC single peak purity of LFV-R was unexpectedly increased from 52.1% to 78.7% after transient transfection into CHO cells and one-step purification with protein A compared to the LFV-M without removal (as shown in Figure 14). In addition, after secondary purification with molecular sieves, LFV-R was concentrated in PBS to facilitate subsequent intravitreal injection. It was found that it still showed good solubility when the concentration reached 19.2 mg / ml, which reflects the good downstream development potential of LFV-R.

[0359] In addition, the ELISA method was used to detect the binding blocking ability of these molecules on VEGFR2 / VEGFR1 and VEGF-A, VEGFR2 and VEGF-C, and VEGFR2 and VEGF-D. The results are shown in Figure 15. It can be seen that LFV-R has the best blocking ability against VEGFR2 and VEGF-A, and is significantly better than the control drug Eylea. The blocking abilities of the four molecules against VEGFR2, VEGF-C, and VEGF-D are relatively close, especially the blocking ability against VEGF-D is significantly better than the control drug OPT.

[0360] In addition, their binding abilities to PLGF and VEGF-B were also tested. The results are shown in FIG16 , which shows that LFV-R has the best binding ability to PlGF and VEGF-B.

[0361] Example 17 - ELISA binding experiment of LFV-R, LFV-Q, LFV-B, LFV-C, etc. to VEGF family molecules

[0362] The binding of LFV-R, LFV-Q, LFV-B, and LFV-C molecules to VEGF-A was detected by ELISA, and VEGFR2-Fc and Eylea with the full extracellular domain were used as controls. The experimental results are shown in Figure 17a. It can be seen that LFV-R has the best binding ability to VEGF-A, which is higher than the control drug Eylea. The binding ability of LFV-R, LFV-Q, LFV-B, and LFV-C molecules to VEGF-A is significantly higher than that of VEGFR2-Fc with the full extracellular domain.

[0363] The binding of LFV-R, LFV-Q, LFV-B, and LFV-C molecules to VEGF-C was detected by ELISA, and VEGFR2-Fc and OPT with the entire extracellular domain were used as controls. The experimental results are shown in Figure 17b. It can be seen that LFV-R and LFV-Q have similar binding abilities to LFV-B, which are higher than the control OPT. The binding abilities of the four molecules LFV-R, LFV-Q, LFV-B, and LFV-C to VEGF-C are all significantly higher than those of VEGFR2-Fc with the entire extracellular domain.

[0364] The binding of LFV-R, LFV-Q, LFV-B, and LFV-C molecules to VEGF-D was detected by ELISA, and VEGFR2-Fc and OPT with the entire extracellular domain were used as controls. The experimental results are shown in Figure 17c. It can be seen that LFV-R and LFV-Q have similar binding abilities to LFV-B, which are significantly higher than the control OPT. The binding abilities of the four molecules LFV-R, LFV-Q, LFV-B, and LFV-C to VEGF-D are all significantly higher than those of VEGFR2-Fc with the entire extracellular domain.

[0365] Example 18- LFV-R binding detection experiment to PDGF family molecules

[0366] The binding of LFV-R molecules to PDGF family molecules was detected using surface plasmon resonance. The specific implementation steps were consistent with those in Example 9 above, i.e., PDGF was first coupled and loaded, and then the fusion protein molecules to be tested were introduced to eliminate non-specific binding of PDGF molecules to the chip. The relevant detection results are shown in Table 18 below:

[0367] Table 18.

[0368]

[0369] It can be seen that LFV-R has a clear binding ability to PDGF-AA, AB, BB, and CC.

[0370] Example 19 - HUVEC cell proliferation inhibition test

[0371] HUVEC cell proliferation was stimulated using a 1:1 mixture of VEGF-A and VEGF-C molecules. LFV-R, Eylea, and OPT molecules were added at varying concentrations. After 72 hours of cell culture, MTS was used for color development to detect the inhibitory effects of the different drugs on cell proliferation. The results, shown in Figure 18a, show that only LFV-R exhibited significant inhibitory activity, achieving complete inhibition at a concentration of approximately 3 μg / ml. OPT exhibited no inhibitory effect, and Eylea exhibited complete inhibition only at ultra-high concentrations (above 30 μg / ml).

[0372] Furthermore, as a potential treatment method, Eylea was mixed with OPT (1:1 mixture) to perform proliferation inhibition test on HUVEC, and LFV-R and LFV-Q were added for comparison. The results are shown in Figure 18b. It can be seen that compared with single use, the mixture of Eylea and OPT can completely inhibit the proliferation of HUVEC cells. Furthermore, LFV-R not only IC 50It is superior to Eylea+OPT and LFV-Q, and shows significantly higher inhibitory efficacy at low concentrations compared to Eylea+OPT and LFV-Q, which will help maintain sustained inhibitory efficacy during the concentration decay period after vitreous administration, thereby improving therapeutic benefits and extending the injection interval.

[0373] Example 20 - Laser-induced treatment of choroidal neovascularization in rats

[0374] Twenty-four Brown-Norway rats were randomly divided into four groups based on body weight, with six animals in each group (half male and half female): model control, LFV-R, LFV-Q, and Eylea. Each group underwent bilateral fundus laser modeling. On day 3 of modeling, a single intravitreal injection of 0.9% sodium chloride, LFV-R, LFV-Q, or Eylea was administered in each eye at a concentration of 10 mg / mL and a dose of 40 μg / eye (4 μL / eye). The day of drug administration was designated as day 1 of the study. Surviving rats in each group were observed daily during the study. Fundus photography and fluorescein angiography were performed before modeling and on day 18 after drug administration to observe neovascularization and leakage in each group, and to compare drug efficacy.

[0375] The results are shown in Figure 19. The LFV-R, LFV-Q, and Eylea treatment groups all showed lower fourth-level spot proportions and fluorescence leakage areas than the model control group. The fourth-level fluorescence spot proportions of the LFV-R and LFV-Q treatment groups showed significant statistical differences compared with the control group and the Eylea group.

[0376] Those skilled in the art will further recognize that the present invention may be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the present invention discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims for an indication of the scope and content of the present invention. <110> Suzhou Guangdu Biotechnology Co., Ltd. Hong Kong Guangdu Biotechnology Co., Ltd. <120> Multispecific ligand binding molecules and their applications <130> IDC216038 <160> 59 <170> PatentIn version 3.5 <210> 1 <211> 1338 <212> PRT <213> people <400> 1Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro20 25 30Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr35 40 45Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro50 55 60Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala65 70 75 80Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr85 90 95Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val100 105 110Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile115 120 125Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu130 135 140Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys ArgVal145 150 155 160Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr165 170 175Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe180 185 190Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu195 200 205Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg210 215 220Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val225 230 235 240Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr245 250 255Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys260 265 270Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His275 280 285Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys290 295 300Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys305 310 315 320Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val325 330 335Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser340 345 350Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val355 360 365Trp Leu LysAsp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu370 375 380Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala385 390 395 400Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys405 410 415Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu420 425 430Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser435 440 445Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile450 455 460Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys465 470 475 480Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser485 490 495Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile500 505 510Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg515 520 525Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val530 535 540Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His545 550 555 560Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser565 570 575Cys Thr Val Asn Lys Phe Leu Tyr ArgAsp Val Thr Trp Ile Leu Leu580 585 590Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys595 600 605Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met610 615 620Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn625 630 635 640Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg645 650 655Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val660 665 670Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro675 680 685Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu690 695 700Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe Ile Glu Arg705 710 715 720Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys Lys Ala Thr Asn Gln725 730 735Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu Thr Val Gln Gly Thr Ser740 745 750Asp Lys Ser Asn Leu Glu Leu Ile Thr Leu Thr Cys Thr Cys Val Ala755 760 765Ala Thr Leu Phe Trp Leu Leu Leu Thr Leu Phe Ile Arg Lys Met Lys770 775 780Arg Ser Ser Ser Glu Ile Lys Thr Asp Tyr Leu Ser Ile Ile Met Asp785790 795 800Pro Asp Glu Val Pro Leu Asp Glu Gln Cys Glu Arg Leu Pro Tyr Asp805 810 815Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg Leu Lys Leu Gly Lys Ser820 825 830Leu Gly Arg Gly Ala Phe Gly Lys Val Val Gln Ala Ser Ala Phe Gly835 840 845Ile Lys Lys Ser Pro Thr Cys Arg Thr Val Ala Val Lys Met Leu Lys850 855 860Glu Gly Ala Thr Ala Ser Glu Tyr Lys Ala Leu Met Thr Glu Leu Lys865 870 875 880Ile Leu Thr His Ile Gly His His Leu Asn Val Val Asn Leu Leu Gly885 890 895Ala Cys Thr Lys Gln Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys900 905 910Lys Tyr Gly Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg Asp Leu Phe915 920 925Phe Leu Asn Lys Asp Ala Ala Leu His Met Glu Pro Lys Lys Glu Lys930 935 940Met Glu Pro Gly Leu Glu Gln Gly Lys Lys Pro Arg Leu Asp Ser Val945 950 955 960Thr Ser Ser Glu Ser Phe Ala Ser Ser Gly Phe Gln Glu Asp Lys Ser965 970 975Leu Ser Asp Val Glu Glu Glu Glu Asp Ser Asp Gly Phe Tyr Lys Glu980 985 990Pro Ile Thr Met Glu Asp Leu Ile Ser Tyr Ser Phe Gln Val Ala Arg995 1000 1005Gly Met Glu PheLeu Ser Ser Arg Lys Cys Ile His Arg Asp Leu1010 1015 1020Ala Ala Arg Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile1025 1030 1035Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr1040 1045 1050Val Arg Lys Gly Asp Thr Arg Leu Pro Leu Lys Trp Met Ala Pro1055 1060 1065Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr Lys Ser Asp Val Trp1070 1075 1080Ser Tyr Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Gly Ser1085 1090 1095Pro Tyr Pro Gly Val Gln Met Asp Glu Asp Phe Cys Ser Arg Leu1100 1105 1110Arg Glu Gly Met Arg Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu1115 1120 1125Ile Tyr Gln Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu1130 1135 1140Arg Pro Arg Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu1145 1150 1155Gln Ala Asn Val Gln Gln Asp Gly Lys Asp Tyr Ile Pro Ile Asn1160 1165 1170Ala Ile Leu Thr Gly Asn Ser Gly Phe Thr Tyr Ser Thr Pro Ala1175 1180 1185Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser Ala Pro Lys Phe1190 1195 1200Asn Ser Gly Ser Ser Asp Asp Val Arg Tyr Val Asn Ala Phe Lys12051210 1215Phe Met Ser Leu Glu Arg Ile Lys Thr Phe Glu Glu Leu Leu Pro1220 1225 1230Asn Ala Thr Ser Met Phe Asp Asp Tyr Gln Gly Asp Ser Ser Thr1235 1240 1245Leu Leu Ala Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser1250 1255 1260Lys Pro Lys Ala Ser Leu Lys Ile Asp Leu Arg Val Thr Ser Lys1265 1270 1275Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro Ser Phe Cys1280 1285 1290His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg Arg Phe Thr1295 1300 1305Tyr Asp His Ala Glu Leu Glu Arg Lys Ile Ala Cys Cys Ser Pro1310 1315 1320Pro Pro Asp Tyr Asn Ser Val Val Leu Tyr Ser Thr Pro Pro Ile1325 1330 1335<210> 2<211> 1356<212> PRT<213> Human<400> 2Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu1 5 10 15Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro20 25 30Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr35 40 45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro50 55 60Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser65 70 75 80Asp GlyLeu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn85 90 95Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser100 105 110Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser115 120 125Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys130 135 140Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser145 150 155 160Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg165 170 175Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile180 185 190Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser195 200 205Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr210 215 220Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu225 230 235 240Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile245 250 255Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu260 265 270Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe275 280 285Leu Ser Thr Leu Thr Ile Asp Gly Val ThrArg Ser Asp Gln Gly Leu290 295 300Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr305 310 315 320Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met325 330 335Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala340 345 350Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly355 360 365Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr370 375 380Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu385 390 395 400Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val405 410 415Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val420 425 430Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr435 440 445Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu450 455 460Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr465 470 475 480Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys485 490 495Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys500505 510Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr515 520 525Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser530 535 540Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln545 550 555 560Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser565 570 575Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro580 585 590Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr595 600 605Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile610 615 620Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr625 630 635 640Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val645 650 655Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn660 665 670Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys675 680 685Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn690 695 700Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg705 710 715 720Asn Leu Thr Ile ArgArg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr725 730 735Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe740 745 750Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu755 760 765Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile770 775 780Ile Leu Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys Thr Gly785 790 795 800Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His805 810 815Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp820 825 830Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val835 840 845Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg Thr850 855 860Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg865 870 875 880Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu885 890 895Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu900 905 910Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu915 920 925Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys ThrLys Gly Ala Arg930 935 940Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Leu Lys945 950 955 960Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly965 970 975Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Pro980 985 990Glu Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr995 1000 1005Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys1010 1015 1020Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu1025 1030 1035Lys Asn Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile1040 1045 1050Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro1055 1060 1065Leu Lys Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr1070 1075 1080Ile Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile1085 1090 1095Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu1100 1105 1110Glu Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro1115 1120 1125Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp1130 1135 1140His Gly Glu ProSer Gln Arg Pro Thr Phe Ser Glu Leu Val Glu1145 1150 1155His Leu Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln Asp Gly Lys1160 1165 1170Asp Tyr Ile Val Leu Pro Ile Ser Glu Thr Leu Ser Met Glu Glu1175 1180 1185Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met Glu1190 1195 1200Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala1205 1210 1215Gly Ile Ser Gln Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro1220 1225 1230Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu1235 1240 1245Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly Met Val1250 1255 1260Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Thr Lys Leu1265 1270 1275Ser Pro Ser Phe Gly Gly Met Val Pro Ser Lys Ser Arg Glu Ser1280 1285 1290Val Ala Ser Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly1295 1300 1305Tyr His Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu1310 1315 1320Ala Glu Leu Leu Lys Leu Ile Glu Ile Gly Val Gln Thr Gly Ser1325 1330 1335Thr Ala Gln Ile Leu Gln Pro Asp Ser Gly Thr Thr Leu Ser Ser13401345 1350Pro Pro Val1355<210> 3<211> 1363<212> PRT<213> Human<400> 3Met Gln Arg Gly Ala Ala Leu Cys Leu Arg Leu Trp Leu Cys Leu Gly1 5 10 15Leu Leu Asp Gly Leu Val Ser Gly Tyr Ser Met Thr Pro Pro Thr Leu20 25 30Asn Ile Thr Glu Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser35 40 45Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala50 55 60Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val65 70 75 80Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu85 90 95Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys Tyr100 105 110Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Ser115 120 125Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe Ile Asn Lys Pro Asp130 135 140Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp Val Pro Cys Leu Val145 150 155 160Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser Gln Ser Ser Val Leu165 170 175Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu180 185 190Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln CysGlu Thr195 200 205Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile210 215 220Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys Ser Leu225 230 235 240Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala245 250 255Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln260 265 270Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His275 280 285Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln His Asp290 295 300Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg305 310 315 320Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu325 330 335Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu Leu Val340 345 350Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp355 360 365Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His Ser Pro His Ala Leu370 375 380Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly Thr Tyr Thr Leu Ala385 390 395 400Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu405 410 415Val ValAsn Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro420 425 430Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr435 440 445Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr450 455 460Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Gln465 470 475 480Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala Val Thr Thr Gln Asp485 490 495Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp Thr Glu Phe Val Glu500 505 510Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asn Ala Asn Val515 520 525Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu530 535 540Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile545 550 555 560Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu Leu565 570 575Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg Trp Tyr Arg580 585 590Leu Asn Leu Ser Thr Leu His Asp Ala His Gly Asn Pro Leu Leu Leu595 600 605Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Ala Ala Ser Leu610 615 620Glu Glu Val Ala Pro Gly Ala Arg HisAla Thr Leu Ser Leu Ser Ile625 630 635 640Pro Arg Val Ala Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln645 650 655Asp Arg Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val660 665 670Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu675 680 685Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala Gly Ala690 695 700His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Glu705 710 715 720Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln Lys Leu Ser Ile Gln725 730 735Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn740 745 750Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser755 760 765Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val770 775 780Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met785 790 795 800Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile805 810 815Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr Leu Ser820 825 830Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His LeuGly835 840 845Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val Val Glu Ala Ser Ala850 855 860Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr Val Ala Val Lys Met865 870 875 880Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu885 890 895Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu900 905 910Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu915 920 925Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys Arg Asp930 935 940Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln Arg Gly Arg Phe945 950 955 960Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg Arg Arg Pro Gly Ser965 970 975Ser Asp Arg Val Leu Phe Ala Arg Phe Ser Lys Thr Glu Gly Gly Ala980 985 990Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro995 1000 1005Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg1010 1015 1020Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu1025 1030 1035Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val Lys Ile1040 1045 1050Cys Asp Phe GlyLeu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr1055 1060 1065Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro1070 1075 1080Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp Val Trp1085 1090 1095Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser1100 1105 1110Pro Tyr Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu1115 1120 1125Arg Asp Gly Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala1130 1135 1140Ile Arg Arg Ile Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala1145 1150 1155Arg Pro Ala Phe Ser Glu Leu Val Glu Ile Leu Gly Asp Leu Leu1160 1165 1170Gln Gly Arg Gly Leu Gln Glu Glu Glu Glu Val Cys Met Ala Pro1175 1180 1185Arg Ser Ser Gln Ser Ser Glu Glu Gly Ser Phe Ser Gln Val Ser1190 1195 1200Thr Met Ala Leu His Ile Ala Gln Ala Asp Ala Glu Asp Ser Pro1205 1210 1215Pro Ser Leu Gln Arg His Ser Leu Ala Ala Arg Tyr Tyr Asn Trp1220 1225 1230Val Ser Phe Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly1235 1240 1245Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr12501255 1260Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser Gly Met Val1265 1270 1275Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His Arg Gln1280 1285 1290Glu Ser Gly Phe Ser Cys Lys Gly Pro Gly Gln Asn Val Ala Val1295 1300 1305Thr Arg Ala His Pro Asp Ser Gln Gly Arg Arg Arg Arg Pro Glu1310 1315 1320Arg Gly Ala Arg Gly Gly Gln Val Phe Tyr Asn Ser Glu Tyr Gly1325 1330 1335Glu Leu Ser Glu Pro Ser Glu Glu Asp His Cys Ser Pro Ser Ala1340 1345 1350Arg Val Thr Phe Phe Thr Asp Asn Ser Tyr1355 1360<210> 4<211> 102<212> PRT<213> Artificial Sequence<220><223> LFV-A Trap polypeptide<400> 4Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln1 5 10 15His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile20 25 30Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg35 40 45Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp50 55 60Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly65 70 75 80Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile85 90 95Met TyrIle Val Val Val100<210> 5<211> 211<212> PRT<213> Artificial Sequence<220><223> LFV-B Trap Polypeptide<400> 5Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln1 5 10 15His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile20 25 30Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg35 40 45Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp50 55 60Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly65 70 75 80Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile85 90 95Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu100 105 110Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu115 120 125Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp130 135 140Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp145 150 155 160Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu165 170 175Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala180 185 190Ala Ser Ser Gly Leu Met Thr Lys Lys AsnSer Thr Phe Val Arg Val195 200 205His Glu Lys210<210> 6<211> 205<212> PRT<213> Artificial sequence<220><223> LFV-C Trap polypeptide<400> 6Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr LysLys Asn Ser Thr Phe Val Arg Val His Glu Lys 195 200 205 <210> 7 <211> 305 <212> PRT <213> Artificial Sequence <220> <223> LFV-D Trap Polypeptide <400> 7 Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln 1 5 10 15 His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile 20 25 30 Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg 35 40 45 Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp 50 55 60 Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly 65 70 75 80 Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile 85 90 95 Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu 100 105 110 Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu 115 120 125 Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp 130 135 140 Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp 145 150 155 160 Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu 165 170 175 Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala 180 185 190 AlaSer Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val195 200 205His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu Val210 215 220Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu Gly225 230 235 240Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu Glu245 250 255Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val260 265 270Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro Ile275 280 285Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val Pro290 295 300Pro305<210> 8<211> 305<212> PRT<213> Artificial Sequence<220><223> LFV-E Trap polypeptide<400> 8Gly Leu Pro Ser Val Ser Leu Asp Leu Pro Arg Leu Ser Ile Gln Lys1 5 10 15Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr Leu Gln Ile Thr Cys Arg20 25 30Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Asn Gln Ser Gly Ser35 40 45Glu Gln Arg Val Glu Val Thr Glu Cys Ser Asp Gly Leu Phe Cys Lys50 55 60Thr Leu Thr Ile Pro Lys Val Ile Gly Asn Asp Thr Gly Ala Tyr Lys65 70 75 80Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser Val Ile TyrVal Tyr Val85 90 95Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly100 105 110Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys115 120 125Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro130 135 140Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys145 150 155 160Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val165 170 175Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr180 185 190Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro195 200 205Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys210 215 220Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr225 230 235 240Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys245 250 255Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile260 265 270Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser275 280 285Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu290 295 300Lys305<210>9 <211> 399 <212> PRT <213> Artificial Sequence <220><223> LFV-F Trap Polypeptide <400> 9 Gly Leu Pro Ser Val Ser Leu Asp Leu Pro Arg Leu Ser Ile Gln Lys 1 5 10 15 Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr Leu Gln Ile Thr Cys Arg 20 25 30 Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Asn Gln Ser Gly Ser 35 40 45 Glu Gln Arg Val Glu Val Thr Glu Cys Ser Asp Gly Leu Phe Cys Lys 50 55 60 Thr Leu Thr Ile Pro Lys Val Ile Gly Asn Asp Thr Gly Ala Tyr Lys 65 70 75 80 Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr Val Tyr Val 85 90 95 Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly 100 105 110 Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys 115 120 125 Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro 130 135 140 Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys 145 150 155 160 Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val 165 170 175 Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr 180 185 190 Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu SerPro195 200 205Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys210 215 220Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr225 230 235 240Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys245 250 255Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile260 265 270Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser275 280 285Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu290 295 300Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu Val Glu Ala305 310 315 320Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu Gly Tyr Pro325 330 335Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu Glu Ser Asn340 345 350His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val Ser Glu355 360 365Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro Ile Ser Lys370 375 380Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val Pro Pro385 390 395<210> 10<211> 205<21Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Ala Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys195 200 205<210> 11<211> 205<212> PRT<213> Artificial Sequence<220><223> LFV-H Trap polypeptide<400> 11Ser Pro Phe Ile AlaSer Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Gln Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Ala Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 11His Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys195 200 205<210> 13<211> 205<212> PRT<213> Artificial Sequence<220><223> LFV-J Trap polypeptide<400> 13SerPro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile 1 5 10 15 Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile 20 25 30 Ser Asn Leu Gln Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe 35 40 45 Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr 50 55 60 Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala 65 70 75 80 Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val 85 90 95 Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys 195 200 205 <210> 14 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> LFV-K Trap polypeptide <400>14Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Gln Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys195 200 205<210> 15<211> 205<212> PRT<21Trap polypeptide <400> 15Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Gln Ser Thr Phe Val Arg Val His Glu Lys195 200 205<210> 16<211> 311<212> PRT<213> Artificial sequence<220><223>LFV-M Trap polypeptide <400> 16Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His1 5 10 15Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro20 25 30Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro35 40 45Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser50 55 60Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val65 70 75 80Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn85 90 95Thr Ile Ile Gly Gly Gly Gly Ser Gly Gly Ser Pro Phe Ile Ala Ser100 105 110Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys115 120 125Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser130 135 140Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg145 150 155 160Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile165 170 175Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser180 185 190Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr195 200 205Asp Val Val Leu Ser Pro SerHis Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys<210> 17<211> 309<212> PRT<213> Artificial Sequence<220><223> LFV-N Trap polypeptide<400> 17Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser ProSer His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly195 200 205Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile210 215 220Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr225 230 235 240Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu245 250 255Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile260 265 270Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala275 280 285Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln290 295 300Thr Asn Thr Ile Ile305<210> 18<211> 211<212> PRT<213> Artificial Sequence<220><223> LFV-PTrap polypeptide <400> 18 Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln 1 5 10 15 His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile 20 25 30 Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg 35 40 45 Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp 50 55 60 Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly 65 70 75 80 Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile 85 90 95 Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu 100 105 110 Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu 115 120 125 Gln Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp 130 135 140 Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp 145 150 155 160 Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu 165 170 175 Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala 180 185 190 Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val 195 200 205 His Glu Lys 210 <210> 19 <211>315<212> PRT<213> Artificial Sequence<220><223> LFV-Q Trap Polypeptide<400> 19Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln1 5 10 15His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile20 25 30Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg35 40 45Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp50 55 60Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly65 70 75 80Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile85 90 95Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu100 105 110Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu115 120 125Gln Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp130 135 140Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp145 150 155 160Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu165 170 175Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala180 185 190Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val195200 205His Glu Lys Gly Gly Gly Gly Ser Gly Arg Pro Phe Val Glu Met Tyr210 215 220Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val225 230 235 240Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys245 250 255Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp260 265 270Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly275 280 285Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn290 295 300Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile305 310 315<210> 20<211> 311<212> PRT<213> Artificial Sequence<220><223> LFV-R Trap polypeptide<400> 20Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His1 5 10 15Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro20 25 30Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro35 40 45Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser50 55 60Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val65 70 75 80Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn8590 95Thr Ile Ile Gly Gly Gly Gly Ser Gly Gly Ser Pro Phe Ile Ala Ser100 105 110Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys115 120 125Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser130 135 140Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg145 150 155 160Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile165 170 175Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser180 185 190Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr195 200 205Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu210 215 220Lys Leu Val Leu Gln Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile225 230 235 240Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu245 250 255Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe260 265 270Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu275 280 285Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr290 295 300Phe Val Arg Val His GluLys305 310<210> 21<211> 315<212> PRT<213> Artificial sequence<220><223> LFV-S Trap polypeptide<400> 21Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln1 5 10 15His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile20 25 30Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg35 40 45Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp50 55 60Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly65 70 75 80Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile85 90 95Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu100 105 110Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu115 120 125Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp130 135 140Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp145 150 155 160Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu165 170 175Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala180 185 190Ala Ser Ser Gly Leu Met Thr Lys Lys Asn SerThr Phe Val Arg Val195 200 205His Glu Lys Gly Gly Gly Gly Ser Gly Arg Pro Phe Val Glu Met Tyr210 215 220Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val225 230 235 240Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys245 250 255Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp260 265 270Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly275 280 285Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn290 295 300Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile305 310 315<210> 22<211> 309<212> PRT<213> Artificial sequence<220><223> LFV-001 Trap polypeptide<400> 22Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr LysTyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Leu Val His Ile Thr Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295300 Arg Val His Glu Lys 305 <210> 23 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> LFV-010 Trap Polypeptide <400> 23 Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val 1 5 10 15 Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30 Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45 Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp 50 55 60 Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Gln 65 70 75 80 Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile 85 90 95 Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu 100 105 110 Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp 115 120 125 Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr 130 135 140 Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val 145 150 155 160 Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp 165 170 175 Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu 180 185 190 Ser Asn Pro Phe Leu Val HisIle Thr Gly Asn Glu Leu Tyr Asp Val 195 200 205 Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu 210 215 220 Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe 225 230 235 240 Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn 245 250 255 Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser 260 265 270 Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr 275 280 285 Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val 290 295 300 Arg Val His Glu Lys 305 <210> 24 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> LFV-002 Trap polypeptide <400> 24 Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val 1 5 10 15 Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30 Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45 Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp 50 55 60 Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn 65 70 75 8— Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys AlaArg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Leu Val His Ile Gly Tyr Arg Ile Tyr Asp Val Val195 200 205Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val210 215 220Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn225 230 235 240Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg245 250 255Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr260 265 270Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys275 280 285Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg290 295 300Val His GluLys305 <210> 25 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> LFV-003 Trap Polypeptide <400> 25 Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val 1 5 10 15 Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30 Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45 Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp 50 55 60 Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn 65 70 75 80 Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile 85 90 95 Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu 100 105 110 Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp 115 120 125 Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr 130 135 140 Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val 145 150 155 160 Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp 165 170 175 Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu 180 185 190 Ser Asn Pro Phe Leu Val His Ile Gly Gly Tyr ArgIle Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys305<210> 26<211> 309<212> PRT<213> Artificial Sequence<220><223> LFV-004 Trap polypeptide<400> 26Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Ile Val His Val Thr Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys305<210>27 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> LFV-005 Trap Polypeptide <400> 27 Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val 1 5 10 15 Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30 Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45 Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp 50 55 60 Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn 65 70 75 80 Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile 85 90 95 Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu 100 105 110 Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp 115 120 125 Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr 130 135 140 Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val 145 150 155 160 Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp 165 170 175 Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu 180 185 190 Ser Asn Pro Phe Ile Val Val Val Val Gly Tyr Arg Ile Tyr AspVal195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys3,05<210> 28<211> 309<212> PRT<213> Artificial sequence<220><223> LFV-006 Trap polypeptide<400> 28Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro2,0 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr ThrAla Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys305<210> 29<211> 309<212>PRT<213> Artificial Sequence<220><223> LFV-007 Trap Polypeptide<400> 29Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 11Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val1St5 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val195 200 It should be noted that there seems to be a small formatting issue in the original text where "11Gln" might be a typo. I've translated it as is based on the rules. If it's an error in the original, you may want to correct it for a more accurate representation.205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys305<210> 30<211> 309<212> PRT<213> Artificial Sequence<220><223> LFV-008 Trap polypeptide<400> 30Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala SerSer Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Ala Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys305<210> 31<211> 330<212> PRT<213>Human <400> 31Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys ThrIle Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 32 <211> 332 <212> PRT <213> Artificial Sequence <220> <223> Recombinant receptor molecule of LFV-A <400> 32 Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln 1 5 10 15 His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile 20 25 30 Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg 35 40 45 Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp 50 55 60 Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly 65 70 75 80 Met Val Phe Cys Glu Ala Lys Ile Asn Asp GluSer Tyr Gln Ser Ile85 90 95Met Tyr Ile Val Val Val Gly Pro Gly Asp Lys Thr His Thr Cys Pro100 105 110Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe115 120 125Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val130 135 140Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe145 150 155 160Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro165 170 175Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr180 185 190Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val195 200 205Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala210 215 220Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg225 230 235 240Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly245 250 255Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro260 265 270Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser275 280 285Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln290 295 300GlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 305 310 315 320 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 33 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Recombinant receptor molecule of LFV-B <400> 33 Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln 1 5 10 15 His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile 20 25 30 Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg 35 40 45 Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp 50 55 60 Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly 65 70 75 80 Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile 85 90 95 Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu 100 105 110 Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu 115 120 125 Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp 130 135 140 Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp 145 150 155 160 Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu 165 170175Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala180 185 190Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val195 200 205His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro210 215 220Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val245 250 255Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu275 280 285Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn370 375 380Tyr Lys Thr Thr Pro Pro ValLeu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 34 <211> 435 <212> PRT <213> Artificial Sequence <220> <223> LFV-C recombinant receptor molecule <400> 34 Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile 1 5 10 15 Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile 20 25 30 Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe 35 40 45 Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr 50 55 60 Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala 65 70 75 80 Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val 85 90 95 Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg AspLeu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Pro Gly195 200 205Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr275 280 285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val325 330 335Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser340 345 350Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu355360 365Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser420 425 430Pro Gly Lys435<210> 35<211> 535<212> PRT<213> Artificial Sequence<220><223> Recombinant receptor molecule of LFV-D<400> 35Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln1 5 10 15His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile20 25 30Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg35 40 45Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp50 55 60Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly65 70 75 80Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile85 90 95Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu100 105 110Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu115 120 125Asn Cys Thr Ala Arg Thr Glu Leu AsnVal Gly Ile Asp Phe Asn Trp130 135 140Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp145 150 155 160Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu165 170 175Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala180 185 190Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val195 200 205His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu Val210 215 220Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu Gly225 230 235 240Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu Glu245 250 255Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val260 265 270Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro Ile275 280 285Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val Pro290 295 300Pro Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro305 310 315 320Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys325 330 335Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val ValVal340 345 350Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp355 360 365Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr370 375 380Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp385 390 395 400Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu405 410 415Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg420 425 430Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys435 440 445Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp450 455 460Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys465 470 475 480Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser485 490 495Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser500 505 510Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser515 520 525Leu Ser Leu Ser Pro Gly Lys530 535<210> 36<211> 535<212> PRT<213> Artificial Sequence<220><223> Recombinant receptor molecule of LFV-E<400> 36Gly Leu Pro Ser Val Ser Leu Asp Leu Pro Arg Leu Ser Ile Gln Lys15 10 15Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr Leu Gln Ile Thr Cys Arg20 25 30Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Asn Gln Ser Gly Ser35 40 45Glu Gln Arg Val Glu Val Thr Glu Cys Ser Asp Gly Leu Phe Cys Lys50 55 60Thr Leu Thr Ile Pro Lys Val Ile Gly Asn Asp Thr Gly Ala Tyr Lys65 70 75 80Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr Val Tyr Val85 90 95Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly100 105 110Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys115 120 125Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro130 135 140Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys145 150 155 160Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val165 170 175Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr180 185 190Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro195 200 205Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys210 215 220Thr Ala Arg Thr Glu Leu Asn Val Gly IleAsp Phe Asn Trp Glu Tyr225 230 235 240Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys245 250 255Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile260 265 270Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser275 280 285Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu290 295 300Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro305 310 315 320Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys325 330 335Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val340 345 350Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp355 360 365Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr370 375 380Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp385 390 395 400Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu405 410 415Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg420 425 430Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys435440 445Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp450 455 460Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys465 470 475 480Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser485 490 495Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser500 505 510Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser515 520 525Leu Ser Leu Ser Pro Gly Lys530 535<210> 37<211> 629<212> PRT<213> Artificial Sequence<220><223> LFV-F recombinant receptor molecule<400> 37Gly Leu Pro Ser Val Ser Leu Asp Leu Pro Arg Leu Ser Ile Gln Lys1 5 10 15Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr Leu Gln Ile Thr Cys Arg20 25 30Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Asn Gln Ser Gly Ser35 40 45Glu Gln Arg Val Glu Val Thr Glu Cys Ser Asp Gly Leu Phe Cys Lys50 55 60Thr Leu Thr Ile Pro Lys Val Ile Gly Asn Asp Thr Gly Ala Tyr Lys65 70 75 80Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr Val Tyr Val85 90 95Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly100 105 110Val Val Tyr IleThr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys115 120 125Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro130 135 140Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys145 150 155 160Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val165 170 175Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr180 185 190Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro195 200 205Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys210 215 220Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr225 230 235 240Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys245 250 255Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile260 265 270Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser275 280 285Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu290 295 300Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu Val Glu Ala305 310 315 320Thr Val Gly Glu Arg Val Arg Ile Pro AlaLys Tyr Leu Gly Tyr Pro325 330 335Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu Glu Ser Asn340 345 350His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val Ser Glu355 360 365Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro Ile Ser Lys370 375 380Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val Pro Pro Gly385 390 395 400Pro Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu405 410 415Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr420 425 430Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val435 440 445Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val450 455 460Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser465 470 475 480Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu485 490 495Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala500 505 510Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro515 520 525Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln530 535540Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala545 550 555 560Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr565 570 575Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu580 585 590Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser595 600 605Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser610 615 620Leu Ser Pro Gly Lys625<210> 38<211> 435<212> PRT<213> Artificial Sequence<220><223> LFV-G recombinant receptor molecule<400> 38Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Ala Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys LeuVal Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Pro Gly195 200 205Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr275 280 285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu ProGln Val 325 330 335 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 340 345 350 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360 365 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 370 375 380 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 385 390 395 400 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 405 410 415 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430 Pro Gly Lys 435 <210> 39 <211> 435 <212> PRT <213> Artificial Sequence <220> <223> LFV-H recombinant receptor molecule <400> 39 Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile 1 5 10 15 Thr Glu Asn Lys Gln Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile 20 25 30 Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe 35 40 45 Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr 50 55 60 Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala 65 70 75 80 Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val 85 90 95 Val Gly Tyr Arg Ile TyrAsp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Pro Gly195 200 205Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr275 280 285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu ProAla Pro Ile 305 310 315 320 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 340 345 350 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360 365 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 370 375 380 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 385 390 395 400 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 405 410 415 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430 Pro Gly Lys 435 <210> 40 <211> 435 <212> PRT <213> Artificial Sequence <220> <223> LFV-I recombinant receptor molecule <400> 4 Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile 1 5 10 15 Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile 20 25 30 Ser Asn Leu Ala Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe 35 40 45 Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr 50 55 60 Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala 65 70 75 80 Lys Ile AsnAsp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Pro Gly195 200 205Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr275 280 285Arg Val Val Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn Gly290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val325 330 335Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser340 345 350Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu355 360 365Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser420Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Pro Gly195 200 205Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val260 265 270His Asn Ala Lys Thr Lys Pro Arg GluGlu Gln Tyr Asn Ser Thr Tyr275 280 285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val325 330 335Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser340 345 350Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu355 360 365Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser420 425 430Pro Gly Lys435<210> 42<211> 435<212> PRT<213> Artificial Sequence<220><223> LFV-K recombinant receptor molecule<400> 42Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe3540 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Gln Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Pro Gly195 200 205Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His245 250 255Glu Asp Pro Glu Val Lys Phe AsnTrp Tyr Val Asp Gly Val Glu Val260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr275 280 285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val325 330 335Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser340 345 350Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu355 360 365Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser420 425 430Pro Gly Lys435<210> 43<211> 435<212> PRT<213> Artificial Sequence<220><223> LFV-L recombinant receptor molecule<400> 43Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu GlySer Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Gln Ser Thr Phe Val Arg Val His Glu Lys Gly Pro Gly195 200 205Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro GluVal Thr Cys Val Val Val Asp Val Ser His245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr275 280 285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val325 330 335Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser340 345 350Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu355 360 365Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser420 425 430Pro Gly Lys435<210> 44<211> 541<212> PRT<213> Artificial Sequence<220><Glu Ile Ile His1 5 10 15Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro20 25 30Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro35 40 45Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser50 55 60Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val65 70 75 80Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn85 90 95Thr Ile Ile Gly Gly Gly Gly Ser Gly Gly Ser Pro Phe Ile Ala Ser100 105 110Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys115 120 125Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser130 135 140Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg145 150 155 160Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile165 170 175Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser180 185 190Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr195 200 205Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu210 215 220Lys Leu Val Leu AsnCys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile225 230 235 240Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu245 250 255Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe260 265 270Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu275 280 285Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr290 295 300Phe Val Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys305 310 315 320Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu325 330 335Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu340 345 350Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys355 360 365Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys370 375 380Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu385 390 395 400Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys405 410 415Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys420 425 430Ala Lys Gly Gln Pro Arg Glu Pro Gln Val TyrThr Leu Pro Pro Ser435 440 445Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys450 455 460Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln465 470 475 480Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly485 490 495Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln500 505 510Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn515 520 525His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys530 535 540<210> 45<211> 539<212> PRT<213> Artificial Sequence<220><223> Recombinant receptor molecule LFV-N<400> 45Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val ValLeu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly195 200 205Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile210 215 220Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr225 230 235 240Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu245 250 255Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile260 265 270Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala275 280 285Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln290 295 300Thr Asn Thr Ile Ile Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp435 440 445Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475 480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly500 505 510Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr515 520 525Thr Gln Lys Ser LeuSer Leu Ser Pro Gly Lys530 535<210> 46<211> 652<212> PRT<213> Artificial sequence<220><223> Recombinant receptor molecule of LFV - O<400> 46Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile1 5 10 15Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile20 25 30Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe35 40 45Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr50 55 60Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala65 70 75 80Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val85 90 95Val Gly Tyr Arg Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met180 185 190Thr Lys Lys Asn Ser ThrPhe Val Arg Val His Glu Lys Gly Pro Gly195 200 205Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr275 280 285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val325 330 335Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser340 345 350Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu355 360 365Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe SerCys Ser Val Met405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser420 425 430Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly435 440 445Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile450 455 460Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr465 470 475 480Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu485 490 495Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile500 505 510Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala515 520 525Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln530 535 540Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu545 550 555 560Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu565 570 575Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His580 585 590Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser595 600 605Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg610 615 620SerAsp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr625 630 635 640Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys645 650<210> 47<211> 441<212> PRT<213> Artificial Sequence<220><223> Recombinant receptor molecule LFV-P<400> 47Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln1 5 10 15His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile20 25 30Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg35 40 45Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp50 55 60Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly65 70 75 80Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile85 90 95Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu100 105 110Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu115 120 125Gln Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp130 135 140Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp145 150 155 160Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu165 170175Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala180 185 190Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val195 200 205His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro210 215 220Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val245 250 255Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu275 280 285Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln325 330 335Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu340 345 350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn370 375 380Tyr Lys Thr Thr Pro Pro ValLeu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 48 <211> 545 <212> PRT <213> Artificial Sequence <220> <223> LFV-Q recombinant receptor molecule <400> 48 Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln 1 5 10 15 His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile 20 25 30 Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg 35 40 45 Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp 50 55 60 Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala Gly 65 70 75 80 Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser Ile 85 90 95 Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val Leu 100 105 110 Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu 115 120 125 Gln Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp 130 135 140 Glu Tyr Pro Ser Ser Lys His Gln His LysLys Leu Val Asn Arg Asp145 150 155 160Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu165 170 175Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala180 185 190Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val195 200 205His Glu Lys Gly Gly Gly Gly Ser Gly Arg Pro Phe Val Glu Met Tyr210 215 220Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val225 230 235 240Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys245 250 255Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp260 265 270Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly275 280 285Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn290 295 300Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Gly Pro Gly Asp Lys305 310 315 320Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro325 330 335Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser340 345 350Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp355360 365Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn370 375 380Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val385 390 395 400Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu405 410 415Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys420 425 430Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr435 440 445Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr450 455 460Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu465 470 475 480Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu485 490 495Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys500 505 510Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu515 520 525Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly530 535 540Lys545 <210> 49 <211> 541 <212> PRT <213> Artificial Sequence <220> <223> LFV-R recombinant receptor molecule <400> 49Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His1 5 10 15Met Thr Glu Gly Arg Glu Leu 360 365Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn370 375 380Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val385 390 395 400Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu405 410 415Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys420 425 430Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr435 440 445Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr450 455 460Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu465 470 475 480Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu485 490 495Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys500 505 510Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu515 520 525Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly530 535 540Lys545 <210> 49 <211> 541 <212> PRT <213> Artificial Sequence <220> <223> LFV-R recombinant receptor molecule <400> 49Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His1 5 10 15Met Thr Glu Gly Arg Glu LeuVal Ile Pro Cys Arg Val Thr Ser Pro20 25 30Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro35 40 45Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser50 55 60Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val65 70 75 80Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn85 90 95Thr Ile Ile Gly Gly Gly Gly Ser Gly Gly Ser Pro Phe Ile Ala Ser100 105 110Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys115 120 125Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser130 135 140Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg145 150 155 160Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile165 170 175Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser180 185 190Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr195 200 205Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu210 215 220Lys Leu Val Leu Gln Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile225 230 235240Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu245 250 255Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe260 265 270Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu275 280 285Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr290 295 300Phe Val Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys305 310 315 320Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu325 330 335Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu340 345 350Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys355 360 365Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys370 375 380Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu385 390 395 400Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys405 410 415Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys420 425 430Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser435 440 445Arg Asp Glu Leu Thr Lys AsnGln Val Ser Leu Thr Cys Leu Val Lys 450 455 460 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 465 470 475 480 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 485 490 495 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 500 505 510 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 515 520 525 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535 540 <210> 50 <211> 545 <212> PRT <213> Artificial Sequence <220> <223> LFV-S recombinant receptor molecule <400> 50 Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln 1 5 10 15 His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile 20 25 3Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu115 120 125Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp130 135 140Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp145 150 155 160Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu165 170 175Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala180 185 190Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val195 200 205His Glu Lys Gly Gly Gly Gly Ser Gly Arg Pro Phe Val Glu Met Tyr210 215 220Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val225 230 235 240Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys245 250 255Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp260 265 270Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly275 280 285Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn290 295 300Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Gly Pro Gly Asp Lys305 310 315 320Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluLeu Leu Gly Gly Pro325 330 335Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser340 345 350Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp355 360 365Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn370 375 380Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val385 390 395 400Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu405 410 415Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys420 425 430Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr435 440 445Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr450 455 460Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu465 470 475 480Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu485 490 495Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys500 505 510Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu515 520 525Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly530 535540 Lys 545 <210> 51 <211> 539 <212> PRT <213> Artificial Sequence <220> <223> LFV-001 Recombinant Receptor Molecule <400> 51 Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val 1 5 10 15 Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30 Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45 Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp 50 55 60 Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn 65 70 75 80 Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile 85 90 95 Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu 100 105 110 Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp 115 120 125 Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr 130 135 140 Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val 145 150 155 160 Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp 165 170 175 Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu 180 185 190 Ser Asn Pro Phe Leu Val His Ile Thr Gly TyrArg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp435 440 445Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475 480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly500 505 510Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr515 520 525Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys530 535<210> 52<211> 539<212> PRT<213> Artificial Sequence<220><223> Recombinant receptor molecule LFV-010<400> 52Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Gln65 70 7580Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Leu Val His Ile Thr Gly Asn Glu Leu Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu MetThr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp435 440 445Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475 480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp GlnGln Gly500 505 510Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr515 520 525Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys530 535<210> 53<211> 538<212> PRT<213> Artificial Sequence<220><223> LFV-002 Recombinant Receptor Molecule<400> 53Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu HisAsp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Leu Val His Ile Gly Tyr Arg Ile Tyr Asp Val Val195 200 205Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val210 215 220Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn225 230 235 240Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg245 250 255Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr260 265 270Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys275 280 285Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg290 295 300Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro Cys305 310 315 320Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro325 330 335Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys340 345 350Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp355 360 365Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu370 375 380Glu Gln Tyr AsnSer Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 385 390 395 400 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 405 410 415 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 420 425 430 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 435 440 445 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 450 455 460 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 465 470 475 480 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 485 490 495 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 500 505 510 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 515 520 525 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535 <210> 54 <211> 539 <212> PRT <213> Artificial Sequence <220> <223> LFV-003 recombinant receptor molecule <400> 54 Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val 1 5 10 15 Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30 Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45 Lys Asp SerGlu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Leu Val His Ile Gly Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met LysLys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp435 440 445Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly500 505 510Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr515 520 525Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys530 535<210> 55<211> 539<212> PRT<213> Artificial Sequence<220><223> LFV-004 Recombinant Receptor Molecule<400> 55Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140LeuArg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Ile Val His Val Thr Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val SerHis Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp435 440 445Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475 480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly500 505 510Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr515 520 525Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys530 535<210> 56<211> 539<212> PRT<213> Artificial Sequence<220><223> Recombinant receptor molecule LFV-005<400> 56Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr GlyAsp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile AspPhe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp435 440 445Glu Leu ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 450 455 460 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 465 470 475 480 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 485 490 495 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 500 505 510 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 515 520 525 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535 <210> 57 <211> 539 <212> PRT <213> Artificial Sequence <220> <223> LFV-006 recombinant receptor molecule <400> 57 Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val 1 5 10 15 Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30 Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45 Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp 50 55 60 Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn 65 70 75 80 Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile 85 90 95 Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu 100 105 110 Gln Pro Phe IleAsn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly ProSer Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp435 440 445Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475 480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly500 505 510Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr515 520 525Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys530 535<210> 58<211>539<212> PRT<213> Artificial Sequence<220><223> LFV-007 Recombinant Receptor Molecule<400> 58Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val195200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val290 295 300Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410 415Asn Lys Ala Leu ProAla Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 420 425 430 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 435 440 445 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 450 455 460 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 465 470 475 480 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 485 490 495 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 500 505 510 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 515 520 525 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535 <210> 59 <211> 539 <212> PRT <213> Artificial Sequence <220> <223> Recombinant receptor molecule LFV-008 <400> 59 Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val 1 5 10 15 Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30 Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45 Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp 50 55 60 Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn 65 70 75 80 Asp Thr Gly Ser TyrVal Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu180 185 190Ser Ala Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val195 200 205Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe225 230 235 240Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn245 250 255Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser260 265 270Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr275 280 285Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn SerThr Phe Val290 295 300Arg Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro305 310 315 320Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro325 330 335Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr340 345 350Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn355 360 365Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg370 375 380Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val385 390 395 400Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser405 410 415Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys420 425 430Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp435 440 445Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe450 455 460Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu465 470 475 480Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe485 490 495Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly500 505 510AsnVal Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr515 520 525Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys530 535

Claims

1. An isolated ligand-binding molecule consisting, from N-terminus to C-terminus, of: The VEGFR1-derived sequence is operably linked to the VEGFR2-derived sequence, and the VEGFR2-derived sequence is operably linked to the Fc region of an immunoglobulin, wherein the VEGFR2-derived sequence is SEQ ID NO: 14, and the VEGFR1-derived sequence consists of amino acids 132-230 of the VEGFR1 protein shown in SEQ ID NO: 1, The ligand binding molecule can act as a trap molecule to bind to and capture VEGF-A, VEGF-C, VEGF-D, PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC.

2. The ligand-binding molecule of claim 1, which has enhanced binding ability to at least three of VEGF-A, VEGF-C, VEGF-D, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC compared to native VEGFR2.

3. The ligand binding molecule of claim 1, which is capable of inhibiting or blocking the binding of at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF-AA, PDGF-AB, PDGF-BB, PlGF and PDGF-CC proteins to the corresponding receptor of the protein.

4. The ligand binding molecule of claim 3, wherein the receptor is selected from the group consisting of a VEGFR-1 receptor, a VEGFR-2 receptor, a VEGFR-3 receptor, a PDGFR-α receptor, and a PDGFR-β receptor.

5. The ligand binding molecule of any one of claims 1 to 4, wherein the VEGFR2-derived sequence is linked to the VEGFR1-derived sequence via a linker. The ligand binding molecule of claim 5 , wherein the linker is GGGGS.

7. The ligand binding molecule of any one of claims 1-4, wherein the immunoglobulin is selected from the group consisting of IgA, IgM, IgE, IgD and IgG isotypes, including IgG1, IgG2, IgG3, IgG4 isotypes. The ligand-binding molecule of claim 7 , wherein the Fc region is the Fc region of human IgG1 or a variant thereof.

9. The ligand-binding molecule of claim 7, further comprising a hinge region.

10. The ligand-binding molecule of claim 7, wherein the Fc region of the immunoglobulin is located at the C-terminus of the ligand-binding molecule and is connected to other parts via a linker.

11. The ligand-binding molecule of claim 7, wherein the Fc region is as shown in the amino acid sequence 104-330 of SEQ ID No: 31, and the constructed ligand molecule has a purity of more than 95%.

12. The ligand-binding molecule of claim 11, consisting of the sequence of SEQ ID No:

49.

13. The ligand binding molecule of claim 3, wherein the VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC are from or derived from humans, mice, rats, or cynomolgus monkeys.

14. The ligand-binding molecule of claim 3 or 4, wherein the receptor is in the form of a monomer, a homodimer, or a heterodimer.

15. The ligand-binding molecule of claim 1, which can be produced with a purity of greater than 95%.

16. A conjugate comprising the ligand binding molecule of any one of claims 1 to 15 conjugated to at least one moiety selected from the group consisting of a modifying moiety, a detectable label, and a purification moiety.

17. The conjugate of claim 16, wherein the modifying moiety is a polyethylene glycol moiety.

18. The conjugate of claim 17, wherein the polyethylene glycol moiety is attached to the amino terminus of the ligand binding molecule.

19. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding the ligand binding molecule of any one of claims 1-15. A vector comprising the nucleic acid molecule of claim 19 .

21. The vector of claim 20, wherein the vector is selected from the group consisting of a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, a liposome vector, and any combination thereof.

22. The vector of claim 21, wherein the vector is a replication-defective adenoviral vector, wherein the nucleic acid molecule is operably linked to a promoter and is flanked by adenoviral polynucleotide sequences.

23. A host cell transformed or transfected with the nucleic acid molecule of claim 19 or the vector of any one of claims 20 to 22. The host cell according to claim 23 , which is a eukaryotic cell. The host cell according to claim 24 , which is a Chinese hamster ovary (CHO) cell or a human cell.

26. A method for preparing a ligand binding molecule, comprising the steps of: - culturing the host cell according to any one of claims 23 to 25 under suitable conditions to express the ligand binding molecule; and - isolating the ligand-binding molecule from the culture supernatant of the host cells.

27. A pharmaceutical composition comprising the ligand-binding molecule of any one of claims 1-15 and a pharmaceutically acceptable carrier.

28. The pharmaceutical composition of claim 27, which is formulated for topical administration, intravitreal injection, or intravitreal implant administration.

29. The pharmaceutical composition of claim 27 or 28, which is in the form of a paste, ointment, gel, aerosol, spray, polymer, film, emulsion or suspension.

30. Use of the ligand binding molecule of any one of claims 1-15 in the preparation of a medicament for treating neovascularization, tissue fibrosis, proliferation, and diseases or conditions caused by neovascularization in a subject, wherein the disease or condition is selected from choroidal vasculopathy or neovascularization, retinal angiogenesis, and macular angiogenesis.

31. The use of claim 30, wherein the medicament is topically administered to the eye of the subject.

32. The use of claim 30 or 31, wherein the medicament is administered by intravitreal injection or by administration through an intravitreal implant.

33. A kit comprising the ligand binding molecule of any one of claims 1-15 or the pharmaceutical composition of any one of claims 27-28 in a container.

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