Human antibodies against Middle East Respiratory Syndrome - Coronavirus spike protein

Abstract

The present invention provides monoclonal antibodies that bind to the Middle East Respiratory Syndrome - Coronavirus (MERS-CoV) spike protein, and methods of using the same. In various embodiments of the present invention, the antibodies are full-length human antibodies that bind to the MERS-CoV spike protein. In some embodiments, the antibodies of the present invention can be used to inhibit or neutralize MERS-CoV activity, and thus provide a means for treating or preventing MERS infection in humans. In some embodiments, the present invention provides a combination of one or more antibodies that bind to the MERS-CoV spike protein for treating MERS infection. In some embodiments, the one or more antibodies bind to different non-competing epitopes contained within the receptor-binding domain of the MERS-CoV spike protein.

Description

[0001] This application is a divisional application of PCT application PCT / US2015 / 031800, filed on May 20, 2015, with the invention title of "Human Antibodies Against the Spike Protein of Middle East Respiratory Syndrome - Coronavirus", which entered the Chinese national phase on November 22, 2016, and has the application number 201580026696.8. Field of the Invention

[0002] The present invention relates to human antibodies and antigen - binding fragments of human antibodies that specifically bind to the spike protein of Middle East Respiratory Syndrome - Coronavirus (MERS - CoV), and to therapeutic and diagnostic methods using these antibodies.

[0003] Statement of Related Art

[0004] Middle East Respiratory Syndrome - Coronavirus (MERS - CoV) is a newly emerging β - coronavirus that causes severe acute respiratory disease. It was first isolated in Saudi Arabia in 2012 (Zaki et al. 2012, NEJM 367:1814 - 1820) and has since spread to approximately 18 countries, with the majority of cases in Saudi Arabia and the United Arab Emirates. As of May 15, 2014, the World Health Organization reported 571 cases of MERS, including 171 deaths. Two cases of MERS infection were recently detected in the United States. The clinical features of MERS - CoV infection in humans range from asymptomatic infection to very severe pneumonia, with the potential for the development of acute respiratory distress syndrome, septic shock, and multi - organ failure leading to death.

[0005] MERS - CoV shares similarities with bat coronaviruses HKU4 and HKU5. The virus uses its spike protein to interact with cell receptors for entry into target cells. Raj et al. demonstrated that the virus binds to dipeptidyl peptidase 4 (DPP4) on human epithelial and endothelial cells via the receptor - binding domain of its spike protein (Raj et al. 2013, Nature 495:251 - 256). Lu et al. showed in 2013 that the MERS - CoV receptor - binding domain consists of a core and a receptor - binding sub - domain that interacts with DPP4 (Lu et al. 2013, Nature 500:227 - 231).

[0006] WO2014 / 045254 describes the isolation and characterization of MERS-CoV, the spike protein, and polyclonal antibodies against the receptor-binding domain of the spike protein. Neutralizing monoclonal antibodies against the receptor-binding domain of the spike protein have been disclosed, for example, by Du et al. (2014, J. Virol.), Ying et al. (2014, J. Virol.), Tang et al. (2014, PNAS), and Jiang et al. (2014, Sci. Transl. Med. Vol. 6, 234ra59).

[0007] To date, there are no vaccines or therapeutic agents for the prevention or treatment of MERS infection. Due to its continued threat to human health and high fatality rate (over 30%), there is an urgent need for prophylactic and therapeutic antiviral therapies for MERS control. Full-length human antibodies that specifically bind to the MERS-CoV spike protein with high affinity and inhibit viral infectivity may be important for the prevention and treatment of MERS infection. Summary of the Invention

[0008] The present invention provides antibodies and antigen-binding fragments thereof that bind to the MERS-CoV spike protein. The antibodies of the present invention can be particularly used to inhibit or neutralize the activity of the MERS-CoV spike protein. In some embodiments, the antibodies are used to block the binding of the virus to its host cell receptor dipeptidyl peptidase 4 (DPP4) and to prevent entry of the MERS-coronavirus into host cells. In some embodiments, the antibodies function by inhibiting cell-to-cell transfer of the virus. In some embodiments, the antibodies are used to prevent, treat, or alleviate at least one symptom in a subject. In some embodiments, the antibodies can be administered prophylactically or therapeutically to a subject having or at risk of having an MERS-CoV infection.

[0009] The antibodies of the present invention can be full-length (e.g., IgG1 or IgG4 antibodies) or can comprise only the antigen-binding portion (e.g., Fab, F(ab’)2, or scFv fragments), and can be modified to affect functionality, e.g., to increase persistence in the host or to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933). In some embodiments, the antibodies can be bispecific.

[0010] In a first aspect, the present invention provides an isolated recombinant monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the MERS-CoV spike protein. In some embodiments, the antibody is a full-length human monoclonal antibody. The antibodies of the present invention and their antigen-binding fragments bind to an epitope within the receptor-binding domain (RBD) of the spike protein of MERS-CoV. In some embodiments, the present invention provides antibodies and their antigen-binding fragments that bind to an amino acid selected from amino acids 367-606 of GenBank accession number AFS88936.1 (SEQ ID NO: 457). In one embodiment, the antibody of the present invention binds to the spike protein of the MERS-CoV isolate EMC / 2012. In some embodiments, the antibody binds to the spike proteins of different MERS-CoV isolates.

[0011] Exemplary anti-MERS-CoV-S antibodies of the present invention are listed in Tables 2 and 3 herein. Table 2 describes the amino acid sequence identifiers of the heavy-chain variable region (HCVR), light-chain variable region (LCVR), heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), and light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of the exemplary anti-MERS-CoV-S antibodies. Table 3 describes the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-MERS-CoV-S antibodies.

[0012] The present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR having an amino acid sequence selected from the HCVR amino acid sequences listed in Table 2, or a sequence that is substantially similar thereto and has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0013] The present invention also provides an antibody or an antigen-binding fragment thereof comprising an LCVR having an amino acid sequence selected from the LCVR amino acid sequences listed in Table 2, or a sequence that is substantially similar thereto and has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0014] The present invention also provides an antibody or an antigen-binding fragment thereof comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR), wherein the sequence pair comprises any HCVR amino acid sequence listed in Table 2 paired with any LCVR amino acid sequence listed in Table 2. According to some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair, wherein the HCVR / LCVR amino acid sequence pair is comprised within any exemplary anti-MERS-CoV-S antibody listed in Table 2. In some embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of: SEQ ID NO:2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 106, 122 / 106, 130 / 106, 138 / 106, 146 / 106, 154 / 162, 170 / 162, 178 / 162, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, 378 / 386, 394 / 402, 410 / 418, 426 / 434, and 442 / 450. In some embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of the following: SEQ ID NO:2 / 10 (e.g., H1H15177P), 18 / 26 (e.g., H1H15188P), 66 / 74 (e.g., H1H15211P), 114 / 106 (e.g., H1H15231P2), 170 / 162 (e.g., H1H15260P2), or 218 / 226 (e.g., H1H15277N).

[0015] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1), wherein the heavy chain CDR1 (HCDR1) comprises an amino acid sequence selected from any HCDR1 amino acid sequence listed in Table 2 or a sequence that is substantially similar thereto and has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2), wherein the heavy chain CDR2 (HCDR2) comprises an amino acid sequence selected from any HCDR2 amino acid sequence listed in Table 2 or a sequence that is substantially similar thereto and has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0017] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3), wherein the amino acid sequence of the heavy chain CDR3 (HCDR3) is selected from any of the HCDR3 amino acid sequences listed in Table 2 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity that is substantially similar thereto.

[0018] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1), wherein the amino acid sequence of the heavy chain CDR1 (LCDR1) is selected from any of the LCDR1 amino acid sequences listed in Table 2 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity that is substantially similar thereto.

[0019] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2), wherein the amino acid sequence of the heavy chain CDR2 (LCDR2) is selected from any of the LCDR2 amino acid sequences listed in Table 2 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity that is substantially similar thereto.

[0020] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3), wherein the amino acid sequence of the heavy chain CDR3 (LCDR3) is selected from any of the LCDR3 amino acid sequences listed in Table 2 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity that is substantially similar thereto.

[0021] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3), wherein the HCDR3 / LCDR3 comprises a pairing of any HCDR3 amino acid sequence listed in Table 2 with any LCDR3 amino acid sequence listed in Table 2. According to some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair, wherein the HCDR3 / LCDR3 amino acid sequence pair is comprised in any exemplary anti-MERS-CoV-S antibody listed in Table 2. In some embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of: SEQ ID NO:8 / 16 (e.g., H1H15177P), 24 / 32 (e.g., H1H15188P), 72 / 80 (e.g., H1H15211P), 120 / 112 (e.g., H1H15231P2), 176 / 168 (e.g., H1H15260P2) and 224 / 232 (e.g., H1H15277N).

[0022] The present invention also provides an antibody or an antigen-binding fragment thereof comprising 6 CDR sets (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), said sets being comprised in any of the exemplary anti-MERS-CoV-S antibodies listed in Table 2. In some embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence sets are selected from the group consisting of: SEQ ID NO:4-6-8-12-14-16 (e.g., H1H15177P), 20-22-24-28-30-32 (e.g., H1H15188P); 68-70-72-76-78-80 (e.g., H1H15211P); 116-118-120-108-110-112 (e.g., H1H15231P2); 172-174-176-164-166-168 (e.g., H1H15260P2) and 220-222-224-228-230-232 (e.g., H1H15277N).

[0023] In related embodiments, the invention provides an antibody or antigen-binding fragment thereof of 6 CDR sets (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), said sets comprising HCVR / LCVR amino acid sequence pairs defined by any of the exemplary anti-MERS-CoV-S antibodies listed in Table 2. For example, the invention includes an antibody or antigen-binding fragment thereof containing the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set, said set comprising HCVR / LCVR amino acid sequence pairs selected from the group consisting of: SEQ ID NO:2 / 10 (e.g., H1H15177P), 18 / 26 (e.g., H1H15188P); 66 / 74 (e.g., H1H15211P); 114 / 106 (e.g., H1H15231P2); 170 / 162 (e.g., H1H15260P2) and 218 / 226 (e.g., H1H15277N). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are known in the art and can be used to identify CDRs in the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to define CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Publicly available databases can be accessed to identify CDR sequences in antibodies.

[0024] The invention includes anti-MERS-CoV-S antibodies having a modified glycosylation pattern. In some embodiments, modifications that remove unwanted glycosylation sites may be useful, or antibodies lacking the fucose moiety on the oligosaccharide chain may increase antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, glycosylation modifications can be made to modify complement-dependent cytotoxicity (CDC).

[0025] The present invention also provides antibodies and antigen-binding fragments thereof that compete with an antibody or an antigen-binding fragment thereof containing the CDRs of HCVR and the CDRs of LCVR for specific binding to MERS-CoV-S, wherein each of HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 2.

[0026] The present invention also provides antibodies and antigen-binding fragments thereof that cross-compete with a reference antibody or an antigen-binding fragment thereof containing the CDRs of HCVR and the CDRs of LCVR for binding to MERS-CoV-S, wherein each of HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 2.

[0027] In some embodiments, the antibody or its antigen-binding fragment can specifically bind to MERS-CoV-S in an agonist manner, i.e., it can enhance or stimulate MERS-CoV-S binding and / or activity; in other embodiments, the antibody can specifically bind to MERS-CoV-S in an antagonist manner, i.e., it can block the binding of MERS-CoV-S to its receptor (DPP4).

[0028] The present invention also provides isolated antibodies and antigen-binding fragments thereof that block the binding of the MERS-CoV spike protein to DPP4. In some embodiments, the antibody or its antigen-binding fragment that blocks the binding of the MERS-CoV spike protein to DPP4 can bind to the same epitope on the MERS-CoV spike protein as DPP4 or can bind to an epitope on the MERS-CoV spike protein different from DPP4. In some embodiments, the present invention provides an antibody or its antigen-binding fragment that blocks the binding of MERS-CoV-S to DPP4 of human, camel or bat.

[0029] In some embodiments, the antibody or antigen-binding fragment of the present invention is bispecific and comprises a first binding specificity for a first epitope in the receptor-binding domain of the MERS-CoV spike protein and a second binding specificity for a second epitope in the receptor-binding domain of the MERS-CoV spike protein, wherein the first and second epitopes are different and non-overlapping.

[0030] In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment having one or more of the following characteristics: (a) is a full-length human monoclonal antibody; (b) is isolated from a hybridoma cell line selected from the group consisting of: HBVX06H05, HBVX11H04, HBVX11D02, HBVZ10E10, HBVY09F08, HBVZ05G02, HBVZ09B06, HBVY01F08, HBVY10G02, HBVY04B06, HBVY07D10, HBVZ08A09, HBVZ05G04, HBVY06C07, HBVY03H06, HBVZ10G06, HBVZ04F10, HBVX11E09, HBVY06H09, HBVZ05B11, HBVY02E05, and HBVZ04C07; (c) interacts with one or more amino acid residues in the receptor-binding domain of the MERS-CoV spike protein, said amino acid residues being selected from amino acid residues 367-606 of SEQ ID NO: 457; (d) binds to the MERS-CoV spike protein with a dissociation constant (K -9 ) of less than 10 D M, as measured in a surface plasmon resonance assay; (e) blocks greater than 90% of the binding of the MERS-CoV spike protein to dipeptidyl peptidase 4 (DPP4); as measured in a blocking ELISA assay; (f) neutralizes greater than 90% of the infectivity of MERS-CoV in human host cells and has an IC 50, as measured in a virus-like particle (VLP) neutralization assay; (g) neutralizing MERS-CoV infectivity, wherein the MERS-CoV comprises an isolate of a virus selected from the group consisting of: EMC / 2012, Jordan-N3 / 2012, England-Qatar / 2012, Al-Hasa_1_2013, Al-Hasa_2_2013, Al-Hasa_3_2013, Al-Hasa_4_2013, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17, Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-hasa_25, Bisha_1, Buraidah_1, England 1, Hafr-Al-batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1, KFU-HKU 1, KFU-HKU 13, Munich, Qatar3, Qatar4, Riyadh_1, Riyadh_2, Riyadh_3, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_9, Riyadh_14, Taif_1, UAE, and Wadi-Ad-Dawasir; and (h) being a bispecific antibody that comprises a first binding specificity for a first epitope in the receptor-binding domain of the MERS-CoV spike protein and a second binding specificity for a second epitope in the receptor-binding domain of the MERS-CoV spike protein, wherein the first and second epitopes are different and non-overlapping.

[0031] In a second aspect, the invention provides a nucleic acid molecule encoding an anti-MERS-CoV-S antibody or a portion thereof. For example, the invention provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 2; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0032] The invention also provides a nucleic acid molecule encoding any of the LCVR amino acid sequences listed in Table 2, and in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0033] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 2; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 3 or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0034] The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 2; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 3 or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0035] The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 2; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 3 or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0036] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 2; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 3 or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0037] The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 2; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 3 or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0038] The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 2; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 3 or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0039] The present invention also provides nucleic acid molecules encoding HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1 - HCDR2 - HCDR3), wherein the HCDR1 - HCDR2 - HCDR3 amino acid sequence set is defined by any of the exemplary anti - MERS - CoV - S antibodies listed in Table 2.

[0040] The present invention also provides nucleic acid molecules encoding LCVR, wherein the LCVR comprises a group of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and the amino acid sequence group of LCDR1-LCDR2-LCDR3 is defined as that of any exemplary anti-MERS-CoV-S antibody listed in Table 2.

[0041] The present invention also provides nucleic acid molecules encoding HCVR and LCVR, wherein the HCVR comprises an amino acid sequence of any HCVR amino acid sequence listed in Table 2, and wherein the LCVR comprises an amino acid sequence of any LCVR amino acid sequence listed in Table 2. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCVR nucleic acid sequence listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any LCVR nucleic acid sequence listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In some embodiments, according to this aspect of the present invention, the nucleic acid molecule encodes HCVR and LCVR, wherein both the HCVR and LCVR are derived from the same anti-MERS-CoV-S antibody listed in Table 2.

[0042] The present invention provides nucleic acid molecules encoding the heavy chain amino acid sequences of any of those listed in Table 2. The present invention also provides nucleic acid molecules encoding the light chain amino acid sequences of any of those listed in Table 2.

[0043] In a related aspect, the present invention provides recombinant expression vectors capable of expressing polypeptides containing the heavy and light chain variable regions of an anti-MERS-CoV-S antibody. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR and / or CDR sequences listed in Table 3. Also included within the scope of the present invention are host cells into which the vector has been introduced, and methods for producing an antibody or a portion thereof by culturing the host cells under conditions permitting the production of the antibody or antibody fragment, and recovering the antibody and antibody fragment so produced.

[0044] In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, wherein the monoclonal antibody or the antigen-binding fragment thereof specifically binds to the MERS-CoV spike protein. In a related aspect, the present invention features a composition that is a combination of an anti-MERS-CoV-S antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-MERS-Co-V-S antibody. Exemplary agents that can be advantageously combined with the anti-MERS-Co-V-S antibody include, but are not limited to, other agents that bind to and / or inhibit MERS-CoV activity (including other antibodies or antigen-binding fragments thereof, etc.) and / or agents that do not directly bind to MERS-CoV-S but inhibit viral activity including host cell infectivity. In some embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first anti-MERS-CoV-S antibody or an antigen-binding fragment thereof; (b) a second anti-MERS-CoV-S antibody or an antigen-binding fragment thereof, wherein the first antibody binds to a first epitope on the MERS-CoV spike protein and the second antibody binds to a second epitope on the MERS-CoV spike protein, and wherein the first and second epitopes are different and non-overlapping; and (c) a pharmaceutically acceptable carrier or diluent. In some embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first anti-MERS-CoV-S antibody or an antigen-binding fragment thereof; (b) a second anti-MERS-CoV-S antibody or an antigen-binding fragment thereof, wherein the first antibody does not cross-compete with the second antibody for binding to the MERS-CoV spike protein; and (c) a pharmaceutically acceptable carrier or diluent. Other combination therapies and co-formulations involving the anti-MERS-CoV-S antibodies of the present invention are disclosed elsewhere herein.

[0045] Fourth aspect, the present invention provides a method of treatment for treating a disease or disorder associated with MERS-CoV, such as a viral infection, in a subject, which uses the anti-MERS-CoV-S antibody or antigen-binding portion of the antibody of the present invention, wherein the method of treatment comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen-binding fragment of the antibody of the present invention. The disease or condition to be treated is any disease or condition that is improved, alleviated, inhibited or prevented by inhibiting MERS-CoV activity. In some embodiments, the present invention provides a method of preventing, treating or alleviating at least one symptom of MERS-CoV infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of the anti-MERS-CoV-S antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the present invention provides a method of alleviating or reducing the severity of at least one symptom or indication of MERS infection in a subject by administering the anti-MERS-CoV-S antibody of the present invention, wherein the at least one symptom or indication is selected from the group consisting of: pulmonary inflammation, alveolar damage, fever, cough, tachypnea, diarrhea, organ failure, pneumonia, septic shock and death. In some embodiments, the present invention provides a method of reducing the viral load in a subject, which comprises administering to the subject an effective amount of the antibody or fragment thereof of the present invention, the antibody or fragment thereof binding to MERS-CoV-S and blocking the binding of MERS-CoV-S to the host cell receptor DPP4. In some embodiments, the antibody or antigen-binding fragment thereof can be administered prophylactically or therapeutically to a subject having MERS infection or at risk of having MERS infection. Subjects at risk include, but are not limited to, immunocompromised individuals, the elderly (greater than 65 years old), children less than 2 years old, travelers to the Middle East (such as Saudi Arabia, the United Arab Emirates, Qatar, etc.), healthcare workers, adults or children in close contact with a person confirmed to have or suspected of having MERS infection, and persons having a potential medical condition (such as a lung infection, heart disease or diabetes). In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is administered in combination with a second therapeutic agent to a subject in need thereof. The second therapeutic agent can be selected from the group consisting of: anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, different antibodies against the MERS-CoV spike protein, antiviral drugs, vaccines against MERS-CoV, dietary supplements such as antioxidants and any other drugs or therapies known in the art. In some embodiments, the second therapeutic agent can be an agent that helps to counteract or reduce any possible side effects (if such side effects occur) associated with the antibody or antigen-binding fragment thereof of the present invention. The antibody or fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially. In one embodiment, the antibody can be used as a single intravenous infusion for the maximum concentration of the antibody in the serum of the subject.An antibody or fragment thereof can be administered at a dose of about 0.1 mg / kg to about 100 mg / kg of the subject's body weight. In some embodiments, the antibodies of the invention can be administered in one or more doses of 50 mg - 600 mg.

[0046] The invention also encompasses the use of the anti-MERS-CoV-S antibodies or antigen-binding fragments thereof of the invention in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from blocking MERS-CoV binding and / or activity.

[0047] Other embodiments will be apparent based on the subsequent detailed description of the invention. Brief Description of the Drawings

[0048] Figure 1 The table of lists the antibody hybridoma supernatants (enumerated by their sample ID in column 1) and their characteristics in binding, blocking, and neutralization assays. As described elsewhere herein.

[0049] Figure 2 The matrix of shows the results of antibody cross-competition assays, where the first anti-MERS-CoV-S antibody (mAb-1) was applied to the MERS RBD-coated sensor tip, followed by treatment with the second anti-MERS-CoV-S antibody (mAb-2). The binding response (values -0.05 to 0.64) for each antibody combination assayed is described. Light gray boxes with black font represent the binding response of self-competition. Antibodies that compete in both directions regardless of the order of antigen binding are highlighted in white font in black boxes. Non-competition indicating different binding regions is represented by white boxes with black font. Antibodies showing a shift greater than 0.18 nm in binding do not cross-compete with another.

[0050] Figure 3 The matrix of shows the results of antibody cross-competition assays, where the first anti-MERS-CoV-S antibody (mAb-1) was applied to the MERS RBD-coated sensor tip, followed by treatment with the second anti-MERS-CoV-S antibody (mAb-2). The binding response (values -0.01 to 0.55) for each antibody combination assayed is described. Light gray boxes with black font represent the binding response of self-competition. Antibodies that compete in both directions regardless of the order of antigen binding are highlighted in white font in black boxes. Non-competition indicating different binding regions is represented by white boxes with black font. Antibodies showing a shift greater than 0.14 nm in binding do not cross-compete with another.

[0051] Figure 4 Shows the quantitative PCR of MERS-CoV transcripts (transcript mRNA upstream of the genomic envelope gene - UpE) in humanized DPP4 mice on day 2 and day 4 post-infection.

[0052] Figure 5 Quantitative PCR of MERS-CoV transcripts (MERS-CoV genome - leader sequence) in humanized DPP4 mice on day 2 and day 4 post-infection.

[0053] Figure 6 Quantification of MERS-CoV virus titers in the lungs of infected mice on day 4 post-infection. Quantify the MERS-CoV levels in mouse lungs and express as pfu / ml of homogenized mouse lungs.

[0054] Figure 7 Quantitative PCR of MERS-CoV transcripts (transcriptional mRNA upstream of the envelope gene - UpE of the MERS-CoV genome) from the lungs of mice treated with 200 μg, 20 μg, or 2 μg of H1H15211P or H1H15277N antibodies or with hIgG isotype control one day prior to MERS-CoV infection. All samples were compared to an hIgG1 isotype control set at 100%.

[0055] Figure 8 Quantitative PCR of MERS-CoV transcripts (MERS-CoV genome - leader sequence) from the lungs of mice treated with 200 μg, 20 μg, or 2 μg of H1H15211P or H1H15277N antibodies or with hIgG isotype control one day prior to MERS-CoV infection. All samples were compared to an hIgG1 isotype control set at 100%.

[0056] Figure 9 Analysis of virus titers in the lungs quantified by plaque assay and reported as pfu / ml (from the lungs of mice treated with 200 μg, 20 μg, or 2 μg of H1H15211P or H1H15277N antibodies or with hIgG isotype control one day prior to MERS-CoV infection). All samples were compared to an hIgG1 isotype control set at 100%.

[0057] Figure 10 Inflammatory scores from histological analysis of the lungs of mice treated with 200 μg, 20 μg, or 2 μg of H1H15211P or H1H15277N antibodies or with hIgG isotype control one day prior to MERS-CoV infection.

[0058] Figure 11Quantitative PCR of MERS-CoV transcripts (transcribed mRNA upstream of the genome of the envelope gene - UpE) from the lungs of mice treated with 200 μg or 500 μg of H1H15211P or hIgG isotype control one day after MERS-CoV infection or with 200 μg of H1H15211P one day before MERS-CoV infection. All samples were compared to the hIgG1 isotype control set as 100%.

[0059] Figure 12 Quantitative PCR of MERS-CoV transcripts (MERS-CoV genome - leader sequence) from the lungs of mice treated with 200 μg or 500 μg of H1H15211P or hIgG isotype control one day after MERS-CoV infection or with 200 μg of H1H15211P one day before MERS-CoV infection. All samples were compared to the hIgG1 isotype set as 100%.

[0060] Figure 13 Analysis of viral titers in the lungs quantified by plaque assay and reported as pfu / ml (from the lungs of mice treated with 200 μg or 500 μg of H1H15211P or hIgG isotype control one day after MERS-CoV infection or with 200 μg of H1H15211P one day before MERS-CoV infection). All samples were compared to the hIgG1 isotype set as 100%.

[0061] Figure 14 Inflammatory scores from histological analysis of the lungs of mice treated with 200 μg or 500 μg of H1H15211P or hIgG isotype control one day after MERS-CoV infection. DETAILED DESCRIPTION OF THE INVENTION

[0063] Before describing the methods of the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, and the scope of the invention will be limited only by the appended claims.

[0064] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

[0065] DEFINITIONS

[0066] The term "MERS-CoV", also known as "MERS coronavirus", refers to the newly emerging Middle East Respiratory Syndrome - Coronavirus, which was first isolated in the Arabian Peninsula in 2012 (Zaki et al. 2012, NEJM 367:1814 - 1820) and identified as the cause of an outbreak of severe acute respiratory illness. It was initially called human coronavirus - EMC (Erasmus Medical Center; hCoV-EMC). It belongs to the beta coronavirus lineage 2c and causes severe respiratory illness, similar to the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) that emerged in China in 2002. The MERS coronavirus has been found to be closely related to coronaviruses found in bats and camels. It binds to the human host cell receptor dipeptidyl peptidase 4 (DPP4) via the viral spike protein. The MERS-CoV spike protein has been found to bind to DPP4 in other species, particularly bats and camels (Raj et al. 2013, Nature 495:251 - 254).

[0067] The term "MERS-CoV-S", also known as "S protein", refers to the spike protein of the Middle East Respiratory Syndrome coronavirus (MERS-CoV). The MERS-CoV spike protein is a type I membrane glycoprotein of 1,353 amino acids that assembles as a trimer and forms the spikes or peplomers on the surface of the enveloped MERS-CoV particles. The protein has two basic functions, host receptor binding and membrane fusion, which contribute to the two halves of the S protein, the N-terminus (S1, amino acid residues 1 - 751) and the C-terminus (S2, amino acid residues 752 - 1353). MERS-CoV-S binds to its cognate receptor dipeptidyl peptidase 4 (DPP4) via a receptor-binding domain (RBD) of approximately 230 amino acids in length present in the S1 subunit. Mou et al. (2013) have shown in J. Virology (Volume 87, pages 9379 - 9383) that the MERS-CoV RBD is located within residues 358 - 588 of the spike protein. The amino acid sequence of the full-length MERS-CoV spike protein is exemplified by the amino acid sequence of the spike protein of the MERS-CoV isolate EMC / 2012 provided in GenBank under accession number AFS88936.1 (SEQ ID NO: 457). The term "MERS-CoV-S" also includes protein variants of the MERS-CoV spike protein isolated from different MERS-CoV isolates such as Jordan-N3 / 2012, England-Qatar / 2012, Al-Hasa_1_2013, Al-Hasa_2_2013, Al-Hasa_3_2013, Al-Hasa_4_2013, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17, Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-Hasa_25, Bisha_1, Buraidah_1, England 1, Hafr-Al-Batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1, KFU-HKU 1, KFU-HKU 13, Munich, Qatar3, Qatar4, Riyadh_1, Riyadh_2, Riyadh_3, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_9, Riyadh_14, Taif_1, UAE, and Wadi-Ad-Dawasir. The term "MERS-CoV-S" includes recombinant MERS-CoV spike proteins or fragments thereof. The term also encompasses MERS-CoV spike proteins or fragments thereof conjugated to, for example, a histidine tag, murine or human Fc, or a signal sequence such as ROR1.For example, the term includes sequences exemplified by the sequence shown in SEQ ID NO: 458, which includes murine Fc (mIgG2a) or human (hIgG1) conjugated to amino acid residues 367 - 606 of the full-length MERS-CoV spike protein at the C-terminus. The term also includes protein variants conjugated to amino acid residues 367 - 606 of the full-length MERS-CoV spike protein, which contain a histidine tag at the C-terminus.

[0068] The term "DPP4" refers to dipeptidyl peptidase 4, a receptor for MERS-CoV. DPP4 is a 766-amino acid type II transmembrane glycoprotein that exists as a dimer on the cell surface. It is an exopeptidase that cleaves dipeptides from hormones and chemokines following a proline residue, thereby regulating the biological activities of hormones and chemokines. In humans, DPP4 is mainly expressed on epithelial cells in the kidney, small intestine, liver, and prostate, on ciliated and non-ciliated cells in the upper and lower respiratory tracts, and on immune cells (i.e., CD4+, CD8+, dendritic cells, and macrophages). Unless specified as from a non-human species, the term "DPP4" as used herein refers to human DPP4.

[0069] The term "MERS infection" or "MERS-CoV infection", also characterized as Middle East Respiratory Syndrome herein, refers to a severe acute respiratory disease caused by the MERS coronavirus and initially reported in Saudi Arabia in 2013. The term includes respiratory infections, often in the lower respiratory tract. Symptoms include high fever, cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (renal failure and renal insufficiency), septic shock, and death in severe cases.

[0070] The term "antibody" as used herein is intended to refer to an immunoglobulin molecule composed of four polypeptide chains (where two heavy chains (H) and two light chains (L) are interconnected by disulfide bonds (i.e., "intact antibody molecule")), as well as its polymers (e.g., IgM) or its antigen-binding fragments. Each heavy chain consists of a heavy chain variable region ("HCVR" or "V H ") and a heavy chain constant region (composed of domains C H 1, C H 2, and C H 3). Each light chain consists of a light chain variable region ("LCVR" or "V L ") and a light chain constant region (C L ). The V H and V L regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present invention, the FRs of the antibody (or its antigen-binding fragment) may be identical to the human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence can be defined based on the alignment analysis of two or more CDRs side by side.

[0071] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature where one or more CDRs can be dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions of antibodies and their antigens based on published crystal structures and concluded that only about 1 / 5 - 1 / 3 of the CDR residues actually contact the antigen. Padlan also found that in many antibodies, one or two of the CDRs have no amino acids contacting the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0072] CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically from regions of the Kabat CDR outside the Chothia CDR based on previous studies (e.g., residues H60 - H65 in CDRH2 are often not required). If a CDR or its residue is omitted, it is usually replaced by an amino acid occupying the corresponding position in another human antibody sequence or the consensus sequence of that sequence. The positions for substitution within the CDR and the amino acids to be substituted can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[0073] The full-length human anti-MERS-CoV-S monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework regions and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences obtained from, for example, publicly available antibody sequence databases. The present invention includes antibodies and their antigen-binding fragments derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such changes in the sequence are collectively referred to herein as "germline mutations"). Those skilled in the art can readily generate numerous antibodies and antigen-binding fragments starting from the heavy and light chain variable regions disclosed herein, which contain one or more individual germline mutations or combinations thereof. In some embodiments, in V H and / or V LAll framework and / or CDR residues within the domain are reverted to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only some residues are reverted to the original germline sequence, such as residues mutated within only the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or residues mutated within only CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Additionally, the antibodies of the present invention can comprise any combination of two or more germline mutations within the framework region and / or CDR region, such as where some individual residues are mutated to the corresponding residues of a specific germline sequence while some other residues different from the original germline sequence are retained or mutated to the corresponding residues of a different germline sequence. Once obtained, one or more desirable characteristics of the antibodies and antigen-binding fragments comprising one or more germline mutations can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced agonistic or antagonistic biological properties (as appropriate), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0074] The present invention also includes full-length human anti-MERS-CoV-S monoclonal antibodies that comprise variants with one or more conservative substitutions of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. For example, the present invention includes anti-MERS-CoV antibodies that have HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0075] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), such as in the CDRs and particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs in which the CDR sequences from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in non-human mammalian cells. The term is not intended to include antibodies isolated from or generated in human subjects.

[0076] As used herein, the term "recombinant" refers to an antibody or antigen-binding fragment thereof of the invention that is generated, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA techniques (which include, for example, DNA splicing and transgenic expression). The term refers to an antibody expressed in a non-human mammalian (including transgenic non-human mammals such as transgenic mice) or cell (such as CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

[0077] The terms "specifically binds" or "binds specifically to", etc. mean that an antibody or antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10 -8 M or less (e.g., a smaller K D indicating tighter binding). Methods for determining whether two molecules specifically bind are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies have been identified by surface plasmon resonance, such as BIACORE TM , which specifically binds to MERS-CoV-S. In addition, multispecific antibodies that bind to one domain in MERS-CoV-S and one or more other antigens or bispecific antibodies that bind to two different regions of MERS-CoV-S are also considered antibodies that "specifically bind" as used herein.

[0078] The term "high affinity" antibody refers to those mAbs that have a binding affinity for MERS-CoV-S of at least 10 -8 M, preferably 10 -9 M; more preferably 10 -10 M, further preferably 10 -11 M, more preferably 10 -12 M as represented by the K D , as measured by surface plasmon resonance such as BIACORE TM or solution affinity ELISA.

[0079] The terms "slow rate", "Koff", or "kd" mean an antibody that dissociates from MERS-CoV with a rate constant of 1x 10 -3 s -1 or less, preferably 1x 10 -4 s -1 or less, as determined by surface plasmon resonance such as BIACORE TM .

[0080] As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. As used herein, the term "antigen-binding fragment" or "antibody fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to bind to the MERS-CoV spike protein.

[0081] In certain embodiments, the antibodies or antibody fragments of the invention can be conjugated to a moiety such as a ligand or a therapeutic moiety ("immunoconjugate") such as an antiviral agent, a second anti-MERS-CoV-S antibody, or any other therapeutic moiety useful for treating an infection caused by MERS-CoV.

[0082] As used herein, "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds MERS-CoV-S or a fragment thereof is substantially free of Abs that specifically bind antigens other than MERS-CoV-S).

[0083] As used herein, the term "blocking antibody" or "neutralizing antibody" (or "antibody that neutralizes MERS-CoV-S activity" or "antagonistic antibody") is intended to mean an antibody whose binding to MERS-CoV-S results in inhibition of at least one biological activity of MERS-CoV. For example, the antibodies of the invention can prevent or block the binding of MERS-CoV to DPP4.

[0084] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that permits analysis of real-time biomolecular interactions by detecting changes in the concentration of a protein within a biosensor matrix, e.g., using a BIACORE TM system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0085] As used herein, the term "K D " is intended to mean the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0086] The term "epitope" refers to the antigenic determinant region that interacts with the specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on the antigen and can have different biological effects. The term "epitope" also refers to the site on an antigen to which B and / or T cells respond. It also refers to the region of the antigen that is bound by an antibody. Epitopes can be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the interaction affinity. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In some embodiments, an epitope can include the determinant region of the chemical reactive surface of a molecule such as an amino acid, sugar side chain, phosphate group, or sulfonyl group, and in some embodiments, can have specific three-dimensional structural features and / or specific charge characteristics.

[0087] As used herein, the term "cross-competing" means an antibody or an antigen-binding fragment thereof that binds to an antigen and inhibits or blocks its binding to another antibody or its antigen-binding fragment. The term also includes competition between two antibodies in both directions, i.e., a first antibody that binds and blocks the binding of a second antibody, and vice versa. In some embodiments, the first antibody and the second antibody can bind to the same epitope. Alternatively, the first and second antibodies can bind to different but overlapping epitopes such that the binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, e.g., by real-time, label-free biolayer interferometry. Cross-competition between two antibodies can be expressed as the binding of the second antibody being less than the background signal due to self-self binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed as, for example, the binding % of the second antibody being less than the baseline self-self background binding (where the first and second antibodies are the same antibody).

[0088] When referring to a nucleic acid or a fragment thereof, the term "substantially identical" or "substantially the same" indicates that there is at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity of nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleic acid insertions or deletions, as measured by known sequence identity algorithms such as FASTA, BLAST, or GAP, as discussed below. Nucleic acid molecules having greater identity to a reference nucleic acid can, in some instances, encode polypeptides having the same or substantially similar amino acid sequences as the polypeptide encoded by the reference nucleic acid molecule.

[0089] When applied to polypeptides, the term "substantially similar" or "substantially the same" means that when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, the two peptide sequences share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, the non-identical residue positions are differentiated by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical characteristics (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially change the functional characteristics of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. The manner in which this adjustment is made is known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical characteristics include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. A "moderately conservative" substitution is any change having a negative value in the PAM250 log-likelihood matrix.

[0090] The sequence similarity of polypeptides is generally measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity in closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutant protein. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; programs in GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between a query and a search sequence (Pearson (2000) supra). When comparing the sequences of the present invention to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0091] The phrase "therapeutically effective amount" means an amount that, when administered, produces a desired effect. The precise amount will depend on the therapeutic objective and will be determined by those skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0092] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention, and / or treatment of a disease or disorder such as a viral infection. The term includes human subjects having or at risk of having MERS infection.

[0093] As used herein, the term "treat" (including "treat", "treating", or "treatment") refers to a reduction or alleviation in the severity of at least one symptom or indication of MERS infection resulting from the administration of a therapeutic agent, such as an antibody of the present invention, to a subject in need thereof. The term includes inhibition of disease progression or worsening of the infection. The term also includes a positive prognosis for the disease, i.e., when a therapeutic agent, such as an antibody of the present invention, is administered, the subject may be free of infection or may have a reduced or no viral titer. The therapeutic agent can be administered to the subject in a therapeutically effective dose.

[0094] The term "prevent, preventing or prevention" refers to the inhibition of the manifestation of MERS infection or any symptom or indication of MERS infection when the antibody of the present invention is administered. The term includes prevention of the spread of infection in a subject exposed to the virus or at risk of MERS infection.

[0095] As used herein, the term "antiviral agent" refers to any anti-infective agent or therapy used to treat, prevent or alleviate an infection in a subject. The term "antiviral agent" includes, but is not limited to, ribavirin, oseltamivir, zanamivir, interferon-α2b, analgesics and corticosteroids. In the context of the present invention, viral infection includes infections caused by human coronaviruses (including, but not limited to, MERS-CoV, HCoV_229E, HCoV_NL63, HCoV-OC43, HCoV_HKU1 and SARS-CoV).

[0096] General description

[0097] Passive immunotherapy for the prevention or treatment of infectious diseases has been used for over a century, often in the form of high-titer convalescent human serum containing neutralizing antibodies (Good et al. 1991; Cancer 68:1415-1421). Nowadays, multiple purified monoclonal antibodies are being used as antimicrobial agents and are currently in preclinical and clinical development (Marasco et al. 2007; Nature Biotechnology 25:1421-1434).

[0098] The inventors describe herein full-length human antibodies and antigen-binding fragments thereof that specifically bind to MERS-CoV-S and modulate the interaction of MERS-CoV-S with DPP4. The anti-MERS-CoV-S antibodies can bind MERS-CoV-S with high affinity. In some embodiments, the antibodies of the invention are blocking antibodies, wherein the antibody can bind to MERS-CoV-S and can block the interaction of MERS-CoV-S with DPP4. In some embodiments, the blocking antibodies of the invention can block the binding of MERS-CoV-S to DPP4 and / or inhibit or neutralize the viral infectivity of host cells. In some embodiments, the blocking antibodies can be used to treat subjects suffering from MERS infection. In some embodiments, selected antibodies that do not cross-compete for binding to the spike protein are used in a cocktail combination to reduce the ability of the virus to escape via mutation in response to selective pressure from each component. When administered to a subject in need thereof, the antibody can reduce infection by a virus such as MERS-CoV in the subject. It can be used to reduce the viral load in a subject. It can be used alone or as an adjuvant therapy in combination with other therapeutic moieties for treating viral infections or modalities known in the art. It is also shown herein that these antibodies bind to epitopes on the S protein that have been conserved during the natural evolution of the virus over the past 2 years. In addition, it has been demonstrated with a new transgenic mouse model that the identified antibodies can prophylactically protect mice from infection and alleviate pre-established infections in a post-inoculation treatment regimen. In Example 7, it is shown that administration of anti-MERS-Cov-S antibody 1 day prior to infection was able to reduce MERS-CoV replication to near the level of detection in the live virus assay and by 3 logs in the viral RNA assay. The antibody demonstrated dose-dependent protection, as lower doses of the antibody administered 24 hours prior to infection were able to block MERS-CoV to a lesser extent. In addition, histological analysis of lung tissue demonstrated that mice pretreated with the antibody showed reduced MERS-CoV-induced peribronchial cuffing, alveolar wall thickening, and overall inflammatory foci.

[0099] The full-length amino acid sequence of the full-length MERS-CoV spike protein is shown in SEQ ID NO: 457. In some embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen such as the full-length MERS-CoV spike protein (SEQ ID NO: 457), or mice immunized with a recombinant form of MERS-CoV-S or a modified MERS-CoV-S fragment (e.g., SEQ ID NO: 458), followed by immunization with a secondary immunogen or an immunologically active fragment of MERS-CoV-S.

[0100] The immunogen can be a biologically active and / or immunogenic fragment of MERS-CoV-S or DNA encoding an active fragment thereof. The fragment can be derived from the N-terminus or C-terminus of MERS-CoV-S. In some embodiments of the present invention, the immunogen is the MERS-CoV-S fragment of amino acid residues 367-606 of SEQ ID NO:457.

[0101] The peptide can be modified to include the addition or substitution of some residues for labeling or for the purpose of coupling with a carrier molecule such as KLH. For example, cysteine can be added at the N-terminus or C-terminus of the peptide, or a linker sequence can be added to prepare a peptide for coupling with, for example, KLH for immunization.

[0102] Some anti-MERS-CoV-S antibodies of the present invention are capable of binding to MERS-CoV-S to neutralize its activity, as determined by in vitro or in vivo assays. The ability of the antibodies of the present invention to bind to MERS-CoV-S and neutralize its activity can be measured using any standard method known to those skilled in the art, including the binding assays or activity assays described herein.

[0103] Non-limiting, exemplary in vitro assays for measuring binding and blocking activities are illustrated in Examples 4-5 herein. In Example 4, the binding affinity and dissociation constant of anti-MERS-CoV-S antibodies against MERS-CoV-S were determined by surface plasmon resonance assay. In Example 5, a neutralization assay was used to determine the infectivity of virus-like particles containing the MERS-CoV spike protein.

[0104] Antibodies specifically directed against MERS-CoV-S may not contain other labels or moieties, or they may contain labels or moieties at the N-terminus or C-terminus. In one embodiment, the label or moiety is biotin. In a binding assay, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which the peptide binds. For example, if the surface is coated with avidin, a peptide containing biotin at the N-terminus will be oriented such that the C-terminal portion of the peptide will be away from the surface. In one embodiment, the label can be a radionuclide, a fluorescent dye, or an MRI-detectable label. In some embodiments, such labeled antibodies can be used in diagnostic assays including imaging assays.

[0105] Antigen-binding fragments of antibodies

[0106] Unless otherwise specifically indicated, the term "antibody" as used herein will be understood to encompass antibody molecules (i.e., "full-length antibody molecules") comprising two immunoglobulin heavy chains and two immunoglobulin light chains, and antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex. The term "antigen-binding fragment" or "antibody fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind to the MERS-CoV spike protein. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments comprising CDRs, or isolated CDRs. In some embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of an antibody can be derived, using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques, from, for example, full-length antibody molecules, which techniques involve the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and / or can be readily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthetic. The DNA can be sequenced and chemically manipulated using molecular biotechnology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, generate cysteine residues, modify, add, or delete amino acids, etc.

[0107] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0108] Antigen-binding fragments of an antibody will generally comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to and / or within one or more framework sequences in-frame. In those having a V L domain associated with a V HIn the antigen-binding fragment of the domain, V H and V L domains can be configured relative to each other in any suitable arrangement. For example, the variable region can be dimerized and contain V H -V H 、V H -V L or V L -V L dimers. Alternatively, the antigen-binding fragment of the antibody can contain a monomeric V H or V L domain.

[0109] In some embodiments, the antigen-binding fragment of the antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that can be found within the antigen-binding fragment of the antibodies of the present invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L。In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly connected to each other or can be connected by a full-length or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible linker between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of an antibody of the invention can comprise any of the variable and constant domain constructs listed above either with each other and / or with one or more monomeric V H or V L domains (e.g., via disulfide bonds) non-covalently associated as a homodimer or heterodimer (or other multimer).

[0110] For full-length antibody molecules, the antigen-binding fragment can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will generally comprise at least two different variable domains, where each variable domain is capable of specifically binding a different antigen or a different epitope on the same antigen. Any of the multispecific antibody forms disclosed herein, including exemplary bispecific antibody forms, can be adapted for use in the context of the antigen-binding fragments of the antibodies of the invention using conventional techniques available in the art.

[0111] Preparation of human antibodies

[0112] Methods for generating human antibodies in transgenic mice are known in the art. Any of these known methods can be used in the context of the present invention to prepare human antibodies that specifically bind to the MERS-CoV spike protein.

[0113] An immunogen comprising any of the following can be used to generate antibodies against the MERS-CoV spike protein. In some embodiments, the antibodies of the invention are obtained from mice immunized with full-length native MERS-CoV spike protein, or with DNA encoding its protein or fragment (see, e.g., GenBank accession number AFS88936.1) (SEQ ID NO: 457). Alternatively, the spike protein or its fragment can be produced using standard biochemical techniques, modified, and used as an immunogen. In one embodiment, the immunogen is the receptor-binding domain (S1) of the MERS-CoV spike protein. In some embodiments of the invention, the immunogen is a fragment of the MERS-CoV spike protein that ranges from about amino acid residues 367 - 606 of SEQ ID NO: 457.

[0114] In some embodiments, the immunogen can be a recombinant MERS-CoV spike protein receptor-binding domain peptide expressed in E. coli or any other eukaryotic or mammalian cell such as Chinese hamster ovary (CHO) cells.

[0115] Use techniques (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, ) or any other known method for generating monoclonal antibodies, initially isolate high-affinity chimeric antibodies against MERS-CoV-S, which have human variable regions and murine constant regions. The techniques involve the generation of transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to the endogenous murine constant region loci such that the mice produce antibodies in response to antigenic stimulation that contain murine variable regions and murine constant regions. Isolate the DNA encoding the heavy and light chain variable regions of the antibody and operably link it to the DNA encoding the human heavy and light chain constant regions. Subsequently, express the DNA in cells capable of expressing full-length human antibodies.

[0116] Generally, mice are challenged with the antigen of interest and lymphocytes (such as B-cells) are recovered from the antibody-expressing mice. The lymphocytes can be fused with a myeloma cell line to produce an immortalized hybridoma cell line, and the hybridoma cell line is screened and selected to identify those that produce antibodies specific for the antigen of interest. The DNA encoding the variable regions of the heavy and light chains can be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells, such as CHO cells. Alternatively, the DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0117] Initially, high-affinity chimeric antibodies are isolated, which have human variable regions and murine constant regions. According to the experimental section below, the antibodies are characterized and selected for desired properties including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to produce the full-length human antibodies of the present invention, such as wild-type or modified IgG1 or IgG4. Since the constant regions selected can vary depending on the particular use, the high-affinity antigen-binding and target-specificity characteristics depend on the variable regions.

[0118] Bioequivalents

[0119] The anti-MERS-CoV-S antibodies and antibody fragments of the present invention encompass proteins having an amino acid sequence different from that of the antibodies, but which still retain the ability to bind to the MERS-CoV spike protein. When compared to the parental sequence, such variant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions, but exhibit biological activity essentially equivalent to that of the antibodies. Similarly, when compared to the disclosed sequences, the DNA sequences encoding the antibodies of the present invention encompass sequences containing one or more nucleotide additions, deletions, or substitutions, but which encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present invention.

[0120] Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or substitutes, i.e., when administered at the same molar concentration (single or multiple doses) in a similar experimental setting, their rates and extents of absorption do not show significant differences. Some antibodies will be considered equivalents or substitutes if they are equivalent in their extent of absorption but not in their rate of absorption, provided that this difference in absorption rate is intentional and reflected in the labeling and is not critical for achieving an effective body drug concentration for, e.g., long-term use. They can then be considered bioequivalent and medically non-significant for the particular drug product under study.

[0121] In one embodiment, two antigen-binding proteins are considered bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[0122] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can switch between reference products one or more times and there is no expected increased risk of side effects (including clinically significant changes in immunogenicity or loss of efficacy) for the biological product compared to continuous therapy without such a switch.

[0123] In one embodiment, two antigen-binding proteins are bioequivalent if, under the conditions of use, they act through a common mechanism or mechanisms of action to the extent that the mechanism is known.

[0124] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Measurements of bioequivalence include, for example: (a) in vivo testing in humans or other mammals, where the concentration of the antibody or its metabolite measured in blood, plasma, serum, or other biological fluids is a function of time; (b) in vitro testing that is related to and a reasonable predictor of in vivo bioavailability data in humans; (c) in vivo testing in humans or other mammals, where the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in well-controlled clinical trials establishing the safety, potency, or bioequivalence of the antibody.

[0125] Bioequivalent variants of the antibodies of the invention can be constructed, for example, by generating different substitutions of residues or sequences or deletions of terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bridges upon refolding. In other contexts, bioequivalent antibodies can include antibody variants that contain amino acid alterations that modify the glycosylation properties of the antibody, such as mutations that eliminate or remove glycosylation.

[0126] Anti-MERS-CoV-S antibodies comprising Fc variants

[0127] According to some embodiments of the present invention, anti-MERS-CoV-S antibodies are provided that comprise an Fc domain containing one or more mutations that enhance or abrogate antibody binding to the FcRn receptor (e.g., at acidic pH compared to neutral pH). For example, the present invention includes anti-MERS-CoV-S antibodies containing mutations in the C H 2 or C H 3 region, wherein the mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH is from about 5.5 to about 6.0). When administered to an animal, such mutations can result in an increase in the serum half-life of the antibody. Non-limiting examples of such Fc modifications include, for example, at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]), or modifications at position 250 and / or 428; or modifications at position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications comprise 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In another embodiment, the modifications comprise 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0128] For example, the present invention includes anti-MERS-CoV-S antibodies comprising an Fc domain, said Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A, and 434A (e.g., T307A, E380A, and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are expected to be within the scope of the present invention.

[0129] The present invention also includes anti-MERS-CoV-S antibodies comprising a chimeric heavy chain constant region (C H )), wherein said chimeric C H region comprises segments from C H regions derived from more than one immunoglobulin isotype. For example, the antibodies of the present invention may comprise a chimeric C H region, said chimeric C H region containing portions or all of the C H 2 domain in combination with portions or all of the C H 3 domain from a human IgG1, human IgG2, or human IgG4 molecule. According to some embodiments, the antibodies of the present invention comprise a chimeric C H region having a chimeric hinge region. For example, the chimeric hinge may comprise the "upper hinge" amino acid sequence (amino acid residues according to EU numbering positions 216-227) from the hinge region of a human IgG1, human IgG2, or human IgG4 molecule in combination with the "lower hinge" sequence (amino acid residues according to EU numbering positions 228-236) from the hinge region of a human IgG1, human IgG2, or human IgG4 molecule. According to some embodiments, the chimeric hinge region comprises amino acid residues from the upper hinge of human IgG1 or human IgG4 and amino acid residues from the lower hinge of human IgG2. In some embodiments, antibodies comprising a chimeric C H region as described herein may exhibit modified Fc effector functions without negatively affecting the therapeutic or pharmacological properties of the antibody (see, e.g., U.S. Provisional Application No. 61 / 759,578, filed February 1, 2013, the disclosure of which is hereby incorporated by reference in its entirety).

[0130] Biological properties of the antibody

[0131] Generally, the antibodies of the present invention function by binding to the MERS-CoV spike protein. In some embodiments, the antibodies of the present invention bind with high affinity to one or more amino acids in the receptor binding domain (RBD) of the spike protein of MERS-CoV. For example, the present invention includes antibodies with a K D Antibodies and antigen-binding fragments of antibodies that bind to the dimeric MERS-CoV spike protein RBD (e.g., at 25 °C or at 37 °C), as measured by surface plasmon resonance, e.g., using the assay format defined in Example 4 herein. In some embodiments, the antibody or its antigen-binding fragment has a K of less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 250 pM, or less than 100 pM D Bind to dimeric MERS-CoV-S, as measured by surface plasmon resonance, e.g., using the assay format defined in Example 4 herein, or a substantially similar assay.

[0132] The present invention also includes antibodies and antigen-binding fragments thereof that bind to the MERS-CoV spike protein with a dissociation half-life (t1 / 2) greater than about 2.1 minutes, as measured by surface plasmon resonance at 25 °C, e.g., using the assay format defined in Example 4 herein, or a substantially similar assay. In some embodiments, the antibodies or antigen-binding fragments of the present invention bind to the MERS-CoV spike protein with a t1 / 2 greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 150 minutes, greater than about 200 minutes, or greater than about 250 minutes, as measured by surface plasmon resonance at 25 °C, e.g., using the assay format defined in Example 4 herein (e.g., mAb-capture or antigen capture format), or a substantially similar assay.

[0133] The present invention also includes antibodies and antigen-binding fragments thereof that bind to the MERS-CoV spike protein with a dissociation half-life (t1 / 2) greater than 1.5 minutes, as measured by surface plasmon resonance at 37 °C, e.g., using the assay format defined in Example 4 herein, or a substantially similar assay. In some embodiments, the antibodies or antigen-binding fragments of the present invention bind to the MERS-CoV spike protein with a t1 / 2 greater than about 2 minutes, greater than about 5 minutes, greater than about 10 minutes, greater than about 25 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 150 minutes, greater than about 200 minutes, as measured by surface plasmon resonance at 37 °C, e.g., using the assay format defined in Example 4 herein (e.g., mAb-capture or antigen capture format), or a substantially similar assay.

[0134] The invention also includes an antibody or an antigen-binding fragment thereof that blocks the binding of greater than 90% of MERS-CoV-S to DPP4, as determined using an ELISA-based immunoassay, such as shown in Example 2, or a substantially similar assay.

[0135] The invention also includes an antibody or an antigen-binding fragment thereof that neutralizes or inhibits the infectivity of MERS-CoV for its host cells. In some embodiments, the antibody neutralizes the infectivity of MERS-CoV-like pseudoparticles (MERSpp). In some embodiments, in an optimized virus-like pseudoparticle (VLP) neutralization assay, the antibody inhibits the binding of MERS-CoV to greater than 90% of human host cells, such as shown in Example 5, or a substantially similar assay. The antibody neutralizes MERSpp infectivity with an IC50 of 58.9 pM to 2.93 nM.

[0136] In some embodiments, the antibody of the invention binds to a fragment of the receptor-binding domain or domain of the MERS-CoV spike protein. In some embodiments, the antibody of the invention can bind to more than one domain (cross-reactive antibody). In some embodiments, the antibody of the invention can bind to an epitope located in the receptor-binding domain and comprising amino acid residues 367-606 of MERS-CoV-S. In one embodiment, the antibody can bind to an epitope comprising one or more amino acids selected from the group consisting of the amino acid residues 367-606 set forth in SEQ ID NO: 457.

[0137] In some embodiments, the antibody of the invention can function by blocking or inhibiting the DPP4-binding activity associated with the MERS-CoV spike protein by binding to any other region or fragment of the full-length protein (the sequence set forth in SEQ ID NO: 457).

[0138] In some embodiments, the antibody of the invention can be a bispecific antibody. The bispecific antibody of the invention can bind to an epitope in one domain and can also bind to a second epitope in the same or a different domain of the MERS-CoV spike protein. In some embodiments, the bispecific antibody of the invention can bind to two different epitopes in the same domain. In one embodiment, the invention provides an isolated recombinant antibody or an antigen-binding fragment thereof that specifically binds to the MERS-CoV spike protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) is a full-length human monoclonal antibody; (b) interacts with one or more amino acid residues in the receptor-binding domain of the MERS-CoV spike protein, wherein the amino acid residues are selected from amino acid residues 367-606 of SEQ ID NO: 457; (c) binds to the MERS-CoV spike protein with a dissociation constant (KD ) binding, as measured in surface plasmon resonance assays; (d) blocking greater than 90% of the binding of the MERS-CoV spike protein to dipeptidyl peptidase 4, as measured in blocking ELISA assays; (e) neutralizing greater than 90% of the infectivity of MERS-CoV in human host cells and having an IC 50 , as measured in VLP neutralization assays; (f) neutralizing the infectivity of MERS-CoV, wherein the MERS-CoV comprises isolates of viruses selected from the group consisting of: EMC / 2012, Jordan-N3 / 2012, England-Qatar / 2012, Al-Hasa_1_2013, Al-Hasa_2_2013, Al-Hasa_3_2013, Al-Hasa_4_2013, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17, Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-hasa_25, Bisha_1, Buraidah_1, England 1, Hafr-Al-batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1, KFU-HKU 1, KFU-HKU 13, Munich, Qatar3, Qatar4, Riyadh_1, Riyadh_2, Riyadh_3, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_9, Riyadh_14, Taif_1, UAE, and Wadi-Ad-Dawasir; (g) blocking in vivo replication of MERS-CoV in subjects infected with MERS-CoV; and (h) being a bispecific antibody that comprises a first binding specificity for a first epitope in the receptor-binding domain of the MERS-CoV spike protein and a second binding specificity for a second epitope in the receptor-binding domain of the MERS-CoV spike protein, wherein the first and second epitopes are distinct and non-overlapping.

[0139] The antibodies of the invention can have one or more of the above biological properties, or any combination thereof. Other biological properties of the antibodies of the invention will be apparent to those skilled in the art based on the work herein, including the working examples herein.

[0140] Epitope mapping and related techniques

[0141] The present invention includes anti-MERS-CoV-S antibodies that interact with one or more amino acids found within one or more domains of the MERS-CoV spike protein molecule, said amino acids including the N-terminal S1 domain (amino acid residues 1-751) and the C-terminal S2 domain (amino acid residues 752-1353). The epitope to which the antibody binds can consist of a single continuous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the above-described domains of the MERS-CoV spike protein molecule (e.g., a linear epitope within a domain). Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located within one or both of the above-described domains of the spike protein molecule (e.g., a conformational epitope).

[0142] A variety of techniques known to those of skill in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays, as described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutagenesis, peptide blotting (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of the antigen (Tomer (2000) Prot. Sci. 9:487-496) can be employed. Another method for identifying the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the method of hydrogen / deuterium exchange involves deuterium labeling of the protein of interest, followed by binding of the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water and the exchangeable protons within the amino acids protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a rate lower than that of the exchangeable protons within the amino acids that are not part of the interface. As a result, the amino acids that form the protein / antibody interface portion can retain deuterium and thus exhibit a higher mass than the amino acids not included within the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues of the specific amino acids with which the corresponding antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0143] The term "epitope" refers to the site on an antigen to which B and / or T cells respond. B cell epitopes can be formed from either non - contiguous amino acids juxtaposed by the tertiary folding of a protein or contiguous amino acids. Epitopes formed from contiguous amino acids generally remain exposed to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Epitopes typically include at least 3 or more, usually at least 5 or 8 - 10 amino acids in a specific spatial conformation.

[0144] Modified Affinity Profiling (MAP), also known as Antigen - Structure - based Antibody Profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) against the same antigen according to the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (see US2004 / 0101920, which is specifically incorporated herein by reference in its entirety). Each class can reflect a specific epitope that is either completely distinct or partially overlapping from the epitope represented by another class. This technique allows for the rapid filtration of genetically identical antibodies, thereby characterizing antibodies that can be focused on those that are genetically distinct. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desirable properties. MAP can be used to sort the antibodies of the present invention into groups of antibodies that bind different epitopes.

[0145] In some embodiments, the anti - MERS - CoV - S antibody or its antigen - binding fragment binds to an epitope within any one or more of the regions shown in the MERS - CoV spike protein (either in the native form as shown in SEQ ID NO:457 or recombinantly produced as shown in SEQ ID NO:458) or a fragment thereof. In some embodiments, the antibody of the present invention binds to an extracellular region comprising one or more amino acids selected from amino acid residues 367 - 606 of the MERS - CoV spike protein.

[0146] In some embodiments, the antibody of the present invention interacts with at least one amino acid sequence selected from: the amino acid residues from approximately position 358 to approximately position 450 of SEQ ID NO:457; or the amino acid residues from approximately position 451 to approximately position 606.

[0147] The present invention includes anti - MERS - CoV - S antibodies that bind to the same epitope or epitope portion, such as any of the specific exemplary antibodies obtained from the Figure 1 enumerated cell lines. Similarly, the present invention also includes anti - MERS - CoV - S antibodies that compete for binding to the MERS - CoV spike protein or a fragment thereof with any of the specific exemplary antibodies obtained from the Figure 1 enumerated hybridomas. For example, the present invention includes antibodies that compete for binding to the MERS - CoV spike protein or a fragment thereof with one or more of the antibodies obtained from the Figure 1Anti-MERS-CoV-S antibodies obtained from the listed hybridomas that cross-compete for binding to the MERS-CoV spike protein.

[0148] Using conventional methods known in the art, one of ordinary skill in the art can readily determine whether an antibody binds to the same epitope as a reference anti-MERS-CoV-S antibody or competes with it for binding to the same epitope. For example, to determine whether a test antibody binds to the same epitope as the reference anti-MERS-CoV-S antibody of the present invention, under saturating conditions, the reference antibody is allowed to bind to the MERS-CoV spike protein or peptide. Next, the ability of the test antibody to bind to the MERS-CoV spike protein molecule is evaluated. If the test antibody is able to bind to MERS-CoV-S after saturation binding with the reference MERS-CoV-S antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-MERS-CoV-S antibody. On the other hand, if the test antibody is unable to bind to the MERS-CoV spike protein after saturation binding with the reference MERS-CoV spike protein, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-MERS-CoV-S antibody of the present invention.

[0149] To determine whether an antibody competes for binding with a reference anti-MERS-CoV-S antibody, the above binding method is carried out in two directions: in the first direction, the reference antibody is allowed to bind to the MERS-CoV spike protein under saturating conditions, and then the binding of the test antibody to the MERS-CoV-S molecule is evaluated. In the second direction, the test antibody is allowed to bind to the MERS-CoV-S molecule under saturating conditions, and then the binding of the reference antibody to the MERS-CoV-S molecule is evaluated. If in both directions, only the first (saturating) antibody is able to bind to the MERS-CoV-S molecule, it can be concluded that the test antibody competes with the reference antibody for binding to MERS-CoV-S. One of ordinary skill in the art will understand that an antibody that competes for binding with a reference antibody pair does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0150] If each antibody competitively inhibits (blocks) the binding of the other to the antigen, the two antibodies bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50% but preferably 75%, 90%, or even 99% (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if substantially all of the amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies have the same epitope. If some of the amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies have overlapping epitopes.

[0151] Other conventional experiments (e.g., peptide mutagenesis and binding assays) can then be performed to confirm whether the observed loss of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or whether it is due to steric hindrance (or another phenomenon) responsible for the observed binding. Such sorting experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody-binding assay available in the art.

[0152] Immunoconjugates

[0153] The present invention encompasses human anti-MERS-CoV-S monoclonal antibodies conjugated to a therapeutic moiety such as a toxoid or antiviral agent for the treatment of MERS infection ("immunoconjugates"). As used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically linked to a radioagent, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody can be linked to the radioagent, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent at any position along the molecule, so long as it is capable of binding its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent can be a second, different antibody directed against the MERS-CoV spike protein. In some embodiments, the antibody can be conjugated to an agent specific for cells infected with the virus. The type of therapeutic moiety that can be conjugated to the anti-MERS-CoV-S antibody should be considered in view of the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art, see, e.g., WO 05 / 103081.

[0154] Multispecific antibodies

[0155] The antibodies of the invention can be monospecific, bispecific or multispecific. Multispecific antibodies can be specific for different epitopes of a target polypeptide or can comprise antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

[0156] Any multispecific antigen-binding molecule of the invention or variants thereof can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) known to those of skill in the art.

[0157] In some embodiments, MERS-CoV-S-specific antibodies are generated in bispecific form ("bispecific"), where variable regions that bind to different domains of the MERS-CoV spike protein are linked together to confer bispecificity within a single binding molecule. Appropriately designed bispecific antibodies can enhance the overall MERS-CoV-spike protein inhibitory potency by increasing specificity and binding affinity. Variable regions having specificity for a single domain (e.g., a segment of the N-terminal domain), or variable regions that can bind to different regions within a domain, are paired on a structural scaffold that allows each region to bind simultaneously to different epitopes or allows each region to bind to different regions within a domain. In one example of a bispecific antibody, a heavy chain variable region (V H ) having specificity for one domain from one binding is recombined with a light chain variable region (V L ) having specificity for a second domain from a series of bindings to identify a non-homologous V H partner that can pair with the original V H without disrupting the original specificity for that V L . In this way, a single V L segment (e.g., V L 1) can be combined with two different V H domains (e.g., V H 1 and V H 2) to generate a bispecific antibody containing two binding "arms" (V H 1-V L 1 and V H 2-V L 1). The use of a single V L segment reduces the complexity of the system and thus simplifies the cloning, expression, and purification steps used to generate bispecific antibodies and increases their efficiency (see, e.g., USSN13 / 022759 and US2010 / 0331527).

[0158] Alternatively, antibodies that bind to a second target that incorporates more than one domain, such as but not limited to a second different anti-MERS-CoV-S antibody, can be prepared in bispecific form using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions that bind to different regions can be joined together with variable regions that bind to relevant sites, such as on the extracellular domain of MERS-CoV-S, to confer bispecificity within a single binding molecule. This bispecific property, when appropriately designed, can function as bifunctional. Variable regions with specificity for the extracellular domain are combined with variable regions with specificity for the outside of the extracellular domain and paired on a structural scaffold that allows each variable region to bind to a different antigen.

[0159] Exemplary bispecific antibody formats that can be used in the context of the present invention involve the use of a first immunoglobulin (Ig) C H 3 domain and a second Ig C H 3 domain, where the first and second Ig C H 3 domains differ from each other by at least one amino acid, and the difference in at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H 3 domain binds protein A and the second IgC H 3 domain contains a mutation that reduces or blocks protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C H 3 may further contain the Y96F modification (by IMGT; Y436F by EU). In the case of IgG1 antibodies, other mutations that can be found within the second C H 3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU). Variations in the bispecific antibody formats described above are contemplated within the scope of the present invention.

[0160] Other exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, bispecific formats based on scFv or diabodies, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Bispecific formats (see, e.g., Klein et al. 2012, mAb 4:6, 1-11 and the references cited therein for a review of the above formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugates, for example, where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valency, and geometry (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0161] Therapeutic administration and formulation

[0162] The present invention provides therapeutic compositions comprising the anti-MERS-CoV-S antibodies or antigen-binding fragments thereof of the present invention. The therapeutic compositions according to the present invention will be co-administered with suitable carriers, excipients, and other agents incorporated to provide improved transfer, delivery, tolerance, etc. A variety of suitable formulations can be found in the compendia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorbent pastes, water-in-oil and oil-in-water emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0163] The dosage of the antibody can vary depending on the age and body size of the subject to be administered, the disease targeted, the condition, the route of administration, etc. When the antibody of the present invention is used to treat a disease or disorder in an adult patient or to prevent the disease, it is advantageous to administer the antibody of the present invention normally at a single dose of about 0.1 to about 60 mg / kg body weight, more preferably about 5 to about 60, about 10 to about 50 or about 20 to about 50 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention can be administered at an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg or about 10 to about 200 mg, up to about 100 mg or up to about 50 mg. In some embodiments, a second or subsequent dose of the antibody or antigen-binding fragment thereof is administered in an amount substantially the same as or less than the initial dose after the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; at least 14 weeks.

[0164] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucosal layers (such as oral mucosa, rectal and intestinal mucosa, etc.) and can be co-administered with other bioactive agents. It can be administered systemically or locally. The pharmaceutical composition can also be delivered in vesicles, particularly liposomes (see, for example, Langer (1990) Science 249:1527-1533).

[0165] The use of nanoparticles for delivering the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles can also be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and uses are described in detail by Arruebo, M., et al 2009 (“Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi:10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles can be developed and conjugated with antibodies included in pharmaceutical compositions to target virus-infected cells. Nanoparticles for drug delivery have been described, for example, in US 8257740 or US 8246995, each of which is incorporated herein in its entirety.

[0166] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, polymeric materials can be used. In another embodiment, the controlled release system can be placed near the target of the composition, so that only a fraction of the systemic dose is required.

[0167] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injection, infusion, etc. These injectable preparations can be prepared by publicly known methods. For example, the injectable preparations can be prepared by dissolving, suspending or emulsifying the above-mentioned antibodies or their salts in a sterile aqueous medium or an oily medium conventionally used for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct) of hydrogenated castor oil], etc. As the oily medium, there are, for example, sesame oil, soybean oil, etc., which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injectables thus prepared are preferably filled in suitable ampoules.

[0168] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, pen delivery devices readily have application in delivering the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the entire pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. Reusable pen delivery devices. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a container within the device. Once the container is empty of the pharmaceutical composition, the entire device is discarded.

[0169] A variety of reusable pens and automatic injection delivery devices have applications in subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are certainly not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices having application in subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are certainly not limited to, SOLOSTAR TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM(Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA TM pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0170] Advantageously, the pharmaceutical composition for oral or parenteral use as described above is prepared in a dosage form in unit doses adapted to match the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The amount of antibody contained in a unit dose, especially in the injectable form, is generally about 5 to about 500 mg per dosage form; preferably, for other dosage forms, about 5 to about 100 mg and about 10 to about 250 mg of antibody are included.

[0171] Therapeutic use of antibodies

[0172] The antibodies of the present invention can be used for treating and / or preventing diseases or disorders or conditions associated with MERS coronavirus such as MERS infection and / or for ameliorating at least one symptom associated with the disease, disorder or condition. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention can be administered at a therapeutic dose to a patient having MERS infection.

[0173] In some embodiments, the antibodies of the present invention are used for treating subjects suffering from severe and acute respiratory infections caused by MERS coronavirus. In some embodiments, the antibodies of the present invention are used for reducing viral titer or reducing viral load in a host. In one embodiment, the antibodies of the present invention are used for preventing or reducing inflammation in the lungs of subjects having MERS. In one embodiment, the antibodies of the present invention are used for preventing or reducing inflammation, alveolar damage and pleural changes in the interstitium, peripheral bronchi or peripheral blood vessels of subjects having MERS.

[0174] One or more antibodies of the present invention can be administered to relieve or prevent or reduce one or more symptoms or conditions of a disease or disorder. The antibodies can be used to ameliorate or reduce the severity of at least one symptom of MERS infection, including but not limited to consisting of: fever, cough, tachypnea, pneumonia, diarrhea, organ failure (such as renal failure and renal insufficiency), septic shock and death.

[0175] The present invention also contemplates prophylactic use of one or more antibodies of the invention in subjects at risk of developing MERS infection such as immunocompromised individuals, the elderly (aged greater than 65 years), children less than 2 years of age, travelers to Middle Eastern countries (e.g., Saudi Arabia, United Arab Emirates, and Qatar), healthcare workers, persons with occupational or recreational exposure to camels or bats, family members in close proximity to MERS patients, adults or children in contact with confirmed or suspected MERS-infected patients, and patients with a medical history (e.g., increased risk of pulmonary infections, heart disease, or diabetes).

[0176] In a further embodiment of the invention, the antibodies of the invention are used to prepare a pharmaceutical composition or a medicament for treating a patient suffering from MERS infection. In another embodiment of the invention, the antibodies of the invention are used as an adjuvant therapy to any other agent or any other therapy known to those skilled in the art for treating or ameliorating MERS infection.

[0177] Combination therapy

[0178] The combination therapy may include the anti-MERS-CoV-S antibody of the invention and any other therapeutic agent that can advantageously be combined with the antibody of the invention or with a bioactive fragment of the antibody of the invention. The antibody of the invention can be synergistically combined with one or more drugs or therapies for treating MERS. In some embodiments, the antibody of the invention can be combined with a second therapeutic agent to reduce viral load in patients with MERS infection or to ameliorate one or more symptoms of the infection.

[0179] The antibody of the invention can be used in combination with the following: anti-inflammatory drugs (e.g., corticosteroids and non-steroidal anti-inflammatory drugs), anti-infective drugs, different antibodies against the MERS-CoV spike protein, antiviral drugs, interferon-α-2b plus intramuscular ribavirin, convalescent plasma, inhibitors of the main viral protease and entry / fusion inhibitors targeting the MERS-CoV spike protein, vaccines against MERS-CoV, antibiotics, dietary supplements such as antioxidants, or any other palliative treatment for MERS infection.

[0180] In some embodiments, the second therapeutic agent is another antibody to the MERS-CoV spike protein. Combinations (“cocktails”) of antibodies having broad neutralizing or inhibitory activity against MERS-CoV are contemplated herein. In some embodiments, non-competing antibodies can be combined and administered to a subject in need thereof to reduce the ability of the MERS virus to escape due to rapid mutation under selective pressure. In some embodiments, the antibodies in the combination bind to different non-overlapping epitopes on the spike protein. The antibodies in the combination can block the binding of MERS-CoV to DPP4 or can prevent / inhibit membrane fusion. The antibodies in the combination can inhibit MERS-CoV activity of one or more MERS-CoV isolates, including but not limited to EMC / 2012, Jordan-N3 / 2012, England-Qatar / 2012, Al-Hasa_1_2013, Al-Hasa_2_2013, Al-Hasa_3_2013, Al-Hasa_4_2013, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17, Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-hasa_25, Bisha_1, Buraidah_1, England 1, Hafr-Al-batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1, KFU-HKU 1, KFU-HKU 13, Munich, Qatar3, Qatar4, Riyadh_1, Riyadh_2, Riyadh_3, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_9, Riyadh_14, Taif_1, UAE, and Wadi-Ad-Dawasir.

[0181] Combinations of the anti-MERS-CoV-S antibodies of the invention are also contemplated herein, wherein the combination comprises one or more non-cross-competing antibodies; in some embodiments, the combination comprises a first antibody having broad neutralizing activity and a second antibody having activity against a narrower range of isolates and not cross-competing with the first antibody.

[0182] As used herein, the term “in combination with” means that other therapeutically active components can be administered before, concurrently with, or after the anti-MERS-CoV-S antibodies of the invention. The term “in combination with” also includes sequential or concomitant administration of the anti-MERS-CoV-S antibodies and a second therapeutic agent.

[0183] Other therapeutic active components can be administered to a subject prior to administration of the anti-MERS-CoV-S antibody of the present invention. For example, if the first antibody is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before or less than 1 minute before the administration of the second antibody, the first component is considered to be administered "before" the second component. In other embodiments, other therapeutic active components can be administered to a subject after administration of the anti-MERS-CoV-S antibody of the present invention. For example, if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after the administration of the second component, the first component is considered to be administered "after" the second component. In other embodiments, other therapeutic active components can be administered to a subject simultaneously with the administration of the anti-MERS-CoV-S antibody of the present invention. For the purposes of the present invention, "simultaneous" administration includes administering, for example, the anti-MERS-CoV-S antibody and other therapeutic active components to a subject in a single dosage form or in separate dosage forms within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form can be administered via the same route (e.g., both the anti-MERS-CoV-S antibody and other therapeutic active components can be administered intravenously, etc.); alternatively, each dosage form can be administered via a different route (e.g., the anti-MERS-CoV-S antibody can be administered intravenously while the other therapeutic activity can be administered orally). For the purposes of the present invention, in any event, administration of the components in a single dosage form, administration of different dosage forms via the same route, or administration of different dosage forms via different routes is considered to be "simultaneous administration". For the purposes of the present invention, administration of the anti-MERS-CoV-S antibody "before", "simultaneously" or "after" the administration of other therapeutic active components is considered to be "combined" administration of the anti-MERS-CoV-S antibody with other therapeutic active components.

[0184] The present invention includes pharmaceutical compositions wherein the anti-MERS-CoV-S antibody of the present invention is co-formulated with one or more other therapeutic active components described elsewhere herein.

[0185] Administration regimen

[0186] According to some embodiments, a single dose of the anti-MERS-CoV-S antibody of the present invention (or a pharmaceutical composition comprising a combination of an anti-MERS-CoV-S antibody and any other therapeutically active agent mentioned herein) can be administered to a subject in need thereof. According to some embodiments of the present invention, multiple doses of the anti-MERS-CoV-S antibody (or a pharmaceutical composition comprising a combination of an anti-MERS-CoV-S antibody and any other therapeutically active agent mentioned herein) can be administered to a subject over a defined time course. The method according to this aspect of the present invention includes sequentially administering multiple doses of the anti-MERS-CoV-S antibody of the present invention to a subject. As used herein, "sequential administration" means that each dose of the anti-MERS-CoV-S antibody is administered to the subject at different points in time, e.g., on different days separated by a predetermined time interval (e.g., hours, days, weeks or months). The present invention includes methods that include sequentially administering a single initial dose of the anti-MERS-CoV-S antibody to a patient, followed by one or more second doses of the anti-MERS-CoV-S antibody, and optionally followed by one or more third doses of the anti-MERS-CoV-S antibody.

[0187] The terms "initial dose", "second dose" and "third dose" refer to the time sequence of administering the anti-MERS-CoV-S antibody of the present invention. Thus, the "initial dose" is the dose administered at the start of the treatment regimen (also referred to as the "baseline dose"); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial, second and third doses can all contain the same amount of the anti-MERS-CoV-S antibody, but generally can differ from each other in terms of the frequency of administration. However, in some embodiments, the amounts of the anti-MERS-CoV-S antibody contained in the initial, second and / or third doses differ from each other during the course of the treatment (e.g., upregulated or downregulated as appropriate). In some embodiments, two or more (e.g., 2, 3, 4 or 5) doses are administered at the start of the treatment regimen as "loading doses", followed by subsequent doses (e.g., "maintenance doses") administered at a less frequent basis.

[0188] In some exemplary embodiments of the present invention, each second and / or third dose is administered 1 - 48 hours immediately after the previous dose (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 administered (e.g., within 24 hours, within 12 hours, within 6 hours, within 4 hours, within 2 hours, or longer) after the previous dose. As used herein, the phrase "immediately after the previous dose" refers to the dose of the anti-MERS-CoV-S antibody administered to a patient immediately before the next dose in a multiple-dose sequence without an intervening dose in the dosing sequence.

[0189] The method according to this aspect of the invention may comprise administering to a patient any number of second and / or third doses of the anti-MERS-CoV-S antibody. For example, in some embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in some embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

[0190] In some embodiments of the invention, the frequency of administration of the second and / or third doses to the patient may vary during the course of the treatment regimen. During treatment, the frequency of administration may also be adjusted by the physician according to the needs of the individual patient following a clinical examination.

[0191] Diagnostic use of the antibody

[0192] The anti-MERS-CoV-S antibodies of the present invention can be used to detect and / or measure MERS-CoV in a sample, for example, for diagnostic purposes. Some embodiments contemplate using one or more antibodies of the present invention in an assay to detect a disease or disorder such as a viral infection. Exemplary diagnostic assays for MERS-CoV include, for example, contacting a sample obtained from a patient with an anti-MERS-CoV-S antibody of the present invention, wherein the anti-MERS-CoV-S antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate MERS-CoV from the patient sample. Alternatively, an unlabeled anti-MERS-CoV-S antibody can be used in a diagnostic application in combination with a second antibody that is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125 I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure MERS-CoV in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0193] Samples that can be used in the MERS-CoV diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient that contains a detectable amount of MERS-CoV spike protein or a fragment thereof under normal or physiological conditions. Generally, the level of MERS-CoV spike protein in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease associated with MERS-CoV) will be measured to initially establish a baseline or standard MERS-CoV level. This MERS-CoV baseline level can then be compared to the MERS-CoV level measured in a sample obtained from an individual suspected of having a MERS-CoV-related condition or symptoms associated with the condition.

[0194] Antibodies that specifically bind to the MERS-CoV spike protein may not contain other labels or moieties, or they may contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In a binding assay, the location of the label (if present) can determine the orientation of the peptide relative to the surface to which the peptide binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be away from the surface. Specific embodiments

[0195] 1. An isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) spike protein.

[0196] 2. The antibody or antigen-binding fragment thereof of embodiment 1, wherein the antibody or antigen-binding fragment thereof has one or more of the following characteristics:

[0197] (a) Is a full-length human monoclonal antibody;

[0198] (b) Interacts with one or more amino acid residues in the receptor-binding domain of the MERS-CoV spike protein, wherein the amino acid residues are selected from amino acid residues 367-606 of SEQ ID NO: 457;

[0199] (c) Binds to the MERS-CoV spike protein with a dissociation constant (K D ) of less than 18.5 nM, as measured in a surface plasmon resonance assay;

[0200] (d) Blocks greater than 90% of the binding of the MERS-CoV spike protein to dipeptidyl peptidase 4 (DPP4), as measured in a blocking ELISA assay;

[0201] (e) Neutralizes greater than 90% of the infectivity of MERS-CoV in human host cells and has an IC 50 of less than 4 nM, as measured in a virus-like particle (VLP) neutralization assay;

[0202] (f) Neutralizing MERS-CoV infectivity, wherein the MERS-CoV comprises isolates of a virus selected from the group consisting of: EMC / 2012, Jordan-N3 / 2012, England-Qatar / 2012, Al-Hasa_1_2013, Al-Hasa_2_2013, Al-Hasa_3_2013, Al-Hasa_4_2013, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17, Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-hasa_25, Bisha_1, Buraidah_1, England 1, Hafr-Al-batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1, KFU-HKU 1, KFU-HKU 13, Munich, Qatar3, Qatar4, Riyadh_1, Riyadh_2, Riyadh_3, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_9, Riyadh_14, Taif_1, UAE and Wadi-Ad-Dawasir; and

[0203] (g) being a bispecific antibody that comprises a first binding specificity for a first epitope in the receptor-binding domain of the MERS-CoV spike protein and a second binding specificity for a second epitope in the receptor-binding domain of the MERS-CoV spike protein, wherein the first and second epitopes are different and non-overlapping.

[0204] 3. The antibody or antigen-binding fragment thereof according to embodiment 2, wherein the antibody or antigen-binding fragment comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3), the heavy-chain CDRs being comprised in any of the heavy-chain variable region (HCVR) sequences listed in Table 2, and the light-chain CDRs being comprised in any of the light-chain variable region (LCVR) sequences listed in Table 2.

[0205] 4. The antibody or antigen-binding fragment thereof according to embodiment 3, which comprises an HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 2.

[0206] 5. The antibody or antigen-binding fragment thereof according to embodiment 3 or 4, which comprises an LCVR having an amino acid sequence selected from the LCVR sequences listed in Table 2.

[0207] 6. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-5, which comprises:

[0208] (a) The HCDR1 domain, which has an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 124, 132, 140, 148, 156, 172, 180, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, and 444;

[0209] (b) The HCDR2 domain, which has an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 126, 134, 142, 150, 158, 174, 182, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 398, 414, 430, and 446;

[0210] (c) The HCDR3 domain, which has an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 128, 136, 144, 152, 160, 176, 184, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, and 448;

[0211] (d) The LCDR1 domain, which has an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 164, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, and 452;

[0212] (e) The LCDR2 domain, which has an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 166, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, and 454; and

[0213] (f) The LCDR3 domain, which has an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 168, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, and 456.

[0214] 7. The antibody or antigen-binding fragment of any one of embodiments 3-6, which comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 106, 122 / 106, 130 / 106, 138 / 106, 146 / 106, 154 / 162, 170 / 162, 178 / 162, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, 378 / 386, 394 / 402, 410 / 418, 426 / 434, and 442 / 450.

[0215] 8. An isolated antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment comprising the CDRs of HCVR and the CDRs of LCVR for binding to MERS-CoV-S, wherein the HCVR has an amino acid sequence selected from the HCVR sequences listed in Table 2; and wherein the LCVR has an amino acid sequence selected from the LCVR sequences listed in Table 2.

[0216] 9. An isolated antibody or antigen-binding fragment thereof that blocks the binding of MERS-CoV-S to DPP4 comprising the CDRs of HCVR and the CDRs of LCVR, wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 122, 130, 138, 146, 154, 170, 178, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, and 442; and wherein the LCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 162, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, and 450.

[0217] 10. An isolated full-length human monoclonal antibody or antigen-binding fragment thereof that blocks the binding of MERS-CoV-S to DPP4, wherein the antibody or antigen-binding fragment thereof interacts with the amino acid sequence comprising amino acid residues 367-606 of SEQ ID NO: 457.

[0218] 11. An isolated recombinant human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the MERS-CoV spike protein, wherein the antibody or antigen-binding fragment thereof interacts with the spike protein of a MERS-CoV isolate selected from the group consisting of: EMC / 2012, Jordan-N3 / 2012, England-Qatar / 2012, Al-Hasa_1_2013, Al-Hasa_2_2013, Al-Hasa_3_2013, Al-Hasa_4_2013, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17, Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-hasa_25, Bisha_1, Buraidah_1, England 1, Hafr-Al-batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1, KFU-HKU 1, KFU-HKU13, Munich, Qatar3, Qatar4, Riyadh_1, Riyadh_2, Riyadh_3, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_9, Riyadh_14, Taif_1, UAE, and Wadi-Ad-Dawasir.

[0219] 12. The antibody or antigen-binding fragment thereof of embodiment 11, wherein the antibody or antigen-binding fragment thereof blocks the binding of MERS-CoV to DPP4 on human cells.

[0220] 13. The antibody or antigen-binding fragment thereof according to embodiment 11 or 12, wherein the antibody or antigen-binding fragment thereof is isolated from a hybridoma cell line selected from the group consisting of: HBVX06H05, HBVX11H04, HBVX11D02, HBVZ10E10, HBVY09F08, HBVZ05G02, HBVZ09B06, HBVY01F08, HBVY10G02, HBVY04B06, HBVY07D10, HBVZ08A09, HBVZ05G04, HBVY06C07, HBVY03H06, HBVZ10G06, HBVZ04F10, HBVX11E09, HBVY06H09, HBVZ05B11, HBVY02E05, and HBVZ04C07.

[0221] 14. An isolated recombinant human monoclonal antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment comprising the CDRs of HCVR and the CDRs of LCVR, wherein the HCVR comprises an amino acid sequence selected from the HCVR sequences listed in Table 2; and wherein the LCVR comprises an amino acid sequence selected from the LCVR sequences listed in Table 2.

[0222] 15. The antibody or antigen-binding fragment thereof according to embodiment 14, wherein the antibody or antigen-binding fragment thereof blocks the binding of the MERS-CoV spike protein to DPP4.

[0223] 16. An isolated recombinant human monoclonal antibody or antigen-binding fragment thereof that blocks the binding of MERS-CoV to DPP4 comprising the CDRs of HCVR and the CDRs of LCVR, wherein the HCVR comprises an amino acid sequence selected from the HCVR sequences listed in Table 2; and wherein the LCVR comprises an amino acid sequence selected from the LCVR sequences listed in Table 2.

[0224] 17. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-16, wherein the antibody or antigen-binding fragment thereof prevents MERS-CoV from entering host cells.

[0225] 18. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-17, wherein the antibody or antigen-binding fragment thereof is a multispecific antigen-binding molecule.

[0226] 19. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-18 and a pharmaceutically acceptable carrier or diluent.

[0227] 20. A pharmaceutical composition comprising: (a) a first antibody or an antigen-binding fragment thereof that binds to the MERS-CoV spike protein at a first epitope; (b) a second antibody or an antigen-binding fragment thereof that binds to the MERS-CoV spike protein at a second epitope, and (c) a pharmaceutically acceptable carrier or diluent.

[0228] 21. The pharmaceutical composition of embodiment 20, wherein at least the first antibody or an antigen-binding fragment thereof or the second antibody or an antigen-binding fragment thereof blocks the binding of MERS-CoV-S to DPP4.

[0229] 22. The pharmaceutical composition of embodiment 20 or 21, wherein the first and second epitopes are present in the receptor-binding domain of the MERS-CoV spike protein and are different and non-overlapping.

[0230] 23. The pharmaceutical composition of embodiment 22, wherein the first antibody or an antigen-binding fragment thereof comprises the CDRs of HCVR and LCVR, wherein the HCVR comprises an amino acid sequence selected from the HCVR sequences listed in Table 2; wherein the LCVR comprises an amino acid sequence selected from the LCVR sequences listed in Table 2.

[0231] 24. The pharmaceutical composition of any one of embodiments 20-23, wherein the first antibody or an antigen-binding fragment thereof comprises HCVR and LCVR, wherein the HCVR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, 18, 66, 114, 170, and 218; wherein the LCVR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:10, 26, 74, 106, 162, and 226.

[0232] 25. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of the antibody or an antigen-binding fragment thereof of any one of embodiments 1-18.

[0233] 26. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of the antibody or an antigen-binding fragment thereof of any one of embodiments 1-18.

[0234] 27. A vector comprising the polynucleotide sequence of embodiment 25 or 26.

[0235] 28. A cell expressing the vector of embodiment 27.

[0236] 29. Use of the antibody or an antigen-binding fragment thereof of any one of embodiments 1-18 or the pharmaceutical composition of any one of embodiments 19-24 in the preparation of a drug for preventing, treating, or alleviating at least one symptom or indication of MERS-CoV infection.

[0237] 30. Use according to embodiment 29, wherein the at least one symptom or indication is selected from the group consisting of: pulmonary inflammation, alveolar damage, viral load, fever, cough, tachypnea, pneumonia, diarrhea, organ failure, septic shock, and death.

[0238] 31. Use according to embodiment 29 or 30, wherein the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered prophylactically or therapeutically to a subject in need thereof.

[0239] 32. Use according to embodiment 31, wherein the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered prophylactically to a subject selected from the group consisting of: immunocompromised individuals, the elderly (greater than 65 years old), travelers to the Middle East, healthcare workers, persons with a history of medical problems (such as heart problems and diabetes), persons with occupational or recreational exposure to camels or bats, and persons in contact with a person with confirmed or suspected MERS infection.

[0240] 33. Use according to any one of embodiments 29 - 32, wherein the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered in combination with a second therapeutic agent.

[0241] 34. Use according to embodiment 33, wherein the second therapeutic agent is selected from the group consisting of: anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, different antibodies against the MERS-CoV spike protein, vaccines against MERS-CoV, antibiotics, dietary supplements such as antioxidants, and any other palliative therapy for treating MERS infection.

[0242] 35. Use according to any one of embodiments 29 - 34, wherein the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered by subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular, or intracranial administration.

[0243] Examples

[0244] The following examples are described to provide a complete disclosure and description to those skilled in the art of how to make and use the compositions and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, room temperature is about 25 °C, and pressures are at or near atmospheric pressure.

[0245] Example 1: Generation of Human Antibodies Against the MERS-CoV Spike Protein

[0246] In Human antibodies against the MERS-CoV spike protein are generated in mice that contain DNA encoding the variable regions of the human immunoglobulin heavy and κ light chains. The mice are immunized with a DNA construct encoding the full-length MERS-CoV spike protein, followed by a boost immunization with a purified spike protein fragment (amino acids 367 - 606 of GenBank accession number AFS88936.1 (SEQ ID NO: 457)). A codon-optimized cDNA sequence encoding the MERS-CoV S protein (EMC / 2012 - GenBank JX869059) is synthesized by GeneArt and cloned into a standard expression vector. A plasmid encoding a variant of the MERS-CoV S protein in the receptor-binding domain (RBD) is generated by mutagenesis using QuikChange II (Agilent Technologies) according to the manufacturer's instructions.

[0247] The antibody immune response is monitored by a MERS-CoV-S-specific immunoassay. When a satisfactory immune response is obtained, spleen cells are harvested and fused with myeloma cells of the mouse to retain their variability and form a hybridoma cell line. The hybridoma cell lines are screened and selected to identify the cell lines that produce MERS-CoV-S-specific antibodies. Of a total of 696 hybridomas expressing antigen-specific antibodies, 182 were found to block the interaction of DPP4 with the spike protein and 123 blocked MERSpp entry into target cells. Exemplary cell lines generated in this manner are named HBVX06H05, HBVX11H04, HBVX11D02, HBVZ10E10, HBVY09F08, HBVZ05G02, HBVZ09B06, HBVY01F08, HBVY10G02, HBVY04B06, HBVY07D10, HBVZ08A09, HBVZ05G04, HBVY06C07, HBVY03H06, HBVZ10G06, HBVZ04F10, HBVX11E09, HBVY06H09, HBVZ05B11, HBVY02E05, and HBVZ04C07. The cell lines are used to obtain several anti-MERS-CoV-S chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains); exemplary antibodies generated in this manner are named H1M15277N, H2aM15279N, H1M15280N, H2aM15278N, H2aM15271N, H1M15267N, H2bM15292N, H2bM15291N, H1M15290N, H1M15293N, H1M15289N, H1M15269N, H2aM15287N, H1M15288N, H2aM15268N, H2aM15270N, H2aM15281N, and H2aM15272N.

[0248] Table 1 shows the chimeric antibodies obtained from the corresponding hybridoma supernatants

[0249] Table 1

[0250] Ab PID# Clone Name H1M15267N HBVX11H04 H1M15269N HBVY07D10 H1M15277N HBVX06H05 H1M15280N HBVZ09B06 H1M15288N HBVY02E05 H1M15289N HBVY03H06 H1M15290N HBVZ04F10 H1M15293N HBVZ10G06 H2aM15268N HBVY04B06 H2aM15270N HBVY09F08 H2aM15271N HBVY10G02 H2aM15272N HBVZ05G02 H2aM15278N HBVX11D02 H2aM15279N HBVY01F08 H2aM15281N HBVZ10E10 H2aM15287N HBVX11E09 H2bM15291N HBVZ05G04 H2bM15292N HBVZ08A09

[0251] Anti-MERS-CoV-S antibodies were also directly isolated from antigen-positive murine B cells that do not hybridize with myeloma cells, as described in U.S. Patent No. 7,582,298, which is hereby incorporated by reference in its entirety. Using this method, several full-length human anti-MERS-CoV-S antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained; exemplary antibodies generated in this manner were named H4sH15188P, H1H15188P, H1H15211P, H1H15177P, H4sH15211P, H1H15260P2, H1H15259P2, H1H15203P, H4sH15260P2, H4sH15231P2, H1H15237P2, H1H15208P, H1H15228P2, H1H15233P2, H1H15264P2, H1H15231P2, H1H15253P2, H1H15215P, and H1H15249P2.

[0252] The biological properties of the exemplary antibodies generated according to the method of this example are described in detail in the examples described below.

[0253] Example 2: Characterization of Hybridoma Supernatants

[0254] An ELISA binding assay was performed to identify antibody supernatants (obtained from the hybridomas described above) that bind to the MERS-CoV spike protein. A protein consisting of the receptor domain of MERS-CoV-S (amino acids E367 - Y606) expressed together with the Fc portion of the human IgG1 molecule at the C-terminus (MERS RBD-hFc; SEQ ID NO:458) was coated at 2 μg / ml in PBS buffer on a 96-well plate overnight at 4°C. Non-specific binding sites were then blocked using a 0.5% (w / v) BSA solution in PBS. The hybridoma supernatants were diluted 1:50 in the blocking buffer and allowed to bind to the MERS RBD-coated plate for 1 hour at room temperature. After washing, the bound antibodies were detected using an HRP-conjugated anti-hFc polyclonal antibody (Jackson Immunochemical). The samples were developed with 3,3’,5,5’-tetramethylbenzidine solution (TMB; BD Biosciences) to produce a colorimetric reaction and then neutralized with 1 M sulfuric acid, after which the absorbance at 450 nm was measured on a Victor plate reader (Perkin Elmer). The results are shown in Figure 1 . Antibodies were selected for further characterization using the signal intensity at 450 nm.

[0255] On a Biacore 4000 or Biacore T200 instrument, the association and dissociation rate constants (k a and k d ), respectively) of antigen binding to the hybridoma supernatants were determined using a real-time surface plasmon resonance biosensor assay, as well as the equilibrium dissociation constant and dissociation half-life (K D and t 1 / 2 ) (details are described in Example 4). As Figure 1 shown, 21 out of 22 hybridoma antibodies bound to the MERS-CoV spike protein with K D values of 245 pM - 21.5 nM.

[0256] The ability of anti-MERS coronavirus spike protein antibodies to block the binding of the receptor-binding domain of MERS (MERS-RBD) to its cognate binding partner, human dipeptidyl peptidase 4 (hDPP4), was evaluated using an ELISA-based immunoassay. Briefly, expressed human dipeptidyl peptidase 4 with a 6x histidine tag at the C-terminus (hDPP4-6His; R&D catalog #1180-SE) was coated onto 96-well plates at 2 μg / mL in PBS buffer overnight at 4 °C. Nonspecific binding sites were then blocked using a 0.5% (w / v) BSA solution in PBS. The plate was used to measure MERS-RBD (amino acids E367 - Y606) (MERSRBD-hFc; SEQ ID NO:458) expressed together with the Fc portion of human IgG1 molecules at the C-terminus in a MERS RBD-hFc solution pre-equilibrated with dilutions of anti-MERS antibody supernatants. A constant concentration of 300 pM of MERS RBD-hFc was pre-mixed with 10% volume of anti-MERS antibody supernatants and then incubated for 1 hour at room temperature to allow antibody-antigen binding to reach equilibrium. The equilibrated sample solutions were then transferred to the hDPP4-6his-coated plates. After binding at RT for 1 hour, the plates were washed and bound MERS RBD was detected using an HRP-conjugated anti-hFc polyclonal antibody (Jackson Immunochemical). Samples were developed with TMB solution (BD Biosciences, #51-2606KC and #51-2607KC) to generate a colorimetric reaction and then neutralized with 1 M sulfuric acid, after which absorbance was measured at 450 nm on a Victor X5 plate reader (Perkin Elmer). The absorbance measured with a constant concentration of MERS RBD-hFc alone was defined as 0% block and the absorbance measured without MERS RBD-hFc added was defined as 100% block. The percent block was calculated as the ratio of the signal reduction observed in the presence of the antibody,

[0257] The percent block was calculated as follows: the ratio of the signal reduction observed in the presence of the antibody relative to the difference subtracted from 100% block as defined above using the difference between MERS RBD-hFc alone and background (signal from HRP-conjugated anti-hFc antibody only).

[0258] As Figure 1 shown, 17 out of 22 independent anti-MERS-CoV-S antibodies derived from different hybridomas blocked >90% of the binding of MERS-CoV spike protein to DPP4.

[0259] Anti-MERS-CoV-S antibodies derived from different independent hybridomas were tested for neutralization of MERS infectivity (assay details in Example 5). As Figure 1As shown in 50 ,

[0260] Example 3: Amino Acid and Nucleotide Sequences of Heavy and Light Chain Variable Regions

[0261] Table 2 illustrates the amino acid sequence identities of the heavy and light chain variable regions and CDRs of the selected anti-MERS-CoV-S antibodies of the present invention.

[0262] Table 2: Amino Acid Sequence Identities

[0263]

[0264]

[0265] The corresponding nucleic acid sequence identities are set forth in Table 3.

[0266] Table 3: Nucleic Acid Sequence Identities

[0267]

[0268]

[0269] The antibodies described herein generally follow the following nomenclature: an Fc prefix (e.g., "H4xH", "H1M", "H2M", etc.), followed by a numerical identifier (e.g., "15177", "15228", "15268", etc., as shown in Table 2), followed by a "P", "P2", "N", or "B" suffix. Thus, according to this nomenclature, the antibodies can be referred to herein as, for example, "H1H15177P", "H1H15228P2", "H2M15268N", etc. The H4sH, H1M, and H2M prefixes on the antibody names used herein indicate the specific Fc region isotypes of the antibodies. For example, the "H4sH" antibody has a human IgG4 Fc with two or more amino acid alterations as disclosed in US20100331527, the "H1M" antibody has a murine IgG1 Fc, and the "H2M" antibody has a murine IgG2 Fc (a or b isotype) (all variable regions are fully human, as indicated by the initial 'H' of the antibody nomenclature). Those of ordinary skill in the art will understand that an antibody with a specific Fc isotype can be converted into an antibody with a different Fc isotype (e.g., an antibody with a murine IgG1 Fc can be converted into an antibody with a human IgG4, etc.), but in any event, the variable domains (including the CDRs) - which are represented by the numerical identifiers shown in Table 2 - will remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0270] Example 4: Binding of antibody to MERS-CoV-S determined by surface plasmon resonance The association and dissociation rate constants (k a and k d ), equilibrium dissociation constant and dissociation half-life (K D and t 1 / 2 ) of the antigen binding to the hybridoma supernatant were determined using a real-time surface plasmon resonance biosensor assay on a Biacore 4000 or Biacore T200 instrument. The Biacore sensor surface was derivatized with polyclonal rabbit anti-mouse antibody (GE, #BR-1008-38) or monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture approximately 100 - 900 RU of anti-MERS-CoV-S monoclonal antibody, expressed with mouse Fc or human Fc respectively. The MERS-CoV reagents tested for binding to the anti-MERS-CoV-S antibody included recombinant MERS-CoV-S receptor binding domain expressed with C-terminal human IgG1 Fc (MERS RBD-hFc; SEQ ID NO: 458). Different concentrations of MERS-CoV reagent from 3.7 nM - 200 nM were injected at a flow rate of 30 μL / minute on the Biacore 4000 or 50 μL / minute on the Biacore T200 onto the surface captured with anti-MERS-CoV-S monoclonal antibody. The binding of MERS-CoV reagent to the captured monoclonal antibody was monitored for 3 - 5 minutes in HBST running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20), while its dissociation from the antibody was monitored for 7 - 10 minutes. The experiment was conducted at 25 °C. The kinetic binding (k a ) and dissociation (k d ) rate constants were determined by processing the data using Scrubber 2.0c curve fitting software and fitting it to a 1:1 binding model. The binding dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) were then calculated from the kinetic rate constants as: K D (M) = k d / k a and t 1 / 2 (minutes) = [ln2 / (60 * k d )].

[0271] In the initial experiment, the binding dissociation equilibrium constants and dissociation half-lives of 22 hybridoma supernatants were determined (described in Example 2).

[0272] In subsequent experiments, the binding of the purified antibodies to the MERS-CoV spike protein was tested using surface plasmon resonance assays as described above.

[0273] Table 4

[0274]

[0275]

[0276] Table 5

[0277]

[0278]

[0279] As shown in Tables 4 and 5, the antibodies bound to the MERS-CoV spike protein with K D values of 77.5 pM - 18.5 nM at 25 °C and K D values of 127 pM - 13.2 nM at 37 °C. The antibodies showed dissociation half-life (t1 / 2) values of 2.2 - 262 minutes at 25 °C and 1.8 - 244 minutes at 37 °C.

[0280] Example 5: Antibody-mediated neutralization of MERS infectivity

[0281] Materials and methods

[0282] Generation of Pseudoparticles

[0283] MERS pseudoparticles (MERSpp; also known as virus-like particles - VLP) were generated by co-transfecting 293T cells with a plasmid construct expressing the MERS spike protein, HIV gag-pol, and an HIV proviral vector encoding firefly luciferase. The supernatant containing MERSpp was harvested 72 hours post-transfection, clarified by centrifugation, concentrated, aliquoted, and frozen at -80 °C. Control pseudoparticles were generated by replacing the plasmid expressing the MERS-CoV spike protein with a plasmid encoding Vesicular Stomatitis Virus Glycoprotein (VSVg).

[0284] MERSpp-based Assays

[0285] Antibody dilutions were incubated with MERSpp for 1 hour at room temperature. Huh7 cells in complete DMEM medium were detached using 1% EDTA, washed, and incubated with the antibody / MERSpp mixture for 72 hours. The infectivity was determined by Quantification was performed by luciferase assay (Promega, San Luis Obispo, CA, USA) and light production was read in a Tecan X3 plate reader.

[0286] Virus-based Assays

[0287] One day before the experiment, 1x10 4 Vero E6 cells were seeded per well in a 96-well plate. 500 TCID 50 of the MERS-CoV EMC / 2012 strain was mixed with the indicated amounts of antibody in 100 ul of normal Vero E6 growth medium and incubated at room temperature for 30 minutes, and then the mixture was added to the Vero E6 cells. On day 2 post-infection, a luminescent cell viability assay ( Luminescent Cell Viability Assay, Promega) was used to assess cell death according to the manufacturer's instructions. Luminescence was read on a Spectramax M plate reader (Molecular Devices). Data are presented as a percentage of mock-infected controls.

[0288] Results

[0289] Neutralization of MERS-CoV by Anti-MERS-CoV-S Antibodies

[0290] In the initial experiment, neutralization of MERS infectivity by anti-MERS-CoV-S antibodies derived from different independent hybridomas was tested. As Figure 1 shown, 12 out of 22 cell anti-MERS-CoV-S hybridoma antibodies inhibited or neutralized more than 90% of MERS infectivity and had an IC 50 .

[0291] In subsequent experiments, neutralization of MERSpp and live MERS virus (isolate: EMC / 2012) by purified antibodies was tested. The results are shown in Tables 6 and 7.

[0292] Table 6

[0293]

[0294]

[0295] Table 7

[0296] Antibodies IC50 (M) against MERSpp IC50 (M) against Live Virus (EMC2012) H1H15211P 4.74E-11 3.69E-10 H1H15188P 6.20E-11 5.05E-10 H1H15177P 4.57E-11 6.29E-10 H1H15203P 7.65E-11 8.26E-10 H1H15228P2 9.75E-11 8.98E-10 H1H15231P2 1.02E-10 9.20E-10 H1H15237P2 6.60E-11 1.01E-09 H1H15233P2 8.73E-11 1.37E-09 H1H15259P2 7.64E-11 1.63E-09 H1H15264P2 1.34E-10 2.39E-09 H1H15208P 8.27E-11 2.41E-09 H1H15253P2 1.51E-10 2.42E-09 H1H15215P 1.92E-10 2.67E-09 H1H15260P2 6.43E-11 6.70E-09 H1H15249P2 2.27E-10 1.20E-08 H4sH15188P 2.91E-11 NA H4sH15211P 6.08E-11 NA H4sH15260P2 6.24E-11 NA H4sH15231P2 7.33E-11 NA

[0297] The above data indicate that the anti-MERS-CoV-S antibodies of the present invention potently block MERS-CoV entry into susceptible cells and neutralize infectivity.

[0298] Neutralization of Clinical Isolates by Anti-MERS-CoV-S Antibodies

[0299] RNA viruses encode low-fidelity genome replication machinery, which allows them to rapidly adapt to their host environment (Malpica et al. 2002; Genetics 162:1505-1511). However, coronaviruses such as MERS-CoV encode non-structural proteins with 3'-5' ribonuclease exonucleolytic activity, which provides a proofreading function during their replication and greatly increases the coding capacity of the genome without causing a mutational catastrophe (Cotton et al. 2013; Lancet doi:10.1016 / S0140-6736(13)61887-5). Nevertheless, sequencing of multiple clinical isolates during the first two years of the MERS-CoV outbreak has revealed that the MERS-CoV S protein of the virus is evolving (Cotton et al. 2014; MBio 5 doi:10.1128 / mBio.01062-13). For the present study, the sequences of 38 MERS-CoV clinical isolates deposited in NCBI were aligned and compared with the first isolated strain, EMC / 2012. Based on the design of the screening assay, the antibodies of the present invention are expected to bind within the RBD of the MERS-CoV S protein, and thus the sequence comparison focused specifically on amino acids 367-606. Seven different amino acids were identified that differ from the sequenced clinical isolates and the prototype EMC / 2012 sequence, A431P, S457G, S460F, A482V, L506F, D509G, and V534A (Table 8).

[0300] Table 8

[0301] Amino Acid Changes Isolates A431P Riyadh_9 S457G KFU-HKU 1; KFU-HKU 13 S469F Qatar3; Qatar4 A482V Riyadh_9 L506F England 1; England-Qatar / 2012 D509G Bisha_1; Riyadh_1 V534A Riyadh_2

[0302] To test the ability of the antibodies to bind to conserved regions within the RBD and neutralize all MERS-CoV clinical isolates sequenced in July 2014, plasmid constructs encoding all S protein variants were generated using site-directed mutagenesis. These were used to generate MERSpp pseudotypes with modified S proteins and a neutralization assay was performed. The purified antibodies (described in Example 1) were tested for neutralization of MERS infectivity using MERSpp generated with the above mutations. Tables 9 and 10 show the percent neutralization of the MERSpp variants.

[0303] Table 9

[0304]

[0305]

[0306] Table 10

[0307]

[0308]

[0309] These data indicate that the antibodies of the present invention bind to regions of the MERS-CoV spike protein that are conserved during the natural evolution of the virus.

[0310] The anti-MERS-CoV-S antibodies of the present invention are more effective neutralizing agents compared to previously isolated anti-MERS-CoV antibodies

[0311] The potencies of the selected antibodies of the present invention were compared to previously isolated monoclonal antibodies. Three groups used in vitro antibody isolation methods, namely phage display (Tang et al. 2014, PNAS doi:10.1073 / pnas.1402074111; and Ying et al. 2014, J. Virol. doi:10.1128 / JVI.00912-14) and yeast display (Jiang et al. 2014, Sci. Transl. Med. doi:10.1126 / scitranslmed.3008140), to select antibodies that bind to the MERS-CoV S protein and block virus entry. For this study, a group of three antibodies with publicly available variable domain sequences that were reported to neutralize MERS-CoV and bind to different epitopes were selected. The sequences of antibody 3B12 (Tang et al. 2014, PNAS doi:10.1073 / pnas.1402074111), MERS-4, and MERS-27 (Jiang et al. 2014, Sci. Transl. Med. doi:10.1126 / scitranslmed.3008140) were cloned onto the human IgG1 constant domain and subsequently expressed and purified similarly to the selected antibodies of the present invention. The neutralization potencies of all antibodies were tested using pseudoparticles generated with the prototype EMC / 2012 sequence and all of the above-mentioned clinical isolates.

[0312] The neutralization IC50s of the selected antibodies against the MERSpp variants are shown in Table 11. As seen in Table 11, H1H15211P was able to neutralize all isolates with IC 50 values ranging from 1.3E-11M to 6.5E-11M. Similar values were observed for H1H15277N, except for the V534A variant, which showed partial resistance to the antibody. However, it is worth noting that this amino acid change was only observed in a single MERS-CoV isolate (Riyadh_2) isolated in 2012 and belonging to a dead branch of the MERS-CoV phylogenetic tree.

[0313] H1H15211P and H1H277N neutralize MERSpp with an IC 50 value at least 1 log lower than that of the most potent comparator antibody, MERS-4. In addition, both 3B12 and MERS-27 appear to bind to less conserved sites on the MERS-CoV S protein, as pseudoparticles with multiple clinical isolates cannot be neutralized with this antibody.

[0314] Table 11

[0315]

[0316] These data indicate that the antibodies of the present invention can neutralize a broader range of MERS-CoV isolates with improved potency compared to several antibodies isolated based solely on in vitro biochemical properties.

[0317] Example 6: Octet cross-competition between anti-MERS-CoV-S antibodies

[0318] In Binding competition between MERS-CoV S antibodies was determined using real-time, label-free biolayer interferometry on a RED96 biosensor (Pall ForteBio Corp). The complete experiment was carried out with the plate shaken at 1000 rpm in HBS-P Octet buffer (10 mM HEPES, 150 mM NaCl and 0.05% v / v surfactant Tween-20, pH 7.4, 1 mg / mL BSA) at 25 °C. To evaluate whether antibodies were able to compete with each other for binding to their corresponding epitopes on the recombinant expressed MERS spike protein receptor binding domain fused with human Fc tag (MERS RBD-hFc; SEQ ID: 458), a pre-mixed assay format was employed and 50 nM of MERS-CoV-RBD-mFc was pre-incubated with 500 nM of different anti-MERS monoclonal antibodies (subsequently referred to as mAb-2) for at least 2 hours before performing the binding competition assay. Non-specific monoclonal antibodies were incubated with MERS-RBS.mFc as an isotype control. The Octet biosensor coated with anti-hFc polyclonal antibody (Pall ForteBio Corp., catalog #18-5060) was first dipped into a well containing a 50 μg / mL solution for 4 minutes to capture approximately 2 nm of anti-MERS antibody (subsequently referred to as mAb-1). After the capture step, the vacant anti-hFc polyclonal antibody was then saturated by dipping the Octet biosensor into a well containing 100 μg / mL of non-specific fully human monoclonal antibody. Finally, the biosensor was dipped into a well containing the pre-mixed sample of 50 nM MERS-RBD-mFc and 500 nM mAb-2 for 4 minutes. The biosensor was washed in HBS-P Octet buffer between each step of the experiment. The real-time binding response was monitored during the experiment and the binding response was measured at the end of each step. The response of mAb-1 binding to the pre-complex of MERS-RBD-mFc and mAb-2 was compared and the competitive / non-competitive behavior of different anti-MERS monoclonal antibodies was determined.

[0319] Under the experimental conditions used in this example, cross-competition (i.e., competition between antibodies in both directions) was observed, for example, for: (a) H2aM15281N, H1M15290N, H1M15293N and H2bM15291N; (b) H2aM15272N; H2bM15292N and H2aM15271N; and (c) H2aM15268N, H1M15267N, H2M15279N, H1M15289N and H1M15277N( Figure 2 ).

[0320] In another experiment, the binding competition between selected antibodies directly isolated from antigen-positive murine B cells (described in Example 1) was determined. Under the experimental conditions used in this example, cross-competition (i.e., competition between antibodies in both directions) was observed, for example: (a) H1H15215P, H1H15177P, H1H15203P, and H1H15211P; (b) H1H15188P, H1H15208P, H1H15228P2, H1H15233P2, H1H15237P2, H1H15253P2, and H1H15259P2; and (c) H1H15231P2, H1H15249P2, H1H15260P2, and H1H15264P2( Figure 3 ).

[0321] In another experiment, the binding competition between two antibodies, H1H15211P and H1H15277N, was monitored.

[0322] Table 12

[0323]

[0324] Table 12 shows that the two antibodies do not inhibit each other and that the binding of one antibody to the MERS-CoV RBD still permits the binding of the second antibody. These data indicate that the two antibodies bind to discrete, non-overlapping epitopes. Mutations in one epitope as a result of selection pressure by one antibody do not affect the binding of the other.

[0325] Example 7: In Vivo Efficacy of Anti-MERS-CoV-S Antibodies

[0326] Materials and Methods

[0327] Generation of Human DPP4 Knock-in Mice

[0328] Since the MERS spike protein does not interact with murine DPP4, A technique was used to generate a humanized model of MERS-CoV infection (Valenzuela et al., 2003, Nat. Biotechnol. 21: 652-659). Briefly, a large targeting vector (LTVEC) was constructed to contain 82 kb of human DPP4 genomic DNA from exons 2 to 26 containing the 3'UTR to replace the 79 kb murine Dpp4 counterpart sequence. Human BAC RP11-68L22 and murine BAC RP23-362N15 containing the Dpp4 gene were identified using Blast, and the sequences were confirmed using an Illumina benchtop sequencer, the MiSeq. The LTVEC was electroporated into F1 hybrid (129S6SvEvTac / C57BL6NTac) ES cells. Ten days after electroporation, G418-resistant clones were picked and screened for correct targeting by allele loss assay. The method (Poueymirou et al., 2007; Nature Biotechnology 25: 91-99) was used, in which the targeted ES cells were injected into uncompacted 8-cell stage Swiss Webster embryos to generate fully ES cell-derived F0 generation mice carrying the human DPP4 gene allele. All animal procedures were conducted in strict accordance with the recommendations of the NIH Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Regeneron Pharmaceuticals Institutional Animal Care And Use Committee (IACUC). The method is described in detail in U.S. Patent Application No. 62 / 051,626, filed September 17, 2014, which is incorporated herein by reference in its entirety. Transgenic mice were also generated by random insertion of the hDPP4 gene into the murine genome.

[0329] Animal Experiments

[0330] Depending on the experiment, 6-8-week-old humanized DPP4 mice were injected intraperitoneally (i.p.) with the indicated amounts of antibody or sterile saline as a control 1 day before or 1 day after infection. Before intranasal inoculation, the mice were anesthetized by intraperitoneal injection with a mixture of xylazine (0.38 mg / mouse; Lloyd laboratories) and ketamine (1.3 mg / mouse; Henry Schein animal health) (diluted in PBS to generate a total volume of 50 μl per mouse). Once anesthetized, PBS with a total inoculum volume of 50 μl or 2 x 10 5Mice were intranasally inoculated with pfu of MERS-CoV (Jordan). During the experiment, mice were weighed daily before infection and throughout the experiment to assess MERS-CoV-induced weight loss. Mice were euthanized on days 2 and 4 post-infection with a lethal dose of isoflurane (Butler Animal Health Supply). Lungs were harvested for further MERS-CoV replication and pathological analysis.

[0331] MERS-CoV RNA Analysis

[0332] RNA was extracted from mouse lungs using a Magnalyzer (Roche) according to the manufacturer's instructions by homogenizing in 1 ml of (LifeTechnologies Inc). The levels of MERS-CoV RNA were assessed using primer duplexes obtained from LifeTechnologies (targeting the upstream region of the genome of the envelope gene (UpE)), the leader sequence of the nucleocapsid messenger RNA (leader primer) using Fast Virus One-Step Master Mix (Applied Biosystems); and were compared to mouse 18S rRNA (endogenous control). The qPCR reactions in a Fast Optical 96-well Plate (Applied Biosystems) were read on a 7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems), and the data were analyzed using the δCt method, where uninfected controls were set to 1. The percentage of MERS-CoV RNA detected was expressed relative to the RNA levels detected in infected mice treated with isotype-matched control antibody.

[0333] Plaque Assay for MERS-CoV Titers

[0334] Mouse lungs were homogenized for 60 seconds in 1 ml of PBS with glass beads using a Magnalyzer (Roche) at 6000 rpm. The homogenate was then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was analyzed by plaque assay on Vero E6 cells to quantify the residual virus levels after treatment. The plaque assay was performed as described previously, except that the plates were left for 3 days for plaque appearance.

[0335] Histological Analysis

[0336] Histological sections were prepared from fixed, paraffin-embedded tissues and stained with hematoxylin and eosin. Fields of view were examined and analyzed by light microscopy. Inflammation of the stroma, surrounding bronchi, and peripheral blood vessels was scored from 0 - 5. For each experimental group, slides were blinded and scored from 0 - 5 and tabulated to compare strains, time points, and treatments / therapies. Other histological features such as the presence of bronchial epithelium and alveolar damage, pleural changes, and the degree of fibrobronchovascular inflammation were also noted for each group in the text. The overall inflammation score per mouse was averaged for each group and expressed as the average score for all mice in each group and time point.

[0337] Results

[0338] DPP4-humanized mice are susceptible to MERS-CoV infection

[0339] In vivo testing of antiviral molecules requires a small animal model susceptible to MERS-CoV infection. Mice are not susceptible to MERS-CoV infection. Sequence comparison of the mouse and human DPP4 sequences revealed that the amino acids previously identified as contact sites between the MERS-CoV S protein and its receptor differ between the two species. In addition, expression of human DPP4 in mouse cells permits MERSpp entry and MERS-CoV spread, indicating that virus entry is the limiting step for mouse cell infection and the lack of interaction between mouse DPP4 and the MERS-CoV glycoprotein defines species tropism in vitro.

[0340] The inventors hypothesized that making mice express human DPP4 in place of mouse DPP4 would render them susceptible to MERS-CoV and permit in vivo testing of anti-MERS-CoV therapies. Using techniques to replace the 79 kb mouse Dpp4 gene with its 82 kb human ortholog. The resulting mice express full-length human DPP4 under the control of mouse regulatory elements to maintain appropriate expression regulation and protein tissue distribution.

[0341] To test whether the humanized and transgenic mice could support MERS-CoV infection, in an initial experiment, transgenic mice were treated intraperitoneally with 200 μg of anti-MERS-CoV-S antibody or isotype control on day -1 and infected intranasally with MERS-CoV (~10 6 pfu of EMC2012) on day 0. Four days post-infection, lungs were harvested and the RNA levels of the virus were measured by RT-PCR to examine the effect of the antibody on the viral load. Table 13 shows the average levels of viral genomic RNA and replicating RNA, expressed as a percentage of the isotype control. Treatment with H1H15211P resulted in a ~500-fold reduction in viral RNA in infected mice (reduction to ~1 / 500).

[0342] Table 13

[0343] Antibodies Genomic RNA Replicating RNA (Leader) H1H15177P 0.356839562 0.273565089 H1H15211P 0.254493202 0.206006238 H4sH15211P 1.989548316 1.112094283 IgG1 Isotype Control 104.0889287 101.2578723 IgG4 Isotype Control 100 100

[0344] In another experiment, humanized DPP4 (huDPP4) mice at 6 - 8 weeks of age were inoculated intranasally with MERS-CoV. Although no deaths or clinical signs of disease were observed until day 4, on days 2 and 4 post-inoculation, the mice were euthanized and their lungs were dissected. To obtain viral RNA levels, the lungs were homogenized in and analyzed by real-time PCR using primers specific for MERS-CoV ( Figure 4 and 5 ). To obtain virus titers, the lungs were homogenized in PBS, clarified by centrifugation and titrated on Vero E6 cells ( Figure 6 ). Robust MERS-CoV replication in the lungs was evident on days 2 and 4 post-infection. RNA quantification using a primer set specific for the MERS-CoV leader sequence designed to amplify only replicating MERS-CoV demonstrated high levels of MERS-CoV replicating RNA in the lungs collected on day 2, and these levels were maintained until day 4 post-infection ( Figure 5 ). Plaque assays of lung homogenates on Vero E6 cells quantified MERS-CoV (Jordan) levels at ∼7.27x10 4 pfu / g of lung on day 2 post-infection and at ∼3.75x10 5 pfu / g of lung on day 4 post-infection ( Figure 6 ), demonstrating active replication of MERS-CoV in the lungs of huDPP4 mice. These data indicate that humanization of the receptor DPP4 using technology created a robust MERS-CoV model in mice that can be used to evaluate MERS-CoV therapies in vivo.

[0345] Pathological changes were analyzed in the lungs from huDPP4 mice inoculated intranasally with MERS-CoV (Jordan strain) or PBS (mock-infected). On day 2 post-infection, peribronchial inflammation was evident, with alterations in bronchial cellular architecture found throughout the lungs. Minimal peripheral vascular inflammation or effects on alveolar structure were noted at this time point. On day 4 post-infection, significant interstitial infiltration, peripheral vascular dilation and extensive alveolar thickening were observed. Bronchial alterations were also still present. Importantly, this pathology was consistent with the development of interstitial pneumonia and the radiographic findings of significant pulmonary disease seen in humans with MERS-CoV, indicating that this humanized DPP4 in vivo model of MERS-CoV infection recapitulates the pathological sequelae seen in human MERS-CoV infection.

[0346] In further experiments (see below), infected mice were prophylactically and / or therapeutically administered one or more doses of the purified antibodies (as described in Example 1) or antibody combinations to test their efficacy against MERS infection.

[0347] H1H15211P and H1H15277N Protect Humanized DPP4 Mice from MERS-CoV Infection

[0348] After establishing that humanized DPP4 mice were susceptible to MERS-CoV, this model was used to evaluate the in vivo activity of two monoclonal antibodies. Twenty-four hours prior to intranasal infection with 1 x 10 5 pfu of MERS-CoV (Jordan strain), mice were injected intraperitoneally (i.p.) with 200 μg, 20 μg or 2 μg of H1H15211P or H1H15277N or with 200 μg of an hIgG1 isotype control antibody. As Figure 7 and 8 seen, at the 200 μg per mouse dose, both antibodies were able to significantly reduce MERS-CoV-specific RNA levels in the lungs by more than 2 logs compared to the isotype control antibody. The 20 μg dose of H1H15211P was more effective than the same dose of H1H15277N in reducing MERS-CoV RNA levels. The 2 μg administration of each antibody was ineffective in reducing viral RNA levels compared to mice treated with the isotype control. When analyzing MERS-CoV titers in the lungs ( Figure 9 ), it was found that both the 200 μg and 20 μg doses of H1H15211P reduced viral levels to near the levels detected in the assay (2 x 10 3 pfu / ml). H1H15277N was equivalent to H1H15211P at the 200 μg dose, while the 20 μg and 2 μg doses showed dose-dependent inhibition of virus inhibition. These data indicate that H1H15211P and H1H15277N can effectively block MERS-CoV infection in vivo.

[0349] Immunohistochemical analysis was also performed on the lungs of mice treated with H1H15277N, H1H15211P, or hIgG1 isotype control antibody 24 hours before infection and on day 4 after infection. Lungs from mice pretreated with hIgG isotype control mice showed significant lung pathology with increased interstitial inflammation, peripheral vascular cuffing, and alveolar septal thickening. Mice treated with 200 μg of H1H15277N or H1H15211P had reduced inflammation with minimal foci of inflammatory cells in the interstitium and bronchiolar cuffing. In mice pretreated with 20 μg of H1H15277N and H1H15211P, moderate levels of peripheral vascular cuffing and interstitial inflammation were found compared to the higher antibody groups. Relatively, the 2 μg antibody pretreatment group had a pathology similar to that of the hIgG1 isotype control showing significant interstitial inflammation and major peripheral vascular inflammation. Blinded histological scores ( Figure 10 ) reflected reduced inflammation scores in treated mice. These findings demonstrate that after MERS-CoV infection, H1H15277N and REGN3051 confer a dose-dependent reduction in lung pathology, corroborating the viral RNA levels and viral titers determined for these mice.

[0350] Overall, these data indicate that when injected 1 day before infection, H1H15211P and H1H15277N can block MERS-CoV infection and disease in vivo. To our knowledge, H1H15211P and H1H15277N are the earliest full-length human antibodies shown to be effective in an in vivo model of MERS-CoV infection.

[0351] H1H15211P and H1H15277N Can Treat Humanized DPP4 Mice Infected with MERS-CoV

[0352] The ability to inhibit MERS-CoV replication and lung pathology after infection is a desirable trait in potential therapies. To evaluate whether H1H15211P can have a therapeutic effect, huDPP4 mice were infected with MERS-CoV and then 24 hours later were injected intraperitoneally with 500 μg of hIgG isotype control or 500 μg or 200 μg of H1H15211P. On day 4 after infection, the mice were euthanized and the viral RNA, viral titers, and lung pathology of the mouse lungs were analyzed. Both 500 μg and 200 μg doses of H1H15211P were able to reduce the viral RNA levels in the mouse lungs by approximately 10-fold ( Figure 11 and 12 ) compared to mice treated with the control antibody. On day 4 after infection, the titers of the same mouse lungs demonstrated a significant reduction in viral levels with a greater than 2 log reduction in the lungs ( Figure 13 ). These data demonstrate that H1H15211P can significantly inhibit viral replication even when administered 24 hours after inoculation with the virus.

[0353] Histological analysis was performed on mice treated with hIgG control antibody, 500 μg or 200 μg of H1H15211P 24 hours post-infection. Mice treated with the control antibody demonstrated pathology similar to the above control, with significant interstitial inflammation, perivascular cuffing, and thickening of the alveolar septa. Mice treated with 200 μg or 500 μg of H1H15211P had minimal interstitial inflammation throughout the lungs and had reduced and only focal perivascular inflammation. Blinded histological scoring demonstrated a reduced inflammation score for the treated mice ( Figure 14 ). These data demonstrate that even when administered 24 hours post-infection, therapeutic doses of H1H15211P reduce MERS-CoV-induced lung pathology.

[0354] The present invention is not limited to the scope of the specific embodiments described herein. Indeed, various modifications of the present invention as well as those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. These modifications are intended to fall within the scope of the appended claims. 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Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 11 cagactatta gtagctgg 18 <210> 12 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 12 Gln Thr Ile Ser Ser Trp 1 5 <210> 13 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 13 aaggcgtct 9 <210> 14 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 14 Lys Ala Ser 1 <210> 15 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 15 caacagtata atagttattc gtacact 27 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 16 Gln Gln Tyr Asn Ser Tyr Ser Tyr Thr 1 5 <210> 17 <211> 375 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 17 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc 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60 atctcctgca ggtctagtca gagcctcctg catggtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct atttggtttc tcatcgggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaagtcct 300 tggacgttcg gccaagggac caaggtggaa atcaaa 336 <210> 26 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 26 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Gly 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Val Ser His Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 27 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 27 cagagcctcc tgcatggtaa tggatacaac tat 33 <210> 28 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 28 Gln Ser Leu Leu His Gly Asn Gly Tyr Asn Tyr 1 5 10 <210> 29 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 29 ttggtttct 9 <210> 30 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 30 Leu Val Ser 1 <210> 31 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 31 atgcaagctc tacaaagtcc ttggacg 27 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 32 Met Gln Ala Leu Gln Ser Pro Trp Thr 1 5 <210> 33 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 33 gaggtgcagc tggtggagtc tgggggagac ttggtacagc cgggggggtc cctgagagtc 60 tcctgtgcag cctctggatt cacctttagc aactatgaca tgtactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtt attagtggta ttggtgctac cacatattac 180 gcagactccg tgaagggccg gttcaccata tccagagaca attccaagaa cacggtgttt 240 ctgcaaatga atagtctgag agccgaggac acggccgtat attactgtgt gaaaggggga 300 cctatagtgg ctacggatta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 34 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Ile Gly Ala Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Lys Gly Gly Pro Ile Val Ala Thr Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 35 ggattcacct ttagcaacta tgac 24 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 36 Gly Phe Thr Phe Ser Asn Tyr Asp 1 5 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 37 attagtggta ttggtgctac caca 24 <210> 38 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 38 Ile Ser Gly Ile Gly Ala Thr Thr 1 5 <210> 39 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 39 gtgaaagggg gacctatagt ggctacggat tac 33 <210> 40 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 40 Val Lys Gly Gly Pro Ile Val Ala Thr Asp Tyr 1 5 10 <210> 41 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 41 gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggcatca gcagaaacca 120 gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttatta ctgccaacag tataatagtt attcgtggac gttcggccaa 300 gggaccaagg tggaaatcaa a 321 <210> 42 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp His Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 43 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 43 cagagtatta gtagctgg 18 <210> 44 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 44 Gln Ser Ile Ser Ser Trp 1 5 <210> 45 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 45 aaggcgtct 9 <210> 46 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 46 Lys Ala Ser 1 <210> 47 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 47 caacagtata atagttattc gtggacg 27 <210> 48 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 48 Gln Gln Tyr Asn Ser Tyr Ser Trp Thr 1 5 <210> 49 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 49 gaggtgcagc tggtggagtc tgggggagac ttggtacagc ctggagggtc cctaagactc 60 tcctgtacag cctctggatt caccttcagt aattatgaaa tgaactgggt ccgccaggct 120 ccagagaagg ggctggactg ggtttcattc attagtagta gtggtggtgc catatactac 180 gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac tcggctattt atttctgtgc gcgatccgac 300 tccggtggta actcgaggta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 50 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 50 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Asp Trp Val 35 40 45 Ser Phe Ile Ser Ser Ser Gly Gly Ala Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ser Ala Ile Tyr Phe Cys 85 90 95 Ala Arg Ser Asp Ser Gly Gly Asn Ser Arg Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 51 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 51 ggattcacct tcagtaatta tgaa 24 <210> 52 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 52 Gly Phe Thr Phe Ser Asn Tyr Glu 1 5 <210> 53 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 53 attagtagta gtggtggtgc cata 24 <210> 54 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 54 Ile Ser Ser Ser Gly Gly Ala Ile 1 5 <210> 55 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 55 gcgcgatccg actccggtgg taactcgagg tac 33 <210> 56 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 56 Ala Arg Ser Asp Ser Gly Gly Asn Ser Arg Tyr 1 5 10 <210> 57 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 57 gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60 gtcacttgtc gggcgagtca ggatattagc aactggttag tctggtatca gcagaaacca 120 gggaaagccc ctaagttcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctacagcct 240 gaagattttg cgacttacta ttgtcaacag gctaacagtt tccctcctac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 58 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Val Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Trp 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 59 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 59 caggatatta gcaactgg 18 <210> 60 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 60 Gln Asp Ile Ser Asn Trp 1 5 <210> 61 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 61 gctgcatcc 9 <210> 62 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 62 Ala Ala Ser 1 <210> 63 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 63 caacaggcta acagtttccc tcctact 27 <210> 64 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 64 Gln Gln Ala Asn Ser Phe Pro Pro Thr 1 5 <210> 65 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 65 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgtag tttctggatt cacctttagt aattatgaca tgagctgggt ccgccaggct 120 ccagggaggg ggctggagtg ggtctcagct attaggggta gtggttttaa cacatattac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatggg 300 tctatagtga gtatggacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 66 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Arg Gly Ser Gly Phe Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Ile Val Ser Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 67 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 67 ggattcacct ttagtaatta tgac 24 <210> 68 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 68 Gly Phe Thr Phe Ser Asn Tyr Asp 1 5 <210> 69 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 69 attaggggta gtggttttaa caca 24 <210> 70 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 70 Ile Arg Gly Ser Gly Phe Asn Thr 1 5 <210> 71 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 71 gcgaaagatg ggtctatagt gagtatggac tac 33 <210> 72 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 72 Ala Lys Asp Gly Ser Ile Val Ser Met Asp Tyr 1 5 10 <210> 73 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 73 gacatccaga tgacccagtc tccttccgcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 120 gggaaagccc ctaaactcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttatta ctgccaacag tataatagtt attcgtggac gttcggccaa 300 gggaccaagg tggaaatcaa a 321 <210> 74 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 74 Asp Ile Gln Met Thr Gln Ser Pro Ser Ala Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 75 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 75 cagagtatta gtagctgg 18 <210> 76 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 76 Gln Ser Ile Ser Ser Trp 1 5 <210> 77 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 77 aaggcgtct 9 <210> 78 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 78 Lys Ala Ser 1 <210> 79 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 79 caacagtata atagttattc gtggacg 27 <210> 80 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 80 Gln Gln Tyr Asn Ser Tyr Ser Trp Thr 1 5 <210> 81 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 81 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc cgggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc acctatggca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaact attattggta gtggttataa cacatactac 180 tcagactccg tgaagggccg gttcaccatg tccagagaca attccaagag cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtac gaaagaaggc 300 cctataattg gaaccacgaa ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 82 <211> 118 <212> PRT <213>人工序列 <220> <223>合成的 <400> 82 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ile Gly Ser Gly Tyr Asn Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Glu Gly Pro Ile Ile Gly Thr Thr Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 83 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 83 ggattcacct ttagcaccta tggc 24 <210> 84 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 84 Gly Phe Thr Phe Ser Thr Tyr Gly 1 5 <210> 85 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 85 attattggta gtggttataa caca 24 <210> 86 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 86 Ile Ile Gly Ser Gly Tyr Asn Thr 1 5 <210> 87 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 87 acgaaagaag gccctataat tggaaccacg aac 33 <210> 88 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 88 Thr Lys Glu Gly Pro Ile Ile Gly Thr Thr Asn 1 5 10 <210> 89 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 89 gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca acagaaacca 120 gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240 gacgattttg caacttatta ctgccaccaa tataatagtt attcgtggac gttcggccaa 300 gggaccaagg tggaaatcaa a 321 <210> 90 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 90 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Asn Ser Tyr Ser Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 91 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 91 cagagtatta gtagctgg 18 <210> 92 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 92 Gln Ser Ile Ser Ser Trp 1 5 <210> 93 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 93 aaggcgtct 9 <210> 94 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 94 Lys Ala Ser 1 <210> 95 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 95 caccaatata atagttattc gtggacg 27 <210> 96 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 96 His Gln Tyr Asn Ser Tyr Ser Trp Thr 1 5 <210> 97 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 97 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc cctgagactc 60 tcctgtgcag tctttggatt cacctttagc ggctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggtg gtggtggtag cacatactac 180 acagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gtcctactgg 300 aacaacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc a 351 <210> 98 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 98 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Phe Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Gly Gly Gly Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Trp Asn Asn Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 99 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 99 ggattcacct ttagcggcta tgcc 24 <210> 100 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 100 Gly Phe Thr Phe Ser Gly Tyr Ala 1 5 <210> 101 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 101 attagtggtg gtggtggtag caca 24 <210> 102 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 102 Ile Ser Gly Gly Gly Gly Ser Thr 1 5 <210> 103 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 103 gcgtcctact ggaacaacgg tatggacgtc 30 <210> 104 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 104 Ala Ser Tyr Trp Asn Asn Gly Met Asp Val 1 5 10 <210> 105 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 105 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaa 324 <210> 106 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 106 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 107 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 107 cagagcatta gcagctat 18 <210> 108 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 108 Gln Ser Ile Ser Ser Tyr 1 5 <210> 109 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 109 gctgcatcc 9 <210> 110 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 110 Ala Ala Ser 1 <210> 111 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 111 caacagagtt acagtacccc tccgatcacc 30 <210> 112 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 112 Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 <210> 113 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 113 cagctgcagc tgcaggagtc gggcccagga ctagtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agtaatactt actactgggg ctggatccgc 120 cagcccccag ggaaggggct ggagtggatt ggaactatat attatactgg gaacacctac 180 tacaagtcgt ccctcaagag tcgagtcacc atatccgtag acacgtccag gaaccagttc 240 tccctgaagc tgacctctgt gaccgccgca gacacggctg tctattactg tgcgcgacag 300 tttgctgact tgaactacgt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 114 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 114 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20 25 30 Thr Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Thr Ile Tyr Tyr Thr Gly Asn Thr Tyr Tyr Lys Ser Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Arg Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gln Phe Ala Asp Leu Asn Tyr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 115 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 115 ggtggctcca tcagtagtaa tacttactac 30 <210> 116 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 116 Gly Gly Ser Ile Ser Ser Asn Thr Tyr Tyr 1 5 10 <210> 117 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 117 atatattata ctgggaacac c 21 <210> 118 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 118 Ile Tyr Tyr Thr Gly Asn Thr 1 5 <210> 119 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 119 gcgcgacagt ttgctgactt gaactacgtt gactac 36 <210> 120 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 120 Ala Arg Gln Phe Ala Asp Leu Asn Tyr Val Asp Tyr 1 5 10 <210> 121 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 121 gaggtgcagc tggtggagtc ggggggaggc ttggttcagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt ctcctttagc agctatggca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggtc gtggtggtaa cacatactcc 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgttt 240 cttcaaatga atggcctgag agccgaggac tcggcctttt attactgtgc gaaagtgggg 300 acttatagtt cttcgtcccc ctttgactac tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 122 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 122 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Arg Gly Gly Asn Thr Tyr Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Ser Ala Phe Tyr Tyr Cys 85 90 95 Ala Lys Val Gly Thr Tyr Ser Ser Ser Ser Pro Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 123 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 123 ggattctcct ttagcagcta tggc 24 <210> 124 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 124 Gly Phe Ser Phe Ser Ser Tyr Gly 1 5 <210> 125 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 125 attagtggtc gtggtggtaa caca 24 <210> 126 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 126 Ile Ser Gly Arg Gly Gly Asn Thr 1 5 <210> 127 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 127 gcgaaagtgg ggacttatag ttcttcgtcc ccctttgact ac 42 <210> 128 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 128 Ala Lys Val Gly Thr Tyr Ser Ser Ser Ser Pro Phe Asp Tyr 1 5 10 <210> 129 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 129 gaggtgcagc tggtggagtc tgggggaggc ttggttcagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctctaac agctatccca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggt attagtggta gaggtggtaa cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cactctgtat 240 ctgcaaatga acagcctgag agtcgaggac acggccgtat attactgtgc ggaaactgga 300 actgcctttg actactgggg ccagggaacc ctggtcaccg tctcctca 348 <210> 130 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 130 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Asn Ser Tyr 20 25 30 Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Arg Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 131 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 131 ggattcacct ctaacagcta tccc 24 <210> 132 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 132 Gly Phe Thr Ser Asn Ser Tyr Pro 1 5 <210> 133 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 133 attagtggta gaggtggtaa caca 24 <210> 134 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 134 Ile Ser Gly Arg Gly Gly Asn Thr 1 5 <210> 135 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 135 gcggaaactg gaactgcctt tgactac 27 <210> 136 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 136 Ala Glu Thr Gly Thr Ala Phe Asp Tyr 1 5 <210> 137 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 137 cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc aataataatt actactgggg ctggatccgc 120 cagcccccag ggaaggggct ggactggatt gggagtatct attatagtgg gaatacctac 180 tacaacccgt ccctcaagag tcgagtcacc atatccgttg acacgtccaa gaaccagttc 240 tccctgaaga tgagttctgt gaccgccaca gacacggctc tgtattactg tgcgagacag 300 ggagcagatc acaactgggt cgacccctgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 138 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 138 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asn 20 25 30 Asn Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Asp 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Met Ser Ser Val Thr Ala Thr Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Arg Gln Gly Ala Asp His Asn Trp Val Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 139 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 139 ggtggctcca tcagcaataa taattactac 30 <210> 140 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 140 Gly Gly Ser Ile Ser Asn Asn Asn Tyr Tyr 1 5 10 <210> 141 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 141 atctattata gtgggaatac c 21 <210> 142 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 142 Ile Tyr Tyr Ser Gly Asn Thr 1 5 <210> 143 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 143 gcgagacagg gagcagatca caactgggtc gacccc 36 <210> 144 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 144 Ala Arg Gln Gly Ala Asp His Asn Trp Val Asp Pro 1 5 10 <210> 145 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 145 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgttg tctctggatt cacctctaga aactatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtgggag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca atcccaagaa cacgttgtat 240 ctacaaatga acagcctgag agccgaggac acggccctat attactgtgc ggaagatcct 300 ggaacttctt ttgactactg gggccaggga accctggtca ccgtctcctc a 351 <210> 146 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 146 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr Ser Arg Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Glu Asp Pro Gly Thr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 147 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 147 ggattcacct ctagaaacta tgcc 24 <210> 148 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 148 Gly Phe Thr Ser Arg Asn Tyr Ala 1 5 <210> 149 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 149 attagtggta gtggtgggag caca 24 <210> 150 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 150 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 151 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 151 gcggaagatc ctggaacttc ttttgactac 30 <210> 152 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 152 Ala Glu Asp Pro Gly Thr Ser Phe Asp Tyr 1 5 10 <210> 153 <211> 390 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 153 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt aactatggca tacactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcaggt atatactatg atggaagtaa taaatactat 180 ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctatat 240 ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgc gagagatcgg 300 ggtaataacc actactatca taataatccc tactactatt atcacggttt ggacgtctgg 360 ggccaaggga ccacggtcac cgtctcctca 390 <210> 154 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 154 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Tyr Tyr Asp Gly Ser Asn Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Asn Asn His Tyr Tyr His Asn Asn Pro Tyr Tyr 100 105 110 Tyr Tyr His Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser 130 <210> 155 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 155 ggattcacct tcagtaacta tggc 24 <210> 156 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 156 Gly Phe Thr Phe Ser Asn Tyr Gly 1 5 <210> 157 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 157 atatactatg atggaagtaa taaa 24 <210> 158 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 158 Ile Tyr Tyr Asp Gly Ser Asn Lys 1 5 <210> 159 <211> 69 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 159 gcgagagatc ggggtaataa ccactactat cataataatc cctactacta ttatcacggt 60 ttggacgtc 69 <210> 160 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 160 Ala Arg Asp Arg Gly Asn Asn His Tyr Tyr His Asn Asn Pro Tyr Tyr 1 5 10 15 Tyr Tyr His Gly Leu Asp Val 20 <210> 161 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 161 gaaatagttt tgacacagag tcccggcaca ctgtcactct ctcccgggga aagagccacc 60 ttgtcatgta gagcaagtca gtcagtctct agctcttatc tcgcctggta ccagcagaag 120 ccgggacagg cccctagact gctgatctac ggggcaagtt ccagggccac cggaatcccc 180 gaccggttca gtggaagcgg aagcggaacc gattttactt tgacgatttc tagactggag 240 ccagaggatt tcgccgttta ctattgtcaa cagtacggaa gcagcccgtg gacgtttggc 300 cagggcacga aggtagaaat caag 324 <210> 162 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 162 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 163 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 163 agagcaagtc agtcagtctc tagctcttat ctcgcc 36 <210> 164 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 164 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 165 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 165 ggggcaagtt ccagggccac c 21 <210> 166 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 166 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 167 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 167 caacagtacg gaagcagccc gtggacg 27 <210> 168 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 168 Gln Gln Tyr Gly Ser Ser Pro Trp Thr 1 5 <210> 169 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 169 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgctctg tctctggtgg ctccatcact agttcctatt ggagctggat ccggcagccc 120 ccaggaaggg gcctggagtg gattggatat gtctattact acgggaccac caaatacaac 180 ccctccctca agagtcgagt caccacatca atggacacgt ccaagaacca gttctccctg 240 aaactgaact ctgtgaccgc tgcggacacg gccgtttatt actgtgcgag actggaacta 300 ctctttgact actggggcca gggaaccctg gtcactgtct cctca 345 <210> 170 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 170 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Thr Ser Ser 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Val Tyr Tyr Tyr Gly Thr Thr Lys Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Thr Ser Met Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Leu Glu Leu Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 171 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 171 ggtggctcca tcactagttc ctat 24 <210> 172 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 172 Gly Gly Ser Ile Thr Ser Ser Tyr 1 5 <210> 173 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 173 gtctattact acgggaccac c 21 <210> 174 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 174 Val Tyr Tyr Tyr Gly Thr Thr 1 5 <210> 175 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 175 gcgagactgg aactactctt tgactac 27 <210> 176 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 176 Ala Arg Leu Glu Leu Leu Phe Asp Tyr 1 5 <210> 177 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 177 cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcagtg tctctggtgg ctccatcaac aataataatt actactgggg ctggatccgc 120 cagcccccag ggagggggct ggagtggatt gggagtttct tttatagtgg gcccacctac 180 tacaacccgt ccctcaggag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240 tccctgaagc tgaactctgt aaccgccgca gacacggcta tatattactg tgcgagacag 300 gatgggaact actaccccct ctttgactac tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 178 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 178 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Asn Asn Asn 20 25 30 Asn Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Arg Gly Leu Glu 35 40 45 Trp Ile Gly Ser Phe Phe Tyr Ser Gly Pro Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Gln Asp Gly Asn Tyr Tyr Pro Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 179 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 179 ggtggctcca tcaacaataa taattactac 30 <210> 180 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 180 Gly Gly Ser Ile Asn Asn Asn Asn Tyr Tyr 1 5 10 <210> 181 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 181 ttcttttata gtgggcccac c 21 <210> 182 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 182 Phe Phe Tyr Ser Gly Pro Thr 1 5 <210> 183 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 183 gcgagacagg atgggaacta ctaccccctc tttgactac 39 <210> 184 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 184 Ala Arg Gln Asp Gly Asn Tyr Tyr Pro Leu Phe Asp Tyr 1 5 10 <210> 185 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 185 caggtgcagc tgcaggagtc gggcccaggg ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagccc caactacaac 180 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240 aagctgacct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag atcccttaac 300 tggggacccc cttttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 186 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 186 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Pro Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ser Leu Asn Trp Gly Pro Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 187 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 187 ggtggctcca tcagtagtta ctac 24 <210> 188 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 188 Gly Gly Ser Ile Ser Ser Tyr Tyr 1 5 <210> 189 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 189 atctattaca gtgggagccc c 21 <210> 190 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 190 Ile Tyr Tyr Ser Gly Ser Pro 1 5 <210> 191 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 191 gcgagatccc ttaactgggg accccctttt gactac 36 <210> 192 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 192 Ala Arg Ser Leu Asn Trp Gly Pro Pro Phe Asp Tyr 1 5 10 <210> 193 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 193 gaaatagtga tgacgcagtc tccagccacc ctgtctctgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcaaaaacct 120 ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180 aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240 gaagattttg cagtttatta ctgtcagcag tttaataact ggccgtacac ttttggccag 300 gggaccaagc tggagatcaa a 321 <210> 194 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 194 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Asn Trp Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 195 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 195 cagagtgtta gcagcaac 18 <210> 196 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 196 Gln Ser Val Ser Ser Asn 1 5 <210> 197 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 197 ggtgcatcc 9 <210> 198 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 198 Gly Ala Ser 1 <210> 199 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 199 cagcagttta ataactggcc gtacact 27 <210> 200 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 200 Gln Gln Phe Asn Asn Trp Pro Tyr Thr 1 5 <210> 201 <211> 375 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 201 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtccag cctctggatt cacctttagc aactatgcca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtgctac cacaaagtac 180 gcagactccg tgaagggccg gttcaccatt tccagagaca attccaggaa tacgctatat 240 ctgcaaatga acagtctgag agccgaggac acggccgtat attactgtgc gaaggggggt 300 tcggggagtt atttccctta ctactactac ggtttggacg tctggggcca agggaccacg 360 gtcaccgtct cctca 375 <210> 202 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 202 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Pro Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Ala Thr Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Ser Gly Ser Tyr Phe Pro Tyr Tyr Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 203 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 203 ggattcacct ttagcaacta tgcc 24 <210> 204 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 204 Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 <210> 205 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 205 attagtggta gtggtgctac caca 24 <210> 206 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 206 Ile Ser Gly Ser Gly Ala Thr Thr 1 5 <210> 207 <211> 54 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 207 gcgaaggggg gttcggggag ttatttccct tactactact acggtttgga cgtc 54 <210> 208 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 208 Ala Lys Gly Gly Ser Gly Ser Tyr Phe Pro Tyr Tyr Tyr Tyr Gly Leu 1 5 10 15 Asp Val <210> 209 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 209 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga tagtgccacc 60 ctctcctgca gggccagtca gactgttagc agcaacttag cctggtacca gcagaaacct 120 ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180 aggttcagtg gcagtggctc tgggacagag ttcactctca ccatcagcag cctgcagtct 240 gaagattttg cagtttatta ctgtcagcag tattataact ggtggacgtt cggccaaggg 300 accaaggtgg aaatcaaa 318 <210> 210 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 210 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Ser Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 211 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 211 cagactgtta gcagcaac 18 <210> 212 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 212 Gln Thr Val Ser Ser Asn 1 5 <210> 213 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 213 ggtgcatcc 9 <210> 214 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 214 Gly Ala Ser 1 <210> 215 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 215 cagcagtatt ataactggtg gacg 24 <210> 216 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 216 Gln Gln Tyr Tyr Asn Trp Trp Thr 1 5 <210> 217 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 217 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggtc gtggtggtaa cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgttt 240 ctgcaaatga acaccctgag agccgaggac acggccgtat attactgtgc gaaagatagg 300 ggttttgggt tcttcgatat ctggggccgt ggcaccctgg ccactgtctc ctca 354 <210> 218 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 218 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Arg Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Gly Phe Gly Phe Phe Asp Ile Trp Gly Arg Gly Thr 100 105 110 Leu Ala Thr Val Ser Ser 115 <210> 219 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 219 ggattcacct ttagcagcta tgcc 24 <210> 220 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 220 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 221 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 221 attagtggtc gtggtggtaa caca 24 <210> 222 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 222 Ile Ser Gly Arg Gly Gly Asn Thr 1 5 <210> 223 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 223 gcgaaagata ggggttttgg gttcttcgat atc 33 <210> 224 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 224 Ala Lys Asp Arg Gly Phe Gly Phe Phe Asp Ile 1 5 10 <210> 225 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 225 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattagt aactatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagttcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatatta ctgtcaacag tatgataatc tcccattcac tttcggccct 300 gggaccaaaa taaatatcaa a 321 <210> 226 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 226 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Ile Asn Ile Lys 100 105 <210> 227 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 227 caggacatta gtaactat 18 <210> 228 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 228 Gln Asp Ile Ser Asn Tyr 1 5 <210> 229 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 229 gatgcatcc 9 <210> 230 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 230 Asp Ala Ser 1 <210> 231 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 231 caacagtatg ataatctccc attcact 27 <210> 232 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 232 Gln Gln Tyr Asp Asn Leu Pro Phe Thr 1 5 <210> 233 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 233 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtagtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgttt 240 ctgcaaatga acagcctgag agccgaggac acggccgtct attactgtgc ggaaggggga 300 gacgtggatt ttgactactg gggccaggga accctggtca ccgtctcctc a 351 <210> 234 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 234 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Gly Gly Asp Val Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 235 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 235 ggattcacct ttagcagcta tgcc 24 <210> 236 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 236 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 237 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 237 attagtggta gtggtagtag caca 24 <210> 238 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 238 Ile Ser Gly Ser Gly Ser Ser Thr 1 5 <210> 239 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 239 gcggaagggg gagacgtgga ttttgactac 30 <210> 240 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 240 Ala Glu Gly Gly Asp Val Asp Phe Asp Tyr 1 5 10 <210> 241 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 241 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaa 324 <210> 242 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 242 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 243 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 243 cagagcatta gcagctat 18 <210> 244 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 244 Gln Ser Ile Ser Ser Tyr 1 5 <210> 245 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 245 gctgcatcc 9 <210> 246 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 246 Ala Ala Ser 1 <210> 247 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 247 caacagagtt acagtacccc tccgatcacc 30 <210> 248 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 248 Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 <210> 249 <211> 348 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 249 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120 ccagggaagg gactggaatg gattgggtac atctattaca gtgggagcgc caactacaac 180 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240 aagctaagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgtgag agaccgggac 300 ctactccttg accactgggg ccagggaacc ctggtcaccg tctcctca 348 <210> 250 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 250 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Ala Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Arg Asp Arg Asp Leu Leu Leu Asp His Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 251 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 251 ggtggctcca tcagtagtta ctac 24 <210> 252 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 252 Gly Gly Ser Ile Ser Ser Tyr Tyr 1 5 <210> 253 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 253 atctattaca gtgggagcgc c 21 <210> 254 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 254 Ile Tyr Tyr Ser Gly Ser Ala 1 5 <210> 255 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 255 gtgagagacc gggacctact ccttgaccac 30 <210> 256 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 256 Val Arg Asp Arg Asp Leu Leu Leu Asp His 1 5 10 <210> 257 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 257 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggaa aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120 ggccaggctc ccaggctcct catctatggt gcatccacca ggaccactgg tttcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctgcagtct 240 gaagattttg cagtttatta ctgtcagcag tataataact ggccgtacac ttttggccag 300 gggaccatgc tggagatcaa a 321 <210> 258 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 258 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Lys Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Thr Thr Gly Phe Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Met Leu Glu Ile Lys 100 105 <210> 259 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 259 cagagtgtta gcagcaac 18 <210> 260 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 260 Gln Ser Val Ser Ser Asn 1 5 <210> 261 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 261 ggtgcatcc 9 <210> 262 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 262 Gly Ala Ser 1 <210> 263 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 263 cagcagtata ataactggcc gtacact 27 <210> 264 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 264 Gln Gln Tyr Asn Asn Trp Pro Tyr Thr 1 5 <210> 265 <211> 387 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 265 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc acttatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatccctt tctttggtac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accacggacg aatccacgag cacagcctac 240 atggagttga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaggga 300 acgtattacg attctttgac tggttattac acccactact accgtatgga cgtctggggc 360 caagggacca cggtcaccgt ctcctca 387 <210> 266 <211> 129 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 266 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Tyr Asp Ser Leu Thr Gly Tyr Tyr Thr His 100 105 110 Tyr Tyr Arg Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser <210> 267 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 267 ggaggcacct tcagcactta tgct 24 <210> 268 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 268 Gly Gly Thr Phe Ser Thr Tyr Ala 1 5 <210> 269 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 269 atcatccctt tctttggtac agca 24 <210> 270 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 270 Ile Ile Pro Phe Phe Gly Thr Ala 1 5 <210> 271 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 271 gcgagagagg gaacgtatta cgattctttg actggttatt acacccacta ctaccgtatg 60 gacgtc 66 <210> 272 <211> 22 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 272 Ala Arg Glu Gly Thr Tyr Tyr Asp Ser Leu Thr Gly Tyr Tyr Thr His 1 5 10 15 Tyr Tyr Arg Met Asp Val 20 <210> 273 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 273 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 60 atcacttgcc gggcaagtca gaccattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtgggtc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg cgacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaa 324 <210> 274 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 274 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 275 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 275 cagaccatta gcagctat 18 <210> 276 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 276 Gln Thr Ile Ser Ser Tyr 1 5 <210> 277 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 277 gctgcatcc 9 <210> 278 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 278 Ala Ala Ser 1 <210> 279 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 279 caacagagtt acagtacccc tccgatcacc 30 <210> 280 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 280 Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 <210> 281 <211> 381 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 281 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg tttctggagt caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatccctt tctttggtac agcaagctac 180 gcacagaagt tccagggcag agtcacggtt accacggacg aatccacgag cacagcctac 240 atggaggtga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagataat 300 ccggaactaa ctaaggaggg gtactaccac tactacgcta tggacgtctg gggccaaggg 360 accacggtca ccgtctcctc a 381 <210> 282 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 282 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Val Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Phe Phe Gly Thr Ala Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Val Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Asn Pro Glu Leu Thr Lys Glu Gly Tyr Tyr His Tyr Tyr 100 105 110 Ala Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 283 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 283 ggagtcacct tcagcagcta tgct 24 <210> 284 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 284 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 285 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 285 atcatccctt tctttggtac agca 24 <210> 286 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 286 Ile Ile Pro Phe Phe Gly Thr Ala 1 5 <210> 287 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 287 gcgagagata atccggaact aactaaggag gggtactacc actactacgc tatggacgtc 60 <210> 288 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 288 Ala Arg Asp Asn Pro Glu Leu Thr Lys Glu Gly Tyr Tyr His Tyr Tyr 1 5 10 15 Ala Met Asp Val 20 <210> 289 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 289 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 <220>aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaa 324 <210> 290 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 290 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 291 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 291 cagagcatta gcagctat 18 <210> 292 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 292 Gln Ser Ile Ser Ser Tyr 1 5 <210> 293 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 293 gctgcatcc 9 <210> 294 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 294 Ala Ala Ser 1 <210> 295 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 295 caacagagtt acagtacccc tccgatcacc 30 <210> 296 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 296 Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 <210> 297 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 297 gaggtgcagc tgttggagtc tgggggagtc ttggtacagc cgggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctttgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gaggtggtac tacatactac 180 gcagactccg tgatgggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 gtgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatagg 300 gggttcgggg tctttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 298 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 298 Glu Val Gln Leu Leu Glu Ser Gly Gly Val Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Arg Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Val Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Gly Phe Gly Val Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 299 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 299 ggattcacct ttagcagctt tgcc 24 <210> 300 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 300 Gly Phe Thr Phe Ser Ser Phe Ala 1 5 <210> 301 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 301 attagtggta gaggtggtac taca 24 <210> 302 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 302 Ile Ser Gly Arg Gly Gly Thr Thr 1 5 <210> 303 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 303 gcgaaagata gggggttcgg ggtctttgac tac 33 <210> 304 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 304 Ala Lys Asp Arg Gly Phe Gly Val Phe Asp Tyr 1 5 10 <210> 305 <211> 315 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 305 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagggtcacc 60 atcacttgcc aggcgagtca ggtcattaac aattatttaa attcgtatca gcagaaacca 120 gggaaagccc ctaaggtcct gatctgcgat gcatccaatg tggaaacagg ggtcccgtca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagact 240 gaagatattg caacatatta ctgtcaacag tatgataatc tcactttcgg cggagggacc 300 aaggtggagg tcaaa 315 <210> 306 <211> 105 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 306 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Val Ile Asn Asn Tyr 20 25 30 Leu Asn Ser Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Cys Asp Ala Ser Asn Val Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Thr 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Thr Phe 85 90 95 Gly Gly Gly Thr Lys Val Glu Val Lys 100 105 <210> 307 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 307 caggtcatta acaattat 18 <210> 308 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 308 Gln Val Ile Asn Asn Tyr 1 5 <210> 309 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 309 gatgcatcc 9 <210> 310 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 310 Asp Ala Ser 1 <210> 311 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 311 caacagtatg ataatctcac t 21 <210> 312 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 312 Gln Gln Tyr Asp Asn Leu Thr 1 5 <210> 313 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 313 gaggtgcagc tgttggagtc tgggggaggc ttggaacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgatctgggt ccgccaggct 120 ccagggaagg gactggagtg ggtctcaact attagtggga gtggtgttaa cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgcat 240 ctacaaatga acagcctgag agccgaggac acggccgttt atcactgtgc gaaagaggga 300 ttggattgtg ctaatggtgt atgctataac tactacggta tggacgtctg gggccaaggg 360 accacggtca ccgtctcctc a 381 <210> 314 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 314 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Ser Gly Val Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Lys Glu Gly Leu Asp Cys Ala Asn Gly Val Cys Tyr Asn Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 315 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 315 ggattcacct ttagcagcta tgcc 24 <210> 316 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 316 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 317 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 317 attagtggga gtggtgttaa caca 24 <210> 318 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 318 Ile Ser Gly Ser Gly Val Asn Thr 1 5 <210> 319 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 319 gcgaaagagg gattggattg tgctaatggt gtatgctata actactacgg tatggacgtc 60 <210> 320 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 320 Ala Lys Glu Gly Leu Asp Cys Ala Asn Gly Val Cys Tyr Asn Tyr Tyr 1 5 10 15 Gly Met Asp Val 20 <210> 321 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 321 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattaac agctttttaa attggtatca tcagaaacca 120 gggaaagccc ctaaattcct gatctatagt gcatccaatt tgcaaagtgg ggtcccgtca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccattagcag tctgcaacct 240 gaagattttt caacttacta ctgtcaacag agttacagta tcccgctcac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 322 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 322 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Phe 20 25 30 Leu Asn Trp Tyr His Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ser Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 323 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 323 cagagcatta acagcttt 18 <210> 324 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 324 Gln Ser Ile Asn Ser Phe 1 5 <210> 325 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 325 agtgcatcc 9 <210> 326 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 326 Ser Ala Ser 1 <210> 327 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 327 caacagagtt acagtatccc gctcact 27 <210> 328 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 328 Gln Gln Ser Tyr Ser Ile Pro Leu Thr 1 5 <210> 329 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 329 gaggtgcagc tggtggagtc tgggggagcc ttggtacagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt catcttcggt agttatgaga tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg gctttcatac attagtagta gtggtagtac catatactac 180 gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgc gagagaacgg 300 gggcagctcg gccggggagg gtattactac tacggtatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctca 378 <210> 330 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 330 Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Gly Ser Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Gly Gln Leu Gly Arg Gly Gly Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 331 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 331 ggattcatct tcggtagtta tgag 24 <210> 332 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 332 Gly Phe Ile Phe Gly Ser Tyr Glu 1 5 <210> 333 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 333 attagtagta gtggtagtac cata 24 <210> 334 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 334 Ile Ser Ser Ser Gly Ser Thr Ile 1 5 <210> 335 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 335 gcgagagaac gggggcagct cggccgggga gggtattact actacggtat ggacgtc 57 <210> 336 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 336 Ala Arg Glu Arg Gly Gln Leu Gly Arg Gly Gly Tyr Tyr Tyr Tyr Gly 1 5 10 15 Met Asp Val <210> 337 <211> 327 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 337 gaaattgtgt tgacgcggtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccatcagaaa 120 cctggccagg ctcccaggct cctcatgtat ggtacatcca tcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctcc gtggacgttc 300 ggccaaggga ccaaggtgga aatcaaa 327 <210> 338 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 338 Glu Ile Val Leu Thr Arg Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr His Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Met Tyr Gly Thr Ser Ile Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 339 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 339 cagagtgtta gcagcagcta c 21 <210> 340 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 340 Gln Ser Val Ser Ser Ser Tyr 1 5 <210> 341 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 341 ggtacatcc 9 <210> 342 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 342 Gly Thr Ser 1 <210> 343 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 343 cagcagtatg gtagctcacc tccgtggacg 30 <210> 344 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 344 Gln Gln Tyr Gly Ser Ser Pro Pro Trp Thr 1 5 10 <210> 345 <211> 384 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 345 caggtccggc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggaga caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatccctt tctttgttac aactacctac 180 gcacagaatt tccagggcag agtcacgatt accacggacg aatccacgcg cacagcctac 240 atggagctga gcagtctgag atctgaggac tcggccgtgt attactgtgc gagagatcgg 300 ccgtgtatca gctcggctgg tacacgctac cactactgcg ttatggacgt ctggggccaa 360 gggacaacgg tcaccgtctc ctca 384 <210> 346 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 346 Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Phe Phe Val Thr Thr Thr Tyr Ala Gln Asn Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Pro Cys Ile Ser Ser Ala Gly Thr Arg Tyr His Tyr 100 105 110 Cys Val Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 347 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 347 ggagacacct tcagcagcta tgct 24 <210> 348 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 348 Gly Asp Thr Phe Ser Ser Tyr Ala 1 5 <210> 349 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 349 atcatccctt tctttgttac aact 24 <210> 350 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 350 Ile Ile Pro Phe Phe Val Thr Thr 1 5 <210> 351 <211> 63 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 351 gcgagagatc ggccgtgtat cagctcggct ggtacacgct accactactg cgttatggac 60 gtc 63 <210> 352 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 352 Ala Arg Asp Arg Pro Cys Ile Ser Ser Ala Gly Thr Arg Tyr His Tyr 1 5 10 15 Cys Val Met Asp Val 20 <210> 353 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 353 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agttatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaa 324 <210> 354 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 354 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 355 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 355 cagagcatta gcagttat 18 <210> 356 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 356 Gln Ser Ile Ser Ser Tyr 1 5 <210> 357 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 357 gctgcatcc 9 <210> 358 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 358 Ala Ala Ser 1 <210> 359 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 359 caacagagtt acagtacccc tccgatcacc 30 <210> 360 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 360 Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 <210> 361 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 361 caggttcaac tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agccatggta tcagctgggt gcgacaggcc 120 cctggacagg ggtttgagtg gatgggatgg atcggcactt acaatagtaa cacagactat 180 gcacagaact tccagggcag agtcaccatg accacagaca catccacgag cacggcctac 240 atggagctga ggaacctgag atctgacgac acggccgtat attattgtgc gagagaaagg 300 ggtccctatt acggtatgga cgtctggggc caagggacca cggtcaccgt ctcctca 357 <210> 362 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 362 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Phe Glu Trp Met 35 40 45 Gly Trp Ile Gly Thr Tyr Asn Ser Asn Thr Asp Tyr Ala Gln Asn Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Gly Pro Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 363 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 363 ggttacacct ttaccagcca tggt 24 <210> 364 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 364 Gly Tyr Thr Phe Thr Ser His Gly 1 5 <210> 365 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 365 atcggcactt acaatagtaa caca 24 <210> 366 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 366 Ile Gly Thr Tyr Asn Ser Asn Thr 1 5 <210> 367 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 367 gcgagagaaa ggggtcccta ttacggtatg gacgtc 36 <210> 368 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 368 Ala Arg Glu Arg Gly Pro Tyr Tyr Gly Met Asp Val 1 5 10 <210> 369 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 369 gaaattgtgt tggcgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttaac agcaactact tagcctggta tcagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gtagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcaa cagtatggta actcactcac tttcggccct 300 gggaccaaag tggatttcaa a 321 <210> 370 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 370 Glu Ile Val Leu Ala Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Asn 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Asn Ser Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Phe Lys 100 105 <210> 371 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 371 cagagtgtta acagcaacta c 21 <210> 372 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 372 Gln Ser Val Asn Ser Asn Tyr 1 5 <210> 373 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 373 ggtgcatcc 9 <210> 374 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 374 Gly Ala Ser 1 <210> 375 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 375 caacagtatg gtaactcact cact 24 <210> 376 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 376 Gln Gln Tyr Gly Asn Ser Leu Thr 1 5 <210> 377 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 377 gaggtgcagc tgttggagtc tgggggaggc ttggtacaga ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtgata gaggtggtag tatatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgaagtat 240 ctgcaaatgg acagcctgag agccgaggac acggccgtat attactgtgc gcaagatagg 300 gggttcgggg tctttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 378 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 378 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Asp Arg Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Lys Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Asp Arg Gly Phe Gly Val Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 379 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 379 ggattcacct ttagcaatta tgcc 24 <210> 380 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 380 Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 <210> 381 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 381 attagtgata gaggtggtag tata 24 <210> 382 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 382 Ile Ser Asp Arg Gly Gly Ser Ile 1 5 <210> 383 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 383 gcgcaagata gggggttcgg ggtctttgac tac 33 <210> 384 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 384 Ala Gln Asp Arg Gly Phe Gly Val Phe Asp Tyr 1 5 10 <210> 385 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 385 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattggc aactatttaa attggtttca gcagagacca 120 gggaaagccc ctaatctcct gatctacggt gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaggtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatattt ctgtcaacag tatgataatc tccctttcac tttcggccct 300 gggaccaaag tggaaatcaa a 321 <210> 386 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 386 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Asp Asn Leu Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100 105 <210> 387 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 387 caggacattg gcaactat 18 <210> 388 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 388 Gln Asp Ile Gly Asn Tyr 1 5 <210> 389 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 389 ggtgcatcc 9 <210> 390 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 390 Gly Ala Ser 1 <210> 391 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 391 caacagtatg ataatctccc tttcact 27 <210> 392 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 392 Gln Gln Tyr Asp Asn Leu Pro Phe Thr 1 5 <210> 393 <211> 384 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 393 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctaaaga caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatccctt tctttggtac aactacctac 180 gaacagaagt tccagggcag agtcacgatt accacggacg aatccacgcg cacagcctac 240 atggagctga gcagcctgag atctgaggac tcggccgtgt attactgtgc gagagatcgg 300 ccgtgtatca gctcggctgg tacacgctac cactactgcg ttatggacgt ctggggccaa 360 gggacaacgg tcaccgtctc ctca 384 <210> 394 <211> 128 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 394 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Lys Asp Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Phe Phe Gly Thr Thr Thr Tyr Glu Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Pro Cys Ile Ser Ser Ala Gly Thr Arg Tyr His Tyr 100 105 110 Cys Val Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 395 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 395 aaagacacct tcagcagcta tgct 24 <210> 396 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 396 Lys Asp Thr Phe Ser Ser Tyr Ala 1 5 <210> 397 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 397 atcatccctt tctttggtac aact 24 <210> 398 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 398 Ile Ile Pro Phe Phe Gly Thr Thr 1 5 <210> 399 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 399 gcgagagatc ggccgtgtat cagctcggct ggtacacgct accactactg cgt...

Claims

1. An isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the Middle East Respiratory Syndrome Coronavirus spike protein, wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions in the heavy-chain variable region of SEQ ID NO: 66, designated HCDR1, HCDR2, and HCDR3, and three light-chain complementarity-determining regions in the light-chain variable region of SEQ ID NO: 74, designated LCDR1, LCDR2, and LCDR3.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof has one or more of the following characteristics: (a) is a full-length human monoclonal antibody; (b) interacts with one or more amino acid residues in the receptor-binding domain of the Middle East Respiratory Syndrome Coronavirus spike protein, wherein the amino acid residues are selected from amino acid residues 367 - 606 of SEQ ID NO: 457; (c) binds to the Middle East Respiratory Syndrome Coronavirus spike protein with a dissociation constant of less than 18.5 nM, as measured in a surface plasmon resonance assay; (d) blocks the binding of the Middle East Respiratory Syndrome Coronavirus spike protein to dipeptidyl peptidase 4 by greater than 90%, as measured in a blocking ELISA assay; (e) neutralizes more than 90% of Middle East Respiratory Syndrome coronavirus infectivity in human host cells and has an IC of less than 4 nM 50 , said IC 50 measured in a virus-like particle neutralization assay; (f) neutralizes Middle East Respiratory Syndrome Coronavirus infectivity, wherein the Middle East Respiratory Syndrome Coronavirus comprises isolates of viruses selected from the group consisting of: EMC / 2012, Jordan-N3 / 2012, England-Qatar / 2012, Al-Hasa_1_2013, Al-Hasa_2_2013, Al-Hasa_3_2013, Al-Hasa_4_2013, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17, Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-hasa_25, Bisha_1, Buraidah_1, England 1, Hafr-Al-batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1, KFU-HKU 1, KFU-HKU 13, Munich, Qatar3, Qatar4, Riyadh_1, Riyadh_2, Riyadh_3, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_9, Riyadh_14, Taif_1, UAE, and Wadi-Ad-Dawasir; and (g) is a bispecific antibody that comprises a first binding specificity for a first epitope in the receptor-binding domain of the Middle East Respiratory Syndrome Coronavirus spike protein and a second binding specificity for a second epitope in the receptor-binding domain of the Middle East Respiratory Syndrome Coronavirus spike protein, wherein the first and second epitopes are different and non-overlapping.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a set of six complementarity-determining regions of SEQ ID NOs: 68-70-72-76-78-80, designated HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3.

4. The antibody or antigen-binding fragment thereof of claim 3, which comprises a heavy-chain variable region having the amino acid sequence of SEQ ID NO:

66.

5. The antibody or antigen-binding fragment thereof of claim 3, which comprises a light-chain variable region having the amino acid sequence of SEQ ID NO:

74.

6. The antibody or antigen-binding fragment of claim 1, which comprises a heavy-chain variable region having the amino acid sequence of SEQ ID NO: 66 and a light-chain variable region having the amino acid sequence of SEQ ID NO:

74.

7. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody or antigen-binding fragment thereof blocks the binding of Middle East respiratory syndrome coronavirus spike protein to DPP4.

8. The isolated full-length human monoclonal antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof interacts with the amino acid sequence of amino acid residues 367-606 of SEQ ID NO:

457.

9. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof prevents Middle East respiratory syndrome coronavirus from entering host cells.

10. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a multispecific antigen-binding molecule.

11. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of claim 1 or 2 and a pharmaceutically acceptable carrier or diluent.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition comprises: (a) an antibody or antigen-binding fragment thereof according to any one of claims 1-8, said antibody or antigen-binding fragment thereof being referred to as the first antibody or antigen-binding fragment thereof; (b) a second antibody or antigen-binding fragment thereof that binds to the Middle East respiratory syndrome coronavirus spike protein at a second epitope, and (c) a pharmaceutically acceptable carrier or diluent; wherein the first antibody or antigen-binding fragment thereof does not cross-compete with the second antibody or antigen-binding fragment thereof for binding to the Middle East respiratory syndrome coronavirus spike protein.

13. The pharmaceutical composition of claim 12, wherein at least the first antibody or antigen-binding fragment thereof or the second antibody or antigen-binding fragment thereof blocks the binding of Middle East respiratory syndrome coronavirus spike protein to DPP4.

14. The pharmaceutical composition of claim 12 or 13, wherein the first and second epitopes are present in the receptor-binding domain of the Middle East respiratory syndrome coronavirus spike protein and are different and non-overlapping.

15. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the heavy-chain variable region of the antibody or antigen-binding fragment thereof of claim 1 or 2, and a polynucleotide sequence encoding the light-chain variable region of the antibody or antigen-binding fragment thereof of claim 1 or 2.

16. A vector comprising the polynucleotide molecule of claim 15.

17. A cell expressing the vector of claim 16.

18. Use of the antibody of claim 1 or 2 or an antigen-binding fragment thereof or the pharmaceutical composition of claim 11 in the preparation of a medicament for preventing, treating or alleviating at least one symptom or disease of Middle East Respiratory Syndrome Coronavirus infection.

19. The use of claim 18, wherein the at least one symptom or disease is selected from the group consisting of: pulmonary inflammation, alveolar damage, viral load, fever, cough, tachypnea, diarrhea, organ failure, septic shock, and death.

20. The use of claim 19, wherein the pulmonary inflammation is pneumonia.

21. The use of claim 18 or 19, wherein the pharmaceutical composition or the antibody or an antigen-binding fragment thereof is administered prophylactically or therapeutically to a subject in need thereof.

22. The use of claim 21, wherein the pharmaceutical composition or the antibody or an antigen-binding fragment thereof is administered prophylactically to a subject selected from the group consisting of: immunocompromised individuals, adults over 65 years old, travelers to the Middle East, healthcare workers, persons with a history of medical problems, persons with occupational or recreational exposure to camels or bats, and persons in contact with a person with confirmed or suspected Middle East Respiratory Syndrome Coronavirus infection.

23. The use of claim 22, wherein the history of medical problems is a history of heart problems and diabetes.

24. The use of claim 18, wherein the pharmaceutical composition or the antibody or an antigen-binding fragment thereof is administered in combination with a second therapeutic agent.

25. The use of claim 24, wherein the second therapeutic agent is selected from the group consisting of: anti-inflammatory drugs, antiviral drugs, different antibodies against the spike protein of Middle East Respiratory Syndrome Coronavirus, vaccines against Middle East Respiratory Syndrome Coronavirus, antibiotics, dietary supplements, and any other palliative therapy for treating Middle East Respiratory Syndrome Coronavirus infection.

26. The use of claim 25, wherein the anti-inflammatory drug is a corticosteroid.

27. The use of claim 25, wherein the anti-inflammatory drug is a non-steroidal anti-inflammatory drug.

28. The use of claim 25, wherein the dietary supplement is an antioxidant.

29. The use of claim 18, wherein the pharmaceutical composition or the antibody or an antigen-binding fragment thereof is administered by subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular or intracranial administration.

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