FIMH INHIBITING COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

Among the various aspects of the present disclosure is the provision of FimH inhibiting compositions and methods of use thereof. FimH inhibiting compositions that target and inhibit FimH, including monoclonal antibodies, are described. Methods of identifying FimH inhibiting antibodies are also described. Further, a method of treating bacterial infections, including urinary tract infections, is described.

Claims

1. A composition to treat a bacterial infection, the composition comprising an agent that targets a FimH adhesin protein.

2. The composition of claim 1, wherein the agent is an antibody.

3. The composition of claim 2, wherein the antibody targets the FimH adhesin protein of K. pneumonia and E. coli bacteria.

4. The composition of claim 3, wherein the antibody targets lectin domains of the FimH adhesin protein.

5. The composition of claim 3, wherein the antibody comprises: a. a heavy chain protein variable region comprising an amino acid sequence selected from SEQ ID NOS: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, and 119; and b. light chain protein comprising an amino acid sequence independently selected from SEQ ID NOS: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120.

6. The composition of claim 3, wherein the antibody comprises: a. a heavy chain protein variable region encoded by a nucleotide sequence selected from SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59; and b. a light chain protein encoded by a nucleotide sequence independently selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

7. The composition of claim 3, wherein the antibody comprises: a. a heavy chain protein encoded by a nested nucleotide sequence selected from SEQ ID NOS: 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, and 179, and b. a light chain protein encoded by a nested nucleotide sequence independently selected from SEQ ID NOS: 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, and 180.

8. The composition of claim 3, wherein the antibody comprises: a. a heavy chain protein encoded by a plasmid nucleotide sequence selected from SEQ ID NOS: 181, 183, 185, 187, 189, 191, 193, 195, 197,199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, and 239; and b. a light chain protein encoded by a plasmid nucleotide sequence independently selected from SEQ ID NOS: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, and 240.

9. The composition of claim 1, wherein the composition is used to prevent and treat a urinary tract infection (UTI).

10. A method of identifying at least one antibody to treat a bacterial infection, the method comprising performing an ELISA binding assay comprising a plurality of candidate monoclonal antibodies to identify the at least one antibody from the plurality of the candidate monoclonal antibodies that inhibit FimH binding in vitro.

11. The method of claim 10, wherein the ELISA assay further comprises at least one antigen selected from E. coli and K. pneumonia FimH proteins.

12. The method of claim 10, wherein the bacterial infection is a UTI.

13. The method of claim 11, further comprising administering the at least one antibody identified in vitro to a murine UTI model to characterize protection against UTI in vivo.

14. A method of treating a bacterial infection, the method comprising administering a therapeutically effective amount of a compound that targets and inhibits a FimH protein.

15. The method of claim 14, wherein the bacterial infection is a UTI.

16. The method of claim 14, wherein the compound is an antibody.

17. The method of claim 16, wherein the antibody binds to a lectin domain of FimH.

18. The method of claim 17, wherein the antibody comprises: a. a heavy chain protein variable region comprising an amino acid sequence selected from SEQ ID NOS: 241, 243, 245, 247, 249, 251, 253, 255, and 257 and the light chain protein variable region comprises an amino acid sequence independently selected from SEQ ID NOS: 242, 244, 246, 248, 250, 252, 254, 256, and 258.

19. The method of claim 17, wherein the antibody comprises a heavy chain protein variable region encoded by a nucleotide sequence selected from 259, 261, 263, 265, 267, 269, 271, 273, and 275, and the light chain protein variable region is encoded by a nucleotide sequence independently selected from SEQ ID NOS: 260, 262, 264, 266, 268, 270, 272, 274, and 276.

Description

DESCRIPTION OF THE DRAWINGS

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0015] FIG. 1A is a schematic showing the structure of E. coli FimHLD (PDB 1KLF).

[0016] FIG. 1B is a schematic showing the structure of K. pneumoniae FimHLD (PDB 9AT9).

[0017] FIG. 1C is a schematic showing the structure of E. coli FimHLD (PDB 6AOW).

[0018] FIG. 1D is a chart showing ELISA EC50 values for each mAb to the listed protein. White cells with no values indicate EC50 values were above the range measured in the assay.

[0019] FIG. 1E is a set of schematics of E. coli FimHLD and a corresponding chart showing epitope mapping of mAbs (top labels) to a panel of FimH mutants (right labels). Binding classes were determined by shared residues that abrogated mAb binding which are highlighted in purple on the surface of E. coli FimHLD (above) (PDB 1KLF) and the table (below).

[0020] FIG. 2A is a cryo-EM density map of the Fab Kp1 2H04 (teal) complexed with FimHLD (red).

[0021] FIG. 2B is a cryo-EM density map of the Fab Ec1 E7 (cyan) complexed with FimHLD (red).

[0022] FIG. 2C is a cryo-EM density map of the Fabs Kp1 2H04 (teal) and Ec1 E7 (cyan) maps superimposed on each other and complexed with FimHLD (red).

[0023] FIG. 2D is a s cryo-EM density map of the Fab Kp2 2C07 (yellow) complexed with FimHLD (red).

[0024] FIG. 2E is a cryo-EM density map of the Fab Ec3 B7 (green complexed with FimHLD (red).

[0025] FIG. 2F is a schematic showing the binding epitopes of the Fab Kp1 2H04 (teal) on FimHLD.

[0026] FIG. 2G is a schematic showing the binding epitopes of the Fab Ec1 E7 (cyan) on FimHLD.

[0027] FIG. 2H is a schematic showing the binding epitopes of the Fab Kp2 2C07 (yellow) on FimHLD.

[0028] FIG. 2I is a schematic showing the binding epitopes of the Fab Ec3 B7 (green) on FimHLD.

[0029] FIG. 2J is a schematic of a density map overlaid on model residue interactions of Kp1 2H04 (cyan) with FimH P26. mAb heavy chain residues are labeled HC and light chain residues are labeled LC.

[0030] FIG. 2K is a schematic of a density map overlaid on model residue interactions of Ec3 B7 (green) with FimH Y64. mAb heavy chain residues are labeled HC and light chain residues are labeled LC.

[0031] FIG. 3A is a graph showing FimH mAb binding to UT189 (black) bacteria and the FimS LIR mutants UT189 LON (pink) and UT189 LON fimH (teal).

[0032] FIG. 3B is a graph showing UT189 overexpressing conformationally shifted FimH variants: UT189 LON FimH A27V/V163A (fimBE; pink), UT189 LON FimH WT ((fimBE; black), and UT189 LON FimH A62S (fimBE; teal). n=3, error bars represent SEM.

[0033] FIG. 3C is a set of graphs showing representative binding curves of Kp1 2H04, Ec1 F7, and Kp2 2C07 Fab binding FimHLD (left) and FimGNTE (right). Results are from kinetic measurements of dilution series (500 nM, 250 nM, 125 nM, 62.5 nM, 31.2 nM) of one experiment.

[0034] FIG. 3D is a chart showing the observed biolayer interferometry (BLI) binding kinetics to Ec FimGNTEH and Ec FimHLD. N.D. means not determined due to the off rate being below the detection limit.

[0035] FIG. 4A is a chart showing inhibition of FimHLD binding to BSM at a 5:1 molar ratio of mAb to protein (n3).

[0036] FIG. 4B is a graph showing the inhibition of UT189 guinea pig erythrocyte hemagglutination (n2).

[0037] FIG. 4C is a set of representative immunofluorescent images showing mAb inhibition of FimHLD binding to C3H/HeN mouse bladders. mAb was pre-incubated with FimHLD at a 10:1 molar ratio. Sections were stained with DNA dye Hoechst (blue), Ec FimHLD (red) and antibody to uroplakin III (green). n=4-5 bladder sections with n=2 technical replicates.

[0038] FIG. 4D is a set of representative immunofluorescent images showing mAb inhibition of FimHLD binding to C3H/HeN mouse bladders. mAb was pre-incubated with FimHLD at a 10:1 molar ratio. Sections were stained with DNA dye Hoechst (blue), Kp FimHLD (red) and antibody to uroplakin III (green). n=4-5 bladder sections with n=2 technical replicates.

[0039] FIG. 5A is a schematic showing an experimental timeline. 6-7 week old C3H/HeN mice were pretreated with 0.5 mg of mAb 24 h before infection with UT189.

[0040] FIG. 5B is a graph of UT189 CFU in the bladder 24 hpi after infection. For 1B03 and 2E08 n=5 with 1 independent replicate, for B7 n=8 with 1 independent replicate, for 2C07 n=10 with 2 independent replicates, for control IgG, F7, 2H04, 1A02, 2E02 n=13-33 with three independent replicates. Statistical comparisons were made using Kruskal-Wallis test (nonparametric ANOVA) with Dunn's comparisons to the control group correcting for multiple comparisons. *P0.05, **P0.01.

[0041] FIG. 5C is a graph of UT189 CFU in the kidney 24 hpi after infection. For 1B03 and 2E08 n=5 with 1 independent replicate, for B7 n=8 with 1 independent replicate, for 2C07 n=10 with 2 independent replicates, for control IgG, F7, 2H04, 1A02, 2E02 n=13-33 with three independent replicates. Statistical comparisons were made using Kruskal-Wallis test (nonparametric ANOVA) with Dunn's comparisons to the control group correcting for multiple comparisons. *P0.05, **P0.01.

[0042] FIG. 5D is a graph of IBC counts at 6 hpi (n=16 for control IgG, n=14 for Kp1 2H04 with two independent replicates) of IBCs (green) in splayed mouse bladders for control IgG and Kp1 2H04 treatments. A Mann-Whitney U test was used to evaluate statistical significance.

[0043] FIG. 5E is a representative fluorescent image of IBC counts at 6 hpi. 5 magnification images of IBCs (green) in splayed mouse bladders for control IgG treatments. Scale bar represents 200 m.

[0044] FIG. 5F is a representative fluorescent image of IBC counts at 6 hpi. 5 magnification images of IBCs (green) in splayed mouse bladders for Kp1 2H04 treatments. Scale bar represents 200 m.

[0045] FIG. 5G is a graph showing CFU in the bladder 24 hpi from the prophylactic model testing control IgG, 2H04, and 2H04.sub.LALAPG (n=21 for control group, n=20 for 2H04, and n=19 for 2H04.sub.LALAPG with three independent replicates). Statistical comparisons were made using Kruskal-Wallis test (nonparametric ANOVA) with Dunn's comparisons to the control group correcting for multiple comparisons. **P0.01, ****P0.00001.

[0046] FIG. 5H is a graph showing CFU in the kidneys 24 hpi from the prophylactic model testing control IgG, 2H04, and 2H04.sub.LALAPG (n=21 for control group, n=20 for 2H04, and n=19 for 2H04LALAPG with three independent replicates). Statistical comparisons were made using Kruskal-Wallis test (nonparametric ANOVA) with Dunn's comparisons to the control group correcting for multiple comparisons. **P0.01, ****P0.00001.

[0047] FIG. 6A is a schematic of structural regions of FimHLD in the relaxed conformation (PDB 5jr4). The regions are colored as follows: swing loop in blue (residues 22-35) and linker to pilin domain in orange (residues 157-160).

[0048] FIG. 6B is a schematic of structural regions of FimHLD in the tense conformation (PDB 5jqi). The regions are colored as follows: clamp loop in limon (residues 8-16), peripheral -helix in green (residues 59-72), and insertion loop in magenta (residues 109-124).

[0049] FIG. 6C is a topology diagram of FimHLD with -sheet lettered in dark blue.

[0050] FIG. 6D is an image of amino acid sequence alignment of E. coli FmIHLD (EcFmIH), E. coli FimHLD (EcFimH), and K. pneumoniae FimHLD (KpFimH).

[0051] FIG. 7A is an image of a local resolution map of the Kp1 2H04-FimH complex, colored from high resolution (red) to low resolution (blue).

[0052] FIG. 7B is an image of a local resolution map of the Ec3 B7-FimH complex, colored from high resolution (red) to low resolution (blue).

[0053] FIG. 7C is an image of a local resolution map of the Ec1 F7-FimH complex, colored from high resolution (red) to low resolution (blue).

[0054] FIG. 7D is an image of a local resolution map of the Kp2 2C07-FimH complex, colored from high resolution (red) to low resolution (blue).

[0055] FIG. 8 is a flow diagram of the cryoEM processing of Fabs-FimH complexes. For each Fab-FimH complex, data processing is tracked by arrows starting at the top of the diagram and finishing with final density maps at the bottom.

[0056] FIG. 9A is a cryo-EM density map showing a 20 binding angle shift in the Kp1 2H04 (teal) and Ec1 F7 (cyan) Fabs relative to the binding site on FimH (red). Bars represent a 20 angle.

[0057] FIG. 9B is a schematic showing Ec1 F7 (cyan) coordinated to multiple aromatic residues around FimH (red). Density map is overlaid on model. mAb heavy chain residues are labeled HC and light chain residues are labeled LC.

[0058] FIG. 9C is a cryo-EM density map showing composite surface models of Kp1 2H04 (teal), Ec1 F7 (cyan), Kp2 2C07 (yellow), and Ec3 B7 (green) Fabs on FimH (red).

[0059] FIG. 10 is a Western blot showing anti-type 1 pili (FimA 17 kDa) to cell lysates used in bacterial cell ELISA (normalized by OD600).

[0060] FIG. 11A is a set of graphs showing BLI binding curves of Fabs to E. coli FimGnteH. Results are from kinetic measurements of dilution series of one experiment.

[0061] FIG. 11B is a set of graphs showing BLI binding curves of Fabs to E. coli FimGnteH. Results are from kinetic measurements of dilution series of one experiment.

[0062] FIG. 12A is a set of graphs showing BLI binding curves of Fabs to E. coli FimLD. Results are from kinetic measurements of dilution series of one experiment.

[0063] FIG. 12B is a set of graphs showing BLI binding curves of Fabs to E. coli FimLD. Results are from kinetic measurements of dilution series of one experiment.

[0064] FIG. 13 is a graph showing the presence of FimH mAbs in urine during cystitis. 0.5 mg of selected mAbs were injected via IP. Levels of hIgG mAbs in urine (1:10 dilution) at 24 (Pre-IP), 0 (Pre-infection), 3, 6 and 24 hrs for each of the FimH-specific mAbs and IgG control (n=3 to 5 per group). Mann-Whitney test used to determine significance.

[0065] FIG. 14 is a graph showing ELISA binding of Kp1 2H04 (black) and Kp1 2H04LALAPG (pink) mAbs to E. coli FimHLD (n=3).

[0066] FIG. 15A is a graph showing the presence of protective Kp1 2H04 in serum two weeks after intraperitoneal injection. Titers in urine were measured during the two-week infection period. Horizontal dashed lines represent limit of detection (LOD) of UT189 titers in urine and tissues.

[0067] FIG. 15B is a graph showing the presence of protective Kp1 2H04 in serum two weeks after intraperitoneal injection. Bacterial loads in bladder and kidney tissues (B) were assessed at sacrifice from mice pretreated with 0.5 mg of Kp1 2H04 (n=10) or control IgG (n=10) 24 hours before infection with UT189. Horizontal dashed lines represent limit of detection (LOD) of UT189 titers in urine and tissues.

[0068] FIG. 15C is a graph showing the presence of protective Kp1 2H04 in serum two weeks after intraperitoneal injection. hIgG mAb levels in serum (C, 1:100 dilution) of mice pretreated with 0.5 mg of hIgG mAb before infection. Mann-Whitney test was used to determine significance.

[0069] FIG. 15D is a graph showing the presence of protective Kp1 2H04 in serum two weeks after intraperitoneal injection. hIgG mAb levels in urine (1:10 dilution), and bladder (at sacrifice in day 14) homogenates (1:2 dilution) of mice pretreated with 0.5 mg of hIgG mAb before infection. Mann-Whitney test was used to determine significance.

[0070] FIG. 16A contains a top and side view of a cryoEM density map of singular uroplakin complex (singular unit in blue; EMD-36340).

[0071] FIG. 16B contains a top and side view of a cryoEM density map of singular uroplakin complex (singular unit in blue; EMD-36340) modeled with bound FimH lectin domain (red). FimH is modeled to bind to a glycan off N169. Structural clashes between uroplakin complex density and Fab model are noted, despite FimH being in a high-affinity state.

[0072] FIG. 16C contains a top and side view of a cryoEM density map of singular uroplakin complex (singular unit in blue; EMD-36340) modeled with bound FimH lectin domain (red) and FimH lectin domain-Kp1 2H04 Fab complex (Kp1 2H04 Fab; teal). FimH is modeled to bind to a glycan off N169. Structural clashes between uroplakin complex density and Fab model are noted, despite FimH being in a high-affinity state.

[0073] FIG. 17A is a graph showing the binding curves of the top Kp FimH mAbs.

[0074] FIG. 17B is a graph showing the binding curves of the E. coli mAb F7.

[0075] FIG. 17C is a chart showing that Kp FimH mAbs bind with high affinity to Kp and E. coli y1 CUP adhesins.

[0076] FIG. 17D is a chart showing the binding affinity of E. coli mAbs.

[0077] FIG. 18A is a graph showing the in vitro inhibition of Kp Fim1033 (black) and Kp 2H04 (green).

[0078] FIG. 18B is a graph showing the in vitro inhibition of Kp Fim1033 (black) and 1E04 (red).

[0079] FIG. 18C is a chart showing E. coli FimHLD inhibition at a 5:1 (mAb:FimH) molar ratio.

[0080] FIG. 18D is a chart showing TOP52 FimHLD inhibition at a 5:1 (mAb:FimH) molar ratio.

[0081] FIG. 19A is a graph showing the in vitro inhibition of E. coli mAbs.

[0082] FIG. 19B is a chart showing T2b inhibition at a 5:1 (mAb:FimH) molar ratio.

[0083] FIG. 19C is a chart showing TOP52 FimHLD inhibition at a 5:1 (mAb:FimH) molar ratio.

[0084] FIG. 20 is a graph showing the binding of 2H04 with UT189 (triangle; red), UT189 LON (triangle; black), TOP52 (circle; blue), and TOP52 LON (circle; black).

[0085] FIG. 21A is an image of an electron micrograph showing that 2H04 mAb can bind to native UT189 FimH.

[0086] FIG. 21B is an image of an electron micrograph showing that 2H04 mAb can bind to native TOP52 FimH.

[0087] FIG. 22 is a chart and corresponding graph of mAb inhibition on red blood cell hemagglutination.

[0088] FIG. 23A is a schematic of an experimental timeline of C3H/HeN mice pretreated with 0.5 mg of mAb 3 h before infection with UT189.

[0089] FIG. 23B is a set of graphs of UT189 CFU in the bladder (left) and kidneys (right) 3 hours after injection with a mAb.

[0090] FIG. 24A is a schematic of an experimental timeline of C3H/HeN mice pretreated with 0.5 mg of mAb 24 h before infection with UT189.

[0091] FIG. 24B is a graph showing 2H04 and F7 protection against UT189 in vivo with 10.sup.8 CFU inoculum in the bladder.

[0092] FIG. 24C is a graph showing 2H04 and F7 protection against UT189 in vivo with 10.sup.8 CFU inoculum in the kidneys.

[0093] FIG. 25 is a set of 3D diagrams showing 2H04 binding to the base of the FimHLD.

[0094] FIG. 26 is a table showing mAb class and epitope descriptions between tense and relaxed states.

DETAILED DESCRIPTION OF THE INVENTION

[0095] The present disclosure is based, at least in part, on the discovery that FimH inhibiting antibodies can be generated that bind with high affinity to bacterial FimH lectin domains and protect against UTI in vivo. As shown herein, monoclonal antibodies targeting the FimH adhesin that protect against UTI in a murine model are described.

[0096] Monoclonal antibodies that inhibit the function of the FimH adhesin protein on Escherichia coli and Klebsiella pneumonia have been developed, and the monoclonal antibodies are demonstrated to be useful in preventing urinary tract infection in a mouse model.

[0097] One aspect of the present disclosure provides for compositions of FimH inhibiting antibodies.

[0098] The E. coli and K. pneumoniae FimH amino acid sequence is highly conserved among bacterial isolates. In one aspect, monoclonal antibodies (mAbs) to E. coli and/or K. pneumoniae FimH lectin domains can be generated to identify mAbs that inhibit FimH binding and prevent infection in vivo. Using ELISA binding assays, mAbs that bind with high affinity to the antigenic FimH can be identified. In addition, it was found that a subset of mAbs cross-reacted to multiple bacterial FimH proteins and related chaperone usher pili galactose binding adhesin FmIH, which contributes to attachment in chronic UTI. Further, monoclonal antibodies can be identified that inhibited E. coli and/or K. pneumoniae FimH binding in vitro by performing binding inhibition ELISAs. The highest inhibiting mAbs can be tested in an acute murine model of UPEC UTI. One out of three mAbs tested significantly reduced bacterial titers in the urine and bladders of infected mice. Together, these results suggest that monoclonal antibodies inhibiting FimH function are an encouraging antibiotic-sparing therapeutic strategy for K. pneumoniae UTIs.

[0099] In various aspects, it is to be noted that the monoclonal antibodies inhibiting FimH function disclosed herein are useful in the treatment or prevention of UTIs associated with E. coli and K. pneumonia and potentially other bacteria species. In additional aspects, the monoclonal antibodies inhibiting FimH function disclosed herein may also have therapeutic value against additional bacteria-related diseases including, but not limited to, Crohn's disease, sepsis, lung infection, and any other bacterial infection-related disorders.

FimH Modulation Agents

[0100] As described herein, FimH expression has been implicated in various diseases, disorders, and conditions. As such, modulation of FimH (e.g., modulation of bacterial FimH adhesin proteins) can be used for the treatment of such conditions. A FimH modulation agent can modulate FimH response or induce or inhibit FimH. FimH modulation can comprise modulating the expression of FimH on cells, modulating the quantity of cells that express FimH, or modulating the quality of the FimH-expressing cells.

[0101] FimH modulation agents can be any composition or method that can modulate FimH expression on cells (e.g., blocking mannose-binding FimH adhesin proteins). For example, a FimH modulation agent can be an activator, an inhibitor, an agonist, or an antagonist. As another example, the FimH modulation can be the result of gene editing.

[0102] In various aspects, the FimH modulation agent can be an anti-FimH antibody (e.g., a monoclonal antibody to FimH). In various aspects, the monoclonal antibody to FimH comprises a heavy chain protein variable region and a light chain protein variable region configured to bind to and deactivate the binding of the FimH lectin domain protein of K. pneumoniae to exposed mannose residues on the surface of epithelial cells.

[0103] In some aspects, the heavy chain protein variable region comprises the amino acid sequence selected from SEQ ID NOS: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, and 119 and the light chain protein variable region comprises the amino acid sequence selected from SEQ ID NOS: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120 as listed in Table 2. In other aspects, the heavy chain protein variable region is encoded by a nucleotide sequence selected from SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59 and the light chain protein variable region is encoded by a nucleotide sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60, shown listed in Table 1. In additional aspects, the heavy chain protein variable region is encoded by a nested nucleotide sequence (encoding the specific variable region) selected from SEQ ID NOS: 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, and 179 and the light chain protein variable region is encoded by a nested nucleotide sequence (encoding the specific variable region) selected from SEQ ID NOS: 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, and 180, shown listed in Table 3. In additional aspects, the heavy chain protein variable region is encoded by a plasmid nucleotide sequence (encoding the specific variable region and adjoining regions) selected from SEQ ID NOS: 181, 183, 185, 187, 189, 191, 193, 195, 197,199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, and 239, and the light chain protein variable region is encoded by a plasmid nucleotide sequence (encoding the specific variable region and adjoining regions) selected from SEQ ID NOS: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, and 240, shown listed in Table 4.

TABLE-US-00001 TABLE1 Nucleotidesequencesofheavyandlightchainproteinvariableregion SEQIDNO Label Sequence 1 1E04_ ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGTCTGGGGCTGAGCTGGTGAA HC GCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAG CTACTGGGTGCACTGGGTGAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTG GAATGATTCATCCTAATAGTGGTAGTACTAACTACAATGAGAAGTTCAAGAGCAA GGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAG CCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGTTATTACTACGGTAGT AGCTATTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGT CGAC 2 1E04_ ACCGGTGTACATTCCGACATTGTGATGACCCAGTCTCCATCCTCCCTGACTGTGA LC CAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACA GTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTC CTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTT CACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGC TGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCGCTCACGTTC GGTGCTGGGACCAAGCTGGAGCTGAAACGTACG 3 1C08_ ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGTCTGGGGCTGAGCTGGTAAAG HC CCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAAC TACTGGATGCACTGGGTGAAGCAGAGGCCTGGGCAAGGCCTTGAGTGGATTGG AATGATTCATCCTAATAGTGGTACTTCTAACTACAATGAGAAGTTCAAGAGCAAG GCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTTTTACTGTACAAGATCTGACTGGGCCTTTG ACTACTGGGGCCAAGGTACCTCTCTCACAGTCTCCTCAGCGTCGAC 4 1C08_ ACCGGTGTACATTCCGACATTGTGATGACCCAGTCTCCATCCTCCCTGAGTGTGT LC CAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGCCTGTTAAACA GTGGAAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTC CTAAACTGTTGATCTACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCT TCACAGGCAGTGGATCTGGAACCGATTTCACTCTTACCATCAGTAGTGTGCAGG CTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATAGTTATCCGCTCACGTT CGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGCCTTTGTATCATGCTATTGC TTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGCTGN 5 1B02_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGTCTGGAGCTGAGCTGGTGAG HC GCCTGGGTCCTCAGTGAAGATGTCCTGCAAGACTTCTGGATATACATTCACATTC TACGGTATAAACTGGGTGAAGCAGAGGCCTGGACAGGGCCTGGAATGGATTGG ATATATTTATGTTGGAAATGGTTATTCTGAGTACAATGAGAAGTTCAAGGTCAAGG CCACACTGACTTCAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCGGCC TGACATCTGAGGACTCTGCAATCTATTTCTGTGCAAGATCAGATGGTTACAACTA CTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAACGTCGAC 6 1B02_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCAATCCTATCTGCAT LC CTCCAGGGGAGAAAGTCACAATGACTTGCAGGGCCAGCTCAAGTTTACATTACAT GCACTGGTACCAGCAGAAGACAGGATCCTCCCCCAAACCCTGGATTTATGCCAC ATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGAC CTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATACTGCCACTTATTAC TGCCAGCAGTGGAGTAGTAACCCACCCACGTTCGGTGCTGGGACCAAGCTGGA GCTGAAACGTACG 7 1A09_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAA HC GCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAG CTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTG GATTGATTCATCCTAATAGTGGTAGTACTTACTACAATGAGAAGTTCAAGAACAAG GCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATGGGATGATTCCTACT GGTACTTCAAAGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGA C 8 1A09_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCAATCCTGTCTGCAT LC CTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGTTCAAGTGTAAGTTACA TGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCA CATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGA CCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTA CTGCCAGCAGTGGAGTAGCAACCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAACGTACG 9 2A03_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGTCTGGAGCTGAGCTGGTGAG HC GCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACTTTCACTGA CTACTATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTCGAGXXXXXXTG GATTGCAAGGATTTATCCTGGAAGTGGTAATACTTACTACAATGAGAAGTTCAAG GGCAAGGCCACACTGACTGCAGAAAAATCCTCCAGCACTGCCTACATGCAGCTC AGCAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGACGGCTAACTG CGGGATACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCG TCGAC 10 2A03_ ACCGGTGTACATTCCGACATCAAGATGACCCAGTCTCCATCTTCCATGTTTGCAT LC CTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTA TTTATCCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGT GCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGG CAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTA TTGTCTACAGTATGATGAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGA GCTGAAACGTACG 11 2C09_ ACCGGTGTACATTCCGAAGTGATGCTGGTGGAGTCTGGGGAAGACTTAGTGAAA HC CCTGGAGGGTCCCTGAAACTCTCCTGTATAGCCTCTGGATTCACTTTCAGTAGCT ATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCAT ATATTAGTAGTGGTGGTGATTACATCTACTATACAGACACTGTGAAGGGCCGATT CACCATCTCCAGAGACAATGCCAGGAACACCCTGTTCCTACAAATGAGCAGTCT GAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGATACCGGTTACTACGTT TCTCGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCG TCGAC 12 2C09_ ACCGGTGTACATTCCGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACAT LC CAGTTGGAGACAGGGTCAGTGTCACCTGCAAGGCCAGTCAGAATGTGGGAACTA ATGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAAGCACTGATTTACTC GGCATCCTTCCGGAACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGG GACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTAT TTCTGTCATCAATATAACAACTATCCTCTGACGTTCGGTGGAGGCACCAAGCTGG AAATCAAACGTACG 13 2G02_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGTCTGGACCTGTGCTGGTGAAG HC CCTGGGCCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATTCACATTCACTGACT ACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAATGGATTGGAC TTGTTTATCCTTACAATGGTGGTACTTATTACAACCAGAAGTTCAAGGGCAAGGC CACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGGAGCTAAACAGCCT GACTTCTGAGGACTCTGCGGTCTATTACTGTGTACGATTAGGGAACGGTAGTAG CAACGAGTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTC AGCGTCGAC 14 2G02_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCT ACAATTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTA TGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATC TGGGACCTCTTATTCTCTCACAATCAGCAGTATGGAGGCTGAAGATGCTGCCACT TATTACTGTCAACAGTGGAGAACCTACCCGTGGACGTTCGGTGGAGGCACCAAG CTGGAAATCAAACGTACG 15 1D09_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGCCTGGAGCTGAGCTTGTGAAG HC CCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAAC TACTGGATACACTGGATGAAGCAGAGGCCTGGACGAGGCCTTGAGTGGATTGGA AGGATTGAGCCTAATAGCGGTGATACTAAATACAATGAGAAGATCAAGAGCAGG GCCACACTGACTGTAGACAAACCGTCCAGCACAGCCTACATGCAGCTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGATCTGGGTATGATTACC CTGAGGCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGAC 16 1D09_ ACCGGTGTACATTCCGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGT LC CTCCAGGGCAGAGAGCCACCATCTCCTGCAGAGCCAGTGAAGGTGTTGAATATT ATGGCACAACTTTAATGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAAC TCCTCATCTATGCTGCATCCAACGTAGAATCTGGGGTCCCTGCCAGGTTTAGTGG CAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTGTGGAGGAGGATGA TATTGCAATGTATTTCTGTCAGCAAAGTAGGAAGGTTTCTTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAACGTACG 17 2B06_ ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGCCTGGGGCTGAGCTTGTGAAG HC CCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTATACCTTCACCAACT ACTGGATGCACTGGGTGAAACACAGGCCTGGACGAGGCCTTGAGTGGATTGGA AGGATTGATCCTAATAGTGGTGGTACTAAGTACAATGAGAAGTTCAAGAATAAGG CCACACTGACTGTAGACAATCCCTCCAGCACAGGCTACATGCAGCTCAGCAGCC TGACATCTGAGGACTCTGCGGTCTATTATTGTACAAGATCTGGGTATGATTACCC TGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGAC 18 2B06_ ACCGGTGTACATTCCGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGT LC CTCTAGGGCAGAGAGCCACCATCTCCTGCAGAGCCAGTGAAAGTGTTGAATATT ATGGCACAAGTTTAATGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAAG TCCTCATCTATGCTGCATCCAACGTAGATTCTGGGGTCCCTCCCAGGTTTAGTGG CAGTGGGTCTGGGACAGACTTCAGCCTCAACTTCCATCCTGTGGAGGAGGATGA TATTGCAATGTATTTCTGTCAGCAAAGTAGGAAGGTTCCTTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAACGTACG 19 2A02_ ACCGGTGTACATTCCGAGGTCCAACTGCAACAGCCTGGGGCTGAGCTTGTGAAG HC CCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGC TACTGGATGCACTGGGTGAAGCAGAGGCCTGGACGAGGCCTTGAGTGGATTGG AAGGATTGATCCTAATAGTGGTGGTACTAAGTACAATGAGAAGTTCAAGAGCAAG GCCACACTGACTGTAGACAAACCCTCCAGCACAGCCTACATGCAGCTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGATCTGGGTATGATTACC CTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGAC 20 2A02_ ACCGGTGTACATTCCGACATTGTGCTGACCCAATCTCCAGCTCCTTTGGCTGTGT LC CTCTAGGGCAGAGAGCCACCATCTCCTGCAGAGCCAGTGAAGGTGTTGAATATT ATGACACAAGTTTAATGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAACT CCTCATCTATGCTGCATCCAACGTAGAACCTGGGGTTCCTGCCAGGTTTGGTGG CAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTGTGGAGGAGGATGA TATTGCAATGTATTTCTGTCAGCAAAGTAGGAAGGTTCCTTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAACGTACG 21 1B07_ ACCGGTGTACATTCCGAGGTCCAACTGCAACAGCCTGGGGCTGAGCTGGTAAAG HC CCTGGGACTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCT ACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGA ATGATTCATCCTAATAGTGGTAGTACTAACTACAATGAGATGTTCGAGAGCAAGG CCTCACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCT GACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAATCGGATACTCCGGCTG GGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGTCGAC 22 1B07_ ACCGGTGTACATTCCGACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGT LC CAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAATG GTGGAAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTC CTAAAGTGTTGATCAACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCT TCACAGGCAATGGATCTGGAACCGATTTCACTCTTACCATCAGCAGTGTGCAGG CTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATACTTATCCGCTCACGTT CGGTGCTGGGACCAAGCTGGAGCTGAAACGTACG 23 2D04_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGCCTGGAGGTGAGCTGGTGAG HC GCCTGGGTCCTCAGTGAAGATGTCCTGCAAGACTTCTGGATATACATTCACATTC TACGGTATAAACTGGGTGAAGCAGAGGCCTGGACAGGGCCTGGAATGGATTGG ATATATTTATGTTGGAAATGGTTATACTGAGTACAATGAGAAGTTCAAGGTCAAGG CCACACTGACTTCAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCGGCC TGACATCTGAGGACTCTGCAATCTATTTCTGTGCAAGATCCGATGGTTACAACTA CTTCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGAC 24 2D04_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCAATCCTGACTGCAT LC CCCCAGGGGAGAAAGTCACAATGACTTGCAGGGCCAGTTCAAGTGTACATTACA TGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCA CATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGA CCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATACTGCCACTTATTA CTGCCATCAGTGGAGTAGTAACCCACCCACGTTCGGTGCTGGGACAAAGTTGGA AATAAAACGTACG 25 1E07_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAA HC GCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAG CTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTG GATTGATTCATCCTAATAGTGGTAGTACTTACTACAATGAGAAGTTCAAGAACAAG GCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATGGGATGATTCCTACT GGTACTTCAAAGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGA C 26 1E07_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCAATCCTGTCTGCAT LC CTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGTTCAAGTGTAAGTTACA TGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCA CATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGA CCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTA CTGCCAGCAGTGGAGTAGCAACCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAACGTACG 27 2D07_ ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAGG HC CCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACT ACTATATACACTGGCTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGAT TGATTTTTCCTGGAAGTGGTAGTATTTACTGTAATGAGAAGTTCAAGGGCAAGGC TACACTTACTGTAGACAAATCCTCCACCACAGCCTACATGTTGCTCAGCAGCCTG ACCTCTGAGGACTCTGCGGTCTACTTCTGTGCAAGATGGGAGACTACGGCGTGG TACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGAC 28 2D07_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCCCAAATTGTTCTCACCC LC AGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCA GGGCCAGTTTAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCT CCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCCC GCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAGTCAGCAGAGTGG AGGCCGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGGAGTTACCCGTACA CGTTCGGAGGGGGCACCAAGCTGGAAATCAAACGTACG 29 1A02_ ACCGGTGTACATTCCGAGGTCCAACTGCAACAGTCTGGACCTGTGCTGGTGAAG HC CCTGGGCCTTCAATGAAGATATCCTGTAAGGCTTCTGGATTCACATTCACTGACT ACTACATACACTGGGTGAGGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAC TTGTTTCTCCTTACAATGGTGGTACTTACTACAACCAGAAGTTCAAGGGCAAGGC CACATTGACTGTAGACTCATCCTCCAGCACAGCCTACATGGAGCTAAGCAGCCT GACTTCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTGGGCTACTATGGTGAC TGGTACTACTTTGACTACTGGGGCCAAGGCACCCCTCTCACAGTCTCCTCAGCG TCGAC 30 1A02_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTACAT LC CTCCAGGGGAAAAGGTCACCATCACCTGTAGTGTCAGCTCAAGTATAAGTTCCA GCAACTTGAACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTA TGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATC TGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACT TATTACTGTCAACAGTGGAGTAGTTACCCATACACGTTCGGAGGGGGGACCAAG CTGGAAATAAAACGTACG 31 2D10 ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGTCTGGGGCTGAGCTGGTGAA HC GCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAC CTACTGGATGCACTGGATGAAGCAGTGGCCTGGACAAGGCCTTGAGTGGATTGG ATTGATTCATCCTAATAGTGGTAGTACTTACTACAATGAGAAGTTCAAGAGCAAG GCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTTGAGGAGCTATGG GGAGGTTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCC TCAGCGTCGAC 32 2D10 ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCA GCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGTCAACAGTGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCA AGCTGGAAATAAAACGTACG 33 2E08_ ACCGGTGTACATTCCGAGGTCCAGCTGCAGCAGCCTGGGGCTGAGCTGGCAAA HC GCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACAATTTCACCAG CTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTG GATTGATTCATCCTAATAGTGGTGGTACTTACTACAATGAGAAGTTCAAGAGCAA GGCCACACTGACTTTAGACAGATCCTCCAGCACAGCCTACATGCAACTCAGCAG CCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGATTTGAGGAGGAATGG GGAGGGTATTGGTACTTCGATGTCTGGGGCACGGGGACCACGGTCACCGTCTC CTCAGCGTCGAC 34 2E08_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAAAAGGTCACCATCACCTGCAGTGTCAGTTCAAGTATAAGTTCCA GCAACTTGCACTGGTACCAACAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTA TGGCACTTCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATC TGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACT TATTACTGTCAACAGTGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAA CTGGAAATAAAACGTACG 35 2H04_ ACCGGTGTACATTCCGAGGTCCAACTGCAACAGTCTGGGGCTGAGCTGGTAAAG HC CCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGC TACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGG ACTGATTCATCCTAATAGTAGTAGTACTTACTACAATGAGAAGTTCAAGACCAGG GCCACACTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGCAACTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTGGGCTATGGTAACT CCTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAG CGTCGAC 36 2H04_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATTAGTTCCA GCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTATTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGTTTGGAGGCTGAGGATGCTGCCA CTTATTACTGTCAACAGTGGAGAAGTTACCCGTGGACGTTCGGTGGAGGCACCA AGCTGGAAATCAAACGTACG 37 1B03_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGTCTGGGGCTGAGCTGGTAAAG HC CCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGC TACTGGATGCACTGGGTGCAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGG ATTGATTCATCCTATTGGTGGTGGTACTCACTACAATGAGAAGTTCAAGAACAAG GCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACTCGGAACTGGTCCG TACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCG AC 38 1B03_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCA GCACCTTGGACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGGTTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGTCAACAGTGGAGTAGTTACCCGTGGACGTTCGGTGGAGGCACCA AGCTGGAAATCAAACGTACG 39 2H07_ ACCGGTGTACATTCCGAGGTCCAGCTGCAGCAGTCTGGACCTGTGCTGGTGAAG HC CCTGGGCCTTCAGTGAATATATCCTGTAAGGCTTCTGGATTCACATTCACTGACT ACTACATTCACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAC TTGTTTATCCTTACAGTGGTGGTACTTACTACAACCAGAAGTTCAAGGGCAAGGC CACATTGACTGTAGACGCATCCTCCAGCACAGCCTACATGGAGCTAGGCAGCCT GACTTCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTGGGCGACAACTTCTAC TACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGAC 40 2H07_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAACACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCA GCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACATCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGTCAACAGTGGAGAAGTTACCCGTACACGTTCGGAGGGGGGACCA AGCTGGAAATAAAACGTACG 41 1C01_ ACCGGTGTACATTCCGAGGTCCAACTGCAACAGCCTGGACCTGTGCTGGTGAAG HC CCTGGGCCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATTCACATTCACTGACT ACTACATGCACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGTC TTGTTTCTCCTTACAATGGTGGTACTTTCTACAACCAGAAGTTCAAGGGCAAGGC CACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGGAACTAAACAGCCTG ACTTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGTGGGTAATAGCTACGTCC ATTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGT CGAC 42 1C01_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCA GCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGTCAACAGTGGAGTAATTACCCGTACACGTTCGGAGGGGGGACCA AGCTGGAAATAAAACGTACG 43 2C07_ ACCGGTGTACATTCCGAAGTGAAGCTGGTGGAGTCTGGGGAAGGCTTAGTGAAG HC CCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCT ATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGACTGGGTCGCAT ACATTAGTAGTGGTGGTGATCACATCTACTATGCAGACACTGTGAAGGGCCGATT CACCATCTCCAGAGACAATGCCAGGAACACCCTGTACCTGCAAATGAGCAGTCT GAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGATACCGGTTACTACGTC TCTCGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCG TCGAC 44 2C07_ ACCGGTGTACATTCCGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACAT LC CAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGTTACTA ATGTTGCCTGGTATCAACAGAAATCAGGGCAATCTCCTAAAGTAGTGATTTATTC GGCATCCTTCCGGTCCAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGG GACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTAT TTCTGTCACCAATATAACAGCCATCCTCTGACGTTCGGTGGAGGCACCAAGCTG GAAATCAAACGTACG 45 2G04_ ACCGGTGTACATTCCGAGGTCCAGCTGCAGCAGCCTGGACCTGAATTGGTGAAG HC CCCGGGTCCTCACTGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACTGACT ACTTTATAAACTGGGTGAAACAGAGGCCTGGACAGGGACTTGACTGGATTGGAT GGATTTTTCCTGGAAGTGGTAGTACTTACTACAATGACAAGTTCAAGGGCAAGGC CACACTTACTGTAGACAAATCCTCCAGCACTGCCTACATGTTGCTCAGCAGCCTG ACCTCTGAGGCCTCTGCGGTCTATTTCTGTGCAAGATGGGACTCCGATAGTACCT ACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGT CGAC 46 2G04_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAATTCAAGTATAAGTTCCA GCACCTTGCACTGGTACCAGCAGAAGTCACAAACCTCCCCCAAACCCTGGATTT ATGGTACATCCAATTTGGCTTCTGGAGTCCCTATTCGCTTCAGTGGCAGTGGATC TGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACT TATTACTGTCAACAGTGGAGTACTTACCCGTACACGTTCGGAGGTGGCACCAAG CTGGAAATCAAACGTACG 47 1G01_ ACCGGTGTACATTCCGAGGTCCAACTGCAACAGTCTGGAGCTGAGCTGATGAAG HC CCTGGGGCCTCAGTGAAGCTTTCCTGCAAGGCTACTGGCTACACATTCACTGGC TACTGGATAGAGTGGTTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGA GAGATTTTACCTGGAAGTGATAATACTAACTACAATGAGAAGTTCAGGGGCAAGG CCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCACCTCAGCAGCCT GACAACTGAGGACTCTGCCATCTATTACTGTGCAAGAGAAGGGGGTTTCTACTTT GACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGAC 48 1G01_ ACCGGTGTACATTCCCAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCAC LC CTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTATTGGGGCTGTTACAACTAG TAACTACGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATA GGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTG ATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGC AATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACC AAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCG CCCTCGAG 49 2A05_ ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGTCTGGACCTGTGCTGGTGAAG HC CCTGGGCCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATTCACATTCACTGACT ACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAATGGATTGGAC TTGTTTATCCTTACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGC CACATTGACTGTAGACACATCCTCCACCACAGCCTACATGGAGCTAAACAGCCTG ACTTCCGAGGACTCTGCGGTCTATTACTGTGCAAGATTGGGCTCAGGCTACGAG TACTACTTTGACTACTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCAGCGTCGA C 50 2A05_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCA GCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAGGATGCTGCCA CTTATTACTGTCAACAGTGGAGAACTTACCCGTGGACGTTCGGTGGAGGCACCA AGCTGGAAATCAAACGTACG 51 2H05_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGCCTGGACCTGTGCTGGTGAAG HC CCTGGGCCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATTCACATTCACTGACT ACTACATGCACTGGGTGAAGCAGAGCCATGGAGAGAGCCTTGAGTGGATTGGAC TTGTTTCTCCTTACAATGGTGGTACTTTCTACAACCAGAAGTTCAAGGGCAAGGC CACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGGAACTAAACAGCCTG ACTTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGTGGGTAGTAGTTACGTCC ATTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGT CGAC 52 2H05_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCACGGCTGCAT LC CTCCAGGGGAGAGGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCA GCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGTCAACAGTGGAGTAAGTACCCGTACACGTTCGGAGGGGGGACCA AGCTGGAAATAAAACGTACG 53 2B01_ ACCGGTGTACATTCCGAGGTCCAACTGCAACAGTCTGGGGCTGGGCTGGTAAAG HC CCTGGGGCTTCAGTGAAGTTGCCTTGCAAGGCTTCTGGCTACACTTTCACCAGC TACTGGATGCACTGGGTGCAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGG ATTGATTCATCCTATTGGTGGTGGTACTCACTACAATGAGAAGTTCAAGAACAAG GCCACACTGACTGTGGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGC CTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACTCGGAACTGGTCCG TACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCG AC 54 2B01_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCA GCAACTTGCACTGGTACCAGCAGAAGTCAGAGACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGTCAACAGTGGAGTAGTTACCCGTGGACGTTCGGTGGAGGCACCA AGCTGGAAATCAAACGTACG 55 1D01_ ACCGGTGTACATTCCGAGGTCCAGCTGCAGCAGCCTGGACCTGACGTGGTGAA HC GCCTGGGGCTTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTACACCTTCACTGA CTACTATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGG ATGGATTTTTCCTGGAAGTGGTAGTAGTTATTACAATGAGAAGTTCAAGGACAAG GCCACATCTACTGTAGACAAATCCTCCAGCACAGCCTACATGTTGCTCAGCAGCC TGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAAATGGAAGGATTACGGGT GGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCG AC 56 1D01_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAGAAGGTCACCCTCACCTGCAATGTCAGCTCAAGTCTAAGTTCCA GCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGTCAACAGTGGAGGACTTACCCTTACACGTTCGGAGGGGGGACCA AGCTGGAAATAAAACGTACG 57 2E02_ ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGTCTGGACCTGTGCTGGTGAAG HC CCTGGGCCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATTCACATTCACTGACT ACTACATTCACTGGGTGAAGCGGAGCCATGGAAAGAGCCTTGAGTGGATTGGAC TTGTTTCTCCTTACAATGGTGGTACTTTCTACAACCAGAAGTTCAAGGGCAAGGC CACATTGACTGTAGATACATCCTCCAATACAGCCTACATGGAGCTAACCAGCCTG ACTTCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTGGGGGTTAACTGGTACT TCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGAC 58 2E02_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCAT LC CTCCAGGGGAAAAGGTCACCCTCACCTGCAGTGTCAGCTCAAGTATAAGTTCCA GCACCTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGAT CTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGTCAACAGTGGAGTACTTACCCATACACGTTCGGAGGGGGGACCA AGCTGGAAATAAAACGTACG 59 1A05_ ACCGGTGTACATTCCGAGGTCCAACTGCAACAGCCTGGACCTGTACTGGTGAAG HC CCTGGGCCTTCAATGAAGATATCCTGTAAGGCTTCTGGGTACGCTTTCACTGACT ACTTCATACACTGGGTGAGACAGAGCCATGGAAGGAGCCTTGAGTGGATTGGAC TTGTTTCTCCTTATAATGGTGGTACTTACTACAACCAGAAGTTCAAGGGCAAGGC CTCATTGACTGTAGACACATCCTCCAGCACAGCCTACATGGAACTAAGCAGCCTG ACTTCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTGGGCTACTATGGTAACT GGTACTTCTTTGACTACTGGGGCCAAGGCACCCCTCTCACAGTCTCCTCAGCGT CGAC 60 1A05_ ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTACAT LC CTCCAGGGGAAAAGGTCACCATCACCTGTAGTGTCAGCTCAAGTATAAGTTCCA GCAACTTGAACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTA TGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATC TGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACT TATTACTGTCAACAGTGGAGTAGTTACCCATACACGTTCGGAGGGGGGACCAAG CTGGAAATAAAACGTACG

TABLE-US-00002 TABLE2 Aminoacidsequencesofheavyandlightchainproteinvariableregion SEQIDNO Label Sequence 61 1E04_ TGVHSEVQLQQSGAELVKPGASVKLSCKASGYTFTSYWVHWVRQRPGQGLEWIG HC MIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCASYYYGSSYY FDYWGQGTTLTVSSAST 62 1E04_ TGVHSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPP LC KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAG TKLELKRT 63 1C08_ TGVHSEVQLQQSGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIG HC MIHPNSGTSNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVFYCTRSDWAFDY WGQGTSLTVSSAST 64 1C08_ TGVHSDIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPP LC KLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAG TKLELKRTPLYHAIASRMAFIFSSLYKSWXLX 65 1B02_ TGVHSEVQLQQSGAELVRPGSSVKMSCKTSGYTFTFYGINWVKQRPGQGLEWIGYI HC YVGNGYSEYNEKFKVKATLTSDTSSSTAYMQLSGLTSEDSAIYFCARSDGYNYFDY WGQGTTLTVSSTST 66 1B02_ TGVHSQIVLTQSPAILSASPGEKVTMTCRASSSLHYMHWYQQKTGSSPKPWIYATSN LC LASGVPARFSGSGSGTSYSLTISRVEAEDTATYYCQQWSSNPPTFGAGTKLELKRT 67 1A09_ TGVHSEVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGL HC IHPNSGSTYYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARWDDSYWYF KVWGTGTTVTVSSAST 68 1A09_ TGVHSQIVLTQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATS LC NLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPYTFGGGTKLEIKRT 69 2A03_ TGVHSEVQLQQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEXXWIA HC RIYPGSGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARRLTAGYFD VWGTGTTVTVSSAST 70 2A03_ TGVHSDIKMTQSPSSMFASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRAN LC RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELKRT 71 2C09_ TGVHSEVMLVESGEDLVKPGGSLKLSCIASGFTFSSYAMSWVRQTPEKRLEWVAYI HC SSGGDYIYYTDTVKGRFTISRDNARNTLFLQMSSLKSEDTAMYYCTRDTGYYVSRYF DVWGTGTTVTVSSAST 72 2C09_ TGVHSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYS LC ASFRNSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCHQYNNYPLTFGGGTKLEIK RT 73 2G02_ TGVHSEVQLQQSGPVLVKPGPSVKISCKASGFTFTDYYMHWVKQSHGKSLEWIGLV HC YPYNGGTYYNQKFKGKATLTVDTSSSTAYMELNSLTSEDSAVYYCVRLGNGSSNEW YFDVWGTGTTVTVSSAST 74 2G02_ TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSYNLHWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWRTYPWTFGGGTKLEIKR T 75 1D09_ TGVHSEVQLQQPGAELVKPGASVKLSCKASGYTFTNYWIHWMKQRPGRGLEWIGRI HC EPNSGDTKYNEKIKSRATLTVDKPSSTAYMQLSSLTSEDSAVYYCARSGYDYPEAW GQGTTLTVSSAST 76 1D09_ TGVHSDIVLTQSPASLAVSPGQRATISCRASEGVEYYGTTLMQWYQQKPGQPPKLLI LC YAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVSWTFGGGTKL EIKRT 77 2B06_ TGVHSEVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKHRPGRGLEWIGR HC IDPNSGGTKYNEKFKNKATLTVDNPSSTGYMQLSSLTSEDSAVYYCTRSGYDYPDY WGQGTTLTVSSAST 78 2B06_ TGVHSDIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLMQWYQQKPGQPPKVLI LC YAASNVDSGVPPRFSGSGSGTDFSLNFHPVEEDDIAMYFCQQSRKVPWTFGGGTK LEIKRT 79 2A02_ TGVHSEVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGR HC IDPNSGGTKYNEKFKSKATLTVDKPSSTAYMQLSSLTSEDSAVYYCARSGYDYPDY WGQGTTLTVSSAST 80 2A02_ TGVHSDIVLTQSPAPLAVSLGQRATISCRASEGVEYYDTSLMQWYQQKPGQPPKLLI LC YAASNVEPGVPARFGGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKL EIKRT 81 1B07_ TGVHSEVQLQQPGAELVKPGTSVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIG HC MIHPNSGSTNYNEMFESKASLTVDKSSSTAYMQLSSLTSEDSAVYYCARIGYSGWG QGTSVTVSSAST 82 1B07_ TGVHSDIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNGGNQKNYLAWYQQKPGQPP LC KVLINGASTRESGVPDRFTGNGSGTDFTLTISSVQAEDLAVYYCQNDHTYPLTFGAG TKLELKRT 83 2D04_ TGVHSEVQLQQPGGELVRPGSSVKMSCKTSGYTFTFYGINWVKQRPGQGLEWIGYI HC YVGNGYTEYNEKFKVKATLTSDTSSSTAYMQLSGLTSEDSAIYFCARSDGYNYFDY WGQGTTLTVSSAST 84 2D04_ TGVHSQIVLTQSPAILTASPGEKVTMTCRASSSVHYMHWYQQKPGSSPKPWIYATS LC NLASGVPARFSGSGSGTSYSLTISRVEAEDTATYYCHQWSSNPPTFGAGTKLEIKRT 85 1E07_ TGVHSEVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGL HC IHPNSGSTYYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARWDDSYWYF KVWGTGTTVTVSSAST 86 1E07_ TGVHSQIVLTQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATS LC NLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPYTFGGGTKLEIKRT 87 2D07_ TGVHSEVQLQQSGPELVRPGASVKISCKASGYTFTDYYIHWLKQRPGQGLEWIGLIF HC PGSGSIYCNEKFKGKATLTVDKSSTTAYMLLSSLTSEDSAVYFCARWETTAWYFDV WGTGTTVTVSSAST 88 2D07_ TGVHSQIVLTQSPQIVLTQSPAILSASPGEKVTMTCRASLSVSYMHWYQQKPGSSPK LC PWIYATSNLASGVPARFSGSGSGTSYSLTVSRVEAEDAATYYCQQWRSYPYTFGG GTKLEIKRT 89 1A02_ TGVHSEVQLQQSGPVLVKPGPSMKISCKASGFTFTDYYIHWVRQSHGKSLEWIGLV HC SPYNGGTYYNQKFKGKATLTVDSSSSTAYMELSSLTSEDSAVYYCARLGYYGDWYY FDYWGQGTPLTVSSAST 90 1A02_ TGVHSQIVLTQSPALMATSPGEKVTITCSVSSSISSSNLNWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPYTFGGGTKLEIKRT 91 2D10 TGVHSEVQLQQSGAELVKPGASVKLSCKASGYTFTTYWMHWMKQWPGQGLEWIG HC LIHPNSGSTYYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARFEELWGGY WYFDVWGTGTTVTVSSAST 92 2D10 TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPYTFGGGTKLEIKRT 93 2E08_ TGVHSEVQLQQPGAELAKPGASVKLSCKASGYNFTSYWMHWVKQRPGQGLEWIGL HC IHPNSGGTYYNEKFKSKATLTLDRSSSTAYMQLSSLTSEDSAVYYCARFEEEWGGY WYFDVWGTGTTVTVSSAST 94 2E08_ TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPYTFGGGTKLEIKRT 95 2H04_ TGVHSEVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGL HC IHPNSSSTYYNEKFKTRATLTVDKSSSTAYMQLSSLTSEDSAVYYCARLGYGNSYWY FDVWGTGTTVTVSSAST 96 2H04_ TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTS LC NLASGVPIRFSGSGSGTSYSLTISSLEAEDAATYYCQQWRSYPWTFGGGTKLEIKRT 97 1B03_ TGVHSEVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVQQRPGQGLEWIGL HC IHPIGGGTHYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARLGTGPYYFD YWGQGTTLTVSSAST 98 1B03_ TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSSTLDWYQQKSETSPKPWVYGT LC SNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPWTFGGGTKLEIK RT 99 2H07_ TGVHSEVQLQQSGPVLVKPGPSVNISCKASGFTFTDYYIHWVKQSHGKSLEWIGLVY HC PYSGGTYYNQKFKGKATLTVDASSSTAYMELGSLTSEDSAVYYCARLGDNFYYFDY WGQGTTLTVSSAST 100 2H07_ TGVHSQIVLTQSPTLMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSYPYTFGGGTKLEIKR T 101 1C01_ TGVHSEVQLQQPGPVLVKPGPSVKISCKASGFTFTDYYMHWVKQSHGKSLEWIGLV HC SPYNGGTFYNQKFKGKATLTVDTSSSTAYMELNSLTSEDSAVYYCARVGNSYVHYA MDYWGQGTSVTVSSAST 102 1C01_ TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNYPYTFGGGTKLEIKR T 103 2C07_ TGVHSEVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLDWVAYI HC SSGGDHIYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCTRDTGYYVSRYF DVWGTGTTVTVSSAST 104 2C07_ TGVHSDIVMTQSQKFMSTSVGDRVSVTCKASQNVVTNVAWYQQKSGQSPKVVIYS LC ASFRSSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCHQYNSHPLTFGGGTKLEIK RT 105 2G04_ TGVHSEVQLQQPGPELVKPGSSLKISCKASGYTFTDYFINWVKQRPGQGLDWIGWIF HC PGSGSTYYNDKFKGKATLTVDKSSSTAYMLLSSLTSEASAVYFCARWDSDSTYWYF DVWGTGTTVTVSSAST 106 2G04_ TGVHSQIVLTQSPALMAASPGEKVTITCSVNSSISSSTLHWYQQKSQTSPKPWIYGT LC SNLASGVPIRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSTYPYTFGGGTKLEIKR T 107 1G01_ TGVHSEVQLQQSGAELMKPGASVKLSCKATGYTFTGYWIEWLKQRPGHGLEWIGEI HC LPGSDNTNYNEKFRGKATFTADTSSNTAYMHLSSLTTEDSAIYYCAREGGFYFDYW GQGTTLTVSSAST 108 1G01_ TGVHSQAVVTQESALTTSPGETVTLTCRSSIGAVTTSNYANWVQEKPDHLFTGLIGG LC TNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL GQPKSSPSVTLFPPSX 109 2A05_ TGVHSEVQLQQSGPVLVKPGPSVKISCKASGFTFTDYYMHWVKQSHGKSLEWIGLV HC YPYNGGTSYNQKFKGKATLTVDTSSTTAYMELNSLTSEDSAVYYCARLGSGYEYYF DYWGQGTSLTVSSAST 110 2A05_ TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWRTYPWTFGGGTKLEIKR T 111 2H05_ TGVHSEVQLQQPGPVLVKPGPSVKISCKASGFTFTDYYMHWVKQSHGESLEWIGLV HC SPYNGGTFYNQKFKGKATLTVDTSSSTAYMELNSLTSEDSAVYYCARVGSSYVHYA MDYWGQGTSVTVSSAST 112 2H05_ TGVHSQIVLTQSPALTAASPGERVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSKYPYTFGGGTKLEIKRT 113 2B01_ TGVHSEVQLQQSGAGLVKPGASVKLPCKASGYTFTSYWMHWVQQRPGQGLEWIG HC LIHPIGGGTHYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARLGTGPYYF DYWGQGTTLTVSSAST 114 2B01_ TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPWTFGGGTKLEIKR T 115 1D01_ TGVHSEVQLQQPGPDVVKPGASVKISCKASGYTFTDYYINWVKQRPGQGLEWIGWI HC FPGSGSSYYNEKFKDKATSTVDKSSSTAYMLLSSLTSEDSAVYFCAKWKDYGWYFD VWGTGTTVTVSSAST 116 1D01_ TGVHSQIVLTQSPALMAASPGEKVTLTCNVSSSLSSSNLHWYQQKSETSPKPWIYGT LC SNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWRTYPYTFGGGTKLEIK RT 117 2E02_ TGVHSEVQLQQSGPVLVKPGPSVKISCKASGFTFTDYYIHWVKRSHGKSLEWIGLVS HC PYNGGTFYNQKFKGKATLTVDTSSNTAYMELTSLTSEDSAVYYCARLGVNWYFDVW GTGTTVTVSSAST 118 2E02_ TGVHSQIVLTQSPALMAASPGEKVTLTCSVSSSISSSTLHWYQQKSETSPKPWIYGT LC SNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSTYPYTFGGGTKLEIKR T 119 1A05_ TGVHSEVQLQQPGPVLVKPGPSMKISCKASGYAFTDYFIHWVRQSHGRSLEWIGLV HC SPYNGGTYYNQKFKGKASLTVDTSSSTAYMELSSLTSEDSAVYYCARLGYYGNWYF FDYWGQGTPLTVSSAST 120 1A05_ TGVHSQIVLTQSPALMATSPGEKVTITCSVSSSISSSNLNWYQQKSETSPKPWIYGTS LC NLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPYTFGGGTKLEIKRT

TABLE-US-00003 TABLE3 Heavyandlightchainproteinvariableregionencodedbyanested nucleotidesequence SEQIDNO Label Sequence 121 1E04_ NNNNNNNNNNNNNNNNNNNGNANNNNNNGTGAAGTTGTCCTGCAAGGCTTCTG HC GCTACACTTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAG GCCTTGAGTGGATTGGAATGATTCATCCTAATAGTGGTAGTACTAACTACAATGA GAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTA CATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGT TATTACTACGGTAGTAGCTATTACTTTGACTACTGGGGCCAAGGCACCACTCTCA CAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGT GTGGAGGTACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGCTATT NNCCCTGANCAN 122 1E04_ NNNCNNNNNNNTNNNCTTGGTCCCAGCACCGAACGTGAGCGGATAACTATAATC LC ATTCTGACAGTAATAAACTGCCAGGTCTTCAGCCTGCACACTGCTGATGGTGAGA GTGAAATCTGTTCCAGATCCACTGCCTGTGAAGCGATCAGGGACCCCAGATTCC CTAGTGGATGCCCAGTAGATCAACAGTTTAGGAGGCTGCCCTGGTTTCTGCTGG TACCAGGTCAAGTAGTTCTTTTGATTTCCACTGTTTAACAGACTCTGACTGGACTT GCAGCTCATAGTGACCTTCTCTCCTGCTGTCACAGTCAGGGAGGATGGAGACTG GGTCNNNNNAATGTCA 123 1C08_ GNNNNNNNNNNNTNNNNNAGNANGTTCNTGAAGTTGTCCTGCAAGGCTTCTGGC HC TACACTTTCACCAACTACTGGATGCACTGGGTGAAGCATAGGCCTGGACAAGGC CTTGATTGGATTGGAANGATTCATCCTAATAGTGGTACTTCTAACTACAATGAGAA GTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACAT GCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTTTTACTGTACAAGATCT GACTGGGCCTTTGACTACTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCACCC AAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTA ACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTNCCCTGANCA 124 1C08_ NNNNNNNNNNNCNNNNCTTGGTCNNNCACCGAACGTGAGCGGAAACTATGATCA LC TTCTGACAGTAATAAACTGCCAGGTCTTCAGCCTGCACACTACTGATGGTAAGAG TGAAATCGGTTCCAGATCCACTGCCTGTGAAGCGATCAGGGACCCCAGATTCCC TAGTGGATGCCCCGTATATCAACAGTTTAGGAGGCTGCCCTGGTTTCTGCTGGTA CCAGGCCAAGTAGTTCTTTTGATTTCCACTGTTTAACAGGCTCTGACTGGACTTG CAGCTCATAGTGACCTTCTCTCCTGCTGACACACTCAGGGAGGATGGAGACTGG GTNTNTCAATGTCAN 125 1B02_ NNNNNNNNNNNNTNANNNNNGNANTCNNAGTGAAGATGTCCTGCAAGACTTCTG HC GATATACATTCACATTCTACGGTATAAACTGGGTGAAGCAGAGGCCTGGACAGG GCCTGGAATGGATTGGATATATTTATGTTGGAAATGGTTATTCTGAGTACAATGA GAAGTTCAAGGTCAAGGCCACACTGACTTCAGACACATCCTCCAGCACAGCCTA CATGCAGCTCAGCGGCCTGACATCTGAGGACTCTGCAATCTATTTCTGTGCAAGA TCCGATGGTTACAACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCT CCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTG CCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTANTNCCCTG ANCAN 126 1B02_ NNNNCNNNNCNCNNNNCTTGGTCCCAGCACCGAACGTGGGTGGGTTACTACTC LC CACTGCTGGCAGTAATAAGTGGCAGTATCTTCAGCCTCCACTCTGCTGATTGTGA GAGAGTAAGAGGTCCCAGACCCACTGCCACTGAAGCGAGCAGGGACTCCAGAA GCCAGGTTGGATGTGGCATAAATCCAGGGTTTGGGGGAGGATCCTGTCTTCTGC TGGTACCAGTGCATGTAATGTAAACTTGAGCTGGCCCTGCAAGTCATTGTGACTT TCTCCCCTGGAGATGCAGACAGGATTGCTGGAGACTGGGNNNNNCCAATGTCAA NA 127 1A09_ NNNNNNNNNNNNNNNNNNNNNNNANGNTTCAGTGAAGTTGTCCTGCAAGGCTTC HC TGGCTACACTTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACA AGGCCTTGAGTGGATTGGATTGATTCATCCTAATAGTGGTAGTACTTACTACAAT GAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCC TACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAA GATGGGATGATTCCTACTGGTACTTCAAAGTCTGGGGCACAGGGACCTCGGTCA CCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGAT CTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATT NCCCTGANNAN 128 1A09_ NNNNNNNNNNNNTTTNNNCTTGGTCCCCCCTCCGAACGTGTACGGGTTGCTACT LC CCACTGCTGGCAGTAATAAGTGGCAGCATCTTCAGCCTCCACTCTGCTGATTGTG AGAGAGTAAGAGGTCCCAGACCCACTGCCACTGAAGCGAGCAGGGACTCCAGA AGCCAGGTTGGATGTGGCATAAATCCAGGGTTTGGGGGAGGATCCTGGCTTCTG CTGGTACCAGTGCATGTAACTTACACTTGAACTGGCCCTGCAAGTCATTGTGACC TTCTCCCCTGGAGATGCAGACAGGATTGCTGGAGATTGTGTCAGNACAATGTCA N 129 2A03_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTCNGTGAAGCTGTCCTG HC CAAGGCTTCTGGCTACACTTTCACTGACTACTATATAAACTGGGTGAAGCAGAGG CCTGGACAGGGACTTGAGTGGATTGCAAGGATTTATCCTGGAAGTGGTAATACTT ACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGAAAAATCCTCCA GCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCTGTCTATTT CTGTGCAAGACGGCTAACTGCGGGATACTTCGATGTCTGGGGCACAGGGACCA CGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCC CTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGG GCTACTNCCCTGANCAN 130 2A03_ NNNNNNNNNNNCTNNNCTTGGTCCCAGCACCGAACGTGAGCGGAAACTCATCAT LC ACTGTAGACAATAATAAATTCCCATATCTTCATACTCCAGGCTGCTGATGGTGAG AGAATAATCTTGCCCAGATCCACTGCCACTGAACCTTGATGGGACCCCATCTACC AATCTGTTTGCACGATAGATCAGGGTCTTAGGAGATTTCCCTGGTTTCTGCTGGA ACCAGGATAAATAGCTATTAATGTCCTGACTCGCCTTGCAAGTGATAGTGACTCT CTCTCCTAGAGATGCAAACATGGAAGATGGAGACTGTNTNN 131 2C09_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNGNANGGNCCCTGAACTCTCCTGTAC HC AGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCA GAGAAGAGGCTGGAGTGGGTCGCATATATTAGTAGTGGTGGTGATTACATCTAC TATACAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAGGAAC ACCCTGTTCCTACAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACT GTACAAGAGATACCGGTTACTACGTTTCTCGGTACTTCGATGTCTGGGGCACAG GGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCAC TGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGG TCAAGGGCTACTTCCCTGAGCA 132 2C09_ NNNNNNNNNNTGNNTNNNNNCTTNNNNNNNNNNNNNAACGTCAGCAGGATAGTT LC GTTATATTGATGACAGAAATACTCTGCCAAGTCTTCAGACTGCACATTGCTGATG GTGAGAGTGAAATCTGTCCCAGATCCACTGCCTGTGAAGCGATCAGGGACTCCA CTGTTCCGGAAGGATGCCGAGTAAATCAGTGCTTTAGGAGATTGTCCTGGTTTCT GTTGATACCAGGCTACATTAGTTCCCACATTCTGACTGGCCTTGCAGGTGACGCT GACCCTGTCTCCAACTGATGTGGACATGAATTTTGGAGACTGGGTCATCACAATG TCA 133 2G02_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNANCCTTCAGTGAAGATATC HC CTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATGCACTGGGTGAAGCAG AGCCATGGAAAGAGCCTTGAATGGATTGGACTTGTTTATCCTTACAATGGTGGTA CTTATTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTC CAGCACAGCCTACATGGAGCTAAACAGCCTGACTTCTGAGGACTCTGCGGTCTA TTACTGTGTACGATTAGGGAACGGTAGTAGCAACGAGTGGTACTTCGATGTCTG GGGCACAGGGACCACGGTCACTGTCTCCTCAGCCAAAACGACACCCCCATCTGT CTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGG ATGCCTGGTCAAGGGCTACTTCCCTGANCA 134 2G02_ NNNNNCNNNTNANNNNNGTTTGGTGCCTCCCCGAACGTCCACGGGTAAGTTCTC LC CACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATACTGCTGATTGTGA GAGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAG CCAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCT GGTACCAGTGCAAATTGTAGGAACTTATACTTGAGCTGACACTGCAGGTGATGGT GACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGACTGNGTNANNN 135 1D09_ NNNNNNNNNNNGTNANNNTGNNNGCTTCNGTGAAGCTGTCCTGCAAGGCTTCTG HC GCTACACCTTCACCAACTACTGGATACACTGGATGAAGCAGAGGCCTGGACGAG GCCTTGAGTGGATTGGAAGGATTGAGCCTAATAGCGGTGATACTAAATACAATGA GAAGATCAAGAGCAGGGCCACACTGACTGTAGACAAACCGTCCAGCACAGCCTA CATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAG ATCTGGGTATGATTACCCTGAGGCCTGGGGCCAAGGCACCACTCTCACAGTCTC CTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGC CCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTACTNCCCTGA NNA 136 1D09_ NNNNNNNNNNNNNNNNNNNNNTTGGTGCCTCCACCGAACGTCCAAGGAACCTT LC CCTACTTTGCTGACAGAAATACATTGCAATATCATCCTCCTCCACAGGATGGATG TTGAGGCTGAAGTCTGTCCCAGACCCACTGCCACTAAACCTGGCAGGGACCCCA GATTCTACGTTGGATGCAGCATAGATGAGGAGTTTGGGTGGCTGTCCTGGTTTCT GTTGGTACCACTGCATTAAAGTTGTGCCATAATATTCAACACCTTCACTGGCTCT GCAGGAGATGGTGGCTCTCTGCCCTAGAGACACAGCCAAAGAAGCTGGAGATTG GGTCATCACAATGTCAN 137 2B06_ NNNNNNNNNNNNNNNNNNNTGANCCTGNNNCTTCAGTGAAGTTGTCCTGCAAGG HC CTTCTGGCTATACCTTCACCAACTACTGGATGCACTGGGTGAAACACAGGCCTG GACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTGGTGGTACTAAGT ACAATGAGAAGTTCAAGAATAAGGCCACACTGACTGTAGACAATCCCTCCAGCAC AGGCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGT ACAAGATCTGGGTATGATTACCCTGACTACTGGGGCCAAGGCACCACTCTCACA GTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCT GCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTN CCCTGANAAN 138 2B06_ NNNNNNNNNNNNNNTNNNNGCTTGGTGCCTCCNCCGAACGTCCAAGGAACCTT LC CCTACTTTGCTGACAGAAATACATTGCAATATCATCCTCCTCCACAGGATGGAAG TTGAGGCTGAAGTCTGTCCCAGACCCACTGCCACTAAACCTGGGAGGGACCCCA GAATCTACGTTGGATGCAGCATAGATGAGGACTTTGGGTGGCTGTCCTGGTTTCT GTTGGTACCACTGCATTAAACTTGTGCCATAATATTCAACACTTTCACTGGCTCTG CAGGAGATGGTGGCTCTCTGCCCTAGAGACACAGCCAAAGAAGCTGGAGACTG GGTNANNNCAATGTCAA 139 2A02_ NNNNNNNNNNNNNNNNNNNNNNANCNNGNNNGCTTCAGTGAAGCTGTCCTGCA HC AGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGC CTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTGGTGGTACTA AGTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAACCCTCCA GCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATT ATTGTGCAAGATCTGGGTATGATTACCCTGACTACTGGGGCCAAGGCACCACTC TCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTG GATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCT ACTTCCCTGANCA 140 2A02_ NNNNNNNNNATNNNNCTTGGTGCCTCCCCGAACGTCCAAGGAACCTTCCTACTT LC TGCTGACAGAAATACATTGCAATATCATCCTCCTCCACAGGATGGATGTTGAGGC TGAAGTCTGTCCCAGACCCACTGCCACCAAACCTGGCAGGAACCCCAGGTTCTA CGTTGGATGCAGCATAGATGAGGAGTTTGGGTGGCTGTCCTGGTTTCTGTTGGT ACCACTGCATTAAACTTGTGTCATAATATTCAACACCTTCACTGGCTCTGCAGGA GATGGTGGCTCTCTGCCCTAGAGACACAGCCAAAGAAGCTGGAGNNTGTNNNNN N 141 1B07_ NNNNNNNNNNNNNNNNCNTGANNANNATTCNGTGAAGTTGTCCTGCAAGGCTTC HC TGGCTACACTTTCACCATCTACTGGATGCACTGGGTGAAGCATAGGCCTGGACA AGGCCTTGAGTGGATTGTAATGATTCATCCTAATATTGGTATTACTAACTACAATG AGATGTCGAGAGCAAGGCCTCACTGACTGTAGACAAATCCTCCAGCACATCCTA CATGCAACTCANCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGA ATCGGATACTCCGGCTGGGGTCAAGGAACCTCATTCACCGTCTCCTCAGCCAAA ACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTACTNCCCTGANCA 142 1B07_ GNNNNNNCTNNCTNNNCTTGGTCCCAGCACCGAACGTGAGCGGATAAGTATGAT LC CATTCTGACAGTAATAAACTGCCAGGTCTTCAGCCTGCACACTGCTGATGGTAAG AGTGAAATCGGTTCCAGATCCATTGCCTGTGAAGCGATCAGGGACCCCAGATTC CCTAGTGGATGCCCCGTTGATCAACACTTTAGGAGGCTGCCCTGGTTTCTGCTG GTACCAGGCCAAGTAGTTCTTTTGATTTCCACCATTTAACAGACTCTGACTGGAC TTGCAGCTCATAGTGACCTTCTCTCCTGCTGACACACTCAGGGAGGATGGAGAC TGGGTNTNACAATGTCANA 143 2D04_ NNNNNNNNNNNANCTGGNGNNGCCTGGGTCCTCAGTGAAGATGTCCTGCAAGA HC CTTCTGGATATACATTCACATTCTACGGTATAAACTGGGTGAAGCAGAGGCCTGG ACAGGGCCTGGAATGGATTGGATATATTTATGTTGGAAATGGTTATACTGAGTAC AATGAGAAGTTCAAGGTCAAGGCCACACTGACTTCAGACACATCCTCCAGCACA GCCTACATGCAGCTCAGCGGCCTGACATCTGAGGACTCTGCAATCTATTTCTGTG CAAGATCCGATGGTTACAACTACTTCGACTACTGGGGCCAAGGCACCACTCTCA CAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGAT CTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATT NNCCTGANCAN 144 2D04_ NNNNNNNNTNTNGCTNNNNNTTGNNTCCCNGNACCGAACGTGGGTGGGTTACTA LC CTCCACTGATGGCAGTAATAAGTGGCAGTATCTTCAGCCTCCACTCTGCTGATTG TGAGAGAGTAAGAGGTCCCAGACCCACTGCCACTGAAGCGAGCAGGGACTCCA GAAGCCAGGTTGGATGTGGCATAAATCCAGGGTTTGGGGGAGGATCCTGGCTTC TGCTGGTACCAGTGCATGTAATGTACACTTGAACTGGCCCTGCAAGTCATTGTGA CTTTCTCCCCTGGAGATGCAGTCAGGATTGCTGGAGACTGGGTCATCACNATGT CAA 145 1E07_ NNNNNNNNNNNNNNNNNNNNNNNAGNNNNCNNNNNTGAAGTTGTCCTGCAAGG HC CTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTG GACAAGGCCTTGAGTGGATTGGATTGATTCATCCTAATAGTGGTAGTACTTACTA CAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACA GCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGT GCAAGATGGGATGATTCCTACTGGTACTTCAAAGTCTGGGGCACAGGGACCTCG GTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCT GGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGC TACTTCCCTGANCA 146 1E07_ NNNNNNNNNNNNNNCTTGGTCCCCCCTCCGAACGTGTACGGGTTGCTACTCCAC LC TGCTGGCAGTAATAAGTGGCAGCATCTTCAGCCTCCACTCTGCTGATTGTGAGA GAGTAAGAGGTCCCAGACCCACTGCCACTGAAGCGAGCAGGGACTCCAGAAGC CAGGTTGGATGTGGCATAAATCCAGGGTTTGGGGGAGGATCCTGGCTTCTGCTG GTACCAGTGCATGTAACTTACACTTGAACTGGCCCTGCAAGTCATTGTGACCTTC TCCCCTGGAGATGCAGACAGGATTGCTGGAGACTGGGTNNNNNCAATGTCAN 147 2D07_ NNNNCNNNNNNNNNNNNNNTNNNNNNNNNNGNNNGCTTCNGTGAAGATATCCT HC GCAAGGCTTCTGGCTACACCTTCACTGACTACTATATACACTGGCTGAAGCAGAG GCCTGGACAGGGACTTGAGTGGATTGGATTGATTTTTCCTGGAAGTGGTAGTATT TACTGTAATGAGAAGTTCAAGGGCAAGGCCACACTTACTGTAGACAAATCCTCCA CCACAGCCTACATGTTGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTT CTGTGCAAGATGGGAGACTACGGCGTGGTACTTCGATGTCTGGGGCACAGGGA CCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCA AGGGCTACTTCCCTGAGCA 148 2D07_ NNNNNCNNNNNNNNNNNNTTGNNNNNNCTCCGAACGTGTACGGGTAACTCCTC LC CACTGCTGGCAGTAATAAGTGGCAGCATCTTCGGCCTCCACTCTGCTGACTGTG AGAGAGTAAGAGGTCCCAGACCCACTGCCACTGAAGCGGGCAGGGACTCCAGA AGCCAGGTTGGATGTGGCATAAATCCAGGGTTTGGGGGAGGATCCTGGCTTCTG CTGGTACCAGTGCATGTAACTTACACTTAAACTGGCCCTGCAAGTCATTGTGACC TTCTCCCCTGGAGATGCAGACAGGATTGCTGGAGACTGGGTCANNACAATGTCA N 149 1A02_ NNNNNNNGGTGAGCCTGGGCCTTCATGAAGATATCCTGTAAGGCTTCTGGATTC HC ACATTCACTGACTACTACATACACTGGGTGAGGCAGAGCCATGGAAAGAGCCTT GAGTGGATTGGACTTGTTTCTCCTTACAATGGTGGTACTTACTACAACCAGAAGT TCAAGGGCAAGGCCACATTGACTGTAGACTCATCCTCCAGCACAGCCTACATGG AGCTAAGCAGCCTGACTTCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTGG GCTACTATGGTGACTGGTACTACTTTGACTACTGGGGCCAAGGCACCCCTCTCA CAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGAT CTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATT NNCCCTGANCAN 150 1A02_ NNNNNNNNNNNNNNNNTTGGTCCCCCCTCCGAACGTGTATGGGTAACTACTCCA LC CTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGAGA GAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAGCC AGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCTGG TACCAGTTCAAGTTGCTGGAACTTATACTTGAGCTGACACTACAGGTGATGGTGA CCTTTTCCCCTGGAGATGTAGCCATGAGTGCTGGAGACTGGGTGANNNCAATGT CANN 151 2D10_ NNNNNNNNNNNNNNNNNNNNNNNCCNGNNNCTTCAGTGAAGTTGTCCTGCAAG HC GCTTCTGGCTACACTTTCACCACCTACTGGATGCACTGGATGAAGCAGTGGCCT GGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTAATAGTGGTAGTACTTACT ACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCA CAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACT GTGCAAGATTTGAGGAGCTATGGGGAGGTTACTGGTACTTCGATGTCTGGGGCA CAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATC CACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCC TGGTCAAGGGCTATTTCCCTGANCAN 152 2D10_ NNNNNNNNTTNNNNNNNCTTGGTCCCCCTCCGAACGTGTACGGGTAACTACTCC LC ACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGAG AGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAGC CAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCTG GTACCAGTGCAAGTTGCTGGAACTTATACTTGAGCTGACACTGCAGGTGATGGT GACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGACTGGGTNNNNACAAT GTCAN 153 2E08_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNGCTTCAGTGAAGTTGTCCTG HC CAAGGCTTCTGGCTACAATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAG GCCTGGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTAATAGTGGTGGTACT TACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAGATCCTCCA GCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATT ATTGTGCAAGATTTGAGGAGGAATGGGGAGGGTATTGGTACTTCGATGTCTGGG GCACAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCT ATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGAT GCCTGGTCAAGGGCTATTTCCCTGAGCA 154 2E08_ NNNNNNNNNTNTNNNNNNTTGGTNNNCCTCCGAACGTGTACGGGTAACTACTCC LC ACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGAG AGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAGC CAGGTTGGAAGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGTTG GTACCAGTGCAAGTTGCTGGAACTTATACTTGAACTGACACTGCAGGTGATGGTG ACCTTTTCCCCTGGAGATGCAGCCATGAGTGCTGGAGTNTGGGTCANNN 155 2H04_ NNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTCNGTGAAGTTGTCC HC TGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGTGAAGCAG AGGCCTGGACAAGGCCTTGAGTGGATTGGACTGATTCATCCTAATAGTAGTAGTA CTTACTACAATGAGAAGTTCAAGACCAGGGCCACACTGACTGTAGACAAGTCCTC CAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTA TTACTGTGCAAGATTGGGCTATGGTAACTCCTACTGGTACTTCGATGTCTGGGGC ACAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTAT CCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGC CTGGTCAAGGGCTACTTCCCTGANCA 156 2H04_ NNNNNNNNNNNNNNNNGCTTGGTGCCTCCACCGAACGTCCACGGGTAACTTCTC LC CACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCAAACTGCTGATTGTGA GAGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAATAGGGACTCCAGAAG CCAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCT GGTACCAGTGCAAGTTGCTGGAACTAATACTTGAGCTGACACTGCAGGTGATGG TGACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGACTGGGTNNNNCAAT GTCA 157 1B03_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTCNGTGAAGTTGTCCTGCAAGG HC CTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGTGCAGCAGAGGCCTG GACAAGGCCTTGAGTGGATTGGATTGATTCATCCTATTGGTGGTGGTACTCACTA CAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACA GCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGT GCAAGACTCGGAACTGGTCCGTACTACTTTGACTACTGGGGCCAAGGCACCACT CTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCT GGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGC TATTTCCCTGANCA 158 1B03_ NNNNCNTTNNNTNNNNNTTTGGTGCCTCCACCGAACGTCCACGGGTAACTACTC LC CACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGA GAGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAG CCAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCT GGTACCAGTCCAAGGTGCTGGAACTTATACTTGAGCTGACACTGCAGGTGATGG TGACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGACTGGGTCNNNACAA TGTCANNN 159 2H07_ NNNNNNNNNNNNNNNNNNNNNNNNNNANNNNGNANCCTTCAGTGAATATATCCT HC GTAAGGCTTCTGGATTCACATTCACTGACTACTACATTCACTGGGTGAAACAGAG CCATGGAAAGAGCCTTGAGTGGATTGGACTTGTTTATCCTTACAGTGGTGGTACT TACTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCA GCACAGCCTACATGGAGCTAGGCAGCCTGACTTCTGAGGACTCTGCGGTCTATT ACTGTGCAAGATTGGGCGACAACTTCTACTACTTTGACTACTGGGGCCAAGGCA CCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGC CCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAA GGGCTATTTCCCTGAGCAN 160 2H07_ NNNNNNNNNNNTTNNGCTTGGTCNNCCTCCGAACGTGTACGGGTAACTTCTCCA LC CTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGAGA GAATAAGATGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAGCC AGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCTGG TACCAGTGCAAGTTGCTGGAACTTATACTTGAGCTGACACTGCAGGTGATGGTGA CCTTCTCCCCTGGAGATGCAGCCATGAGTGTTGGAGTNTGNGNNANNACAATGT CAN 161 1C01_ GNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTCAGTGAAGATATCCTGTAAG HC GCTTCTGGATTCACATTCACTGACTACTACATGCACTGGGTGAAACAGAGCCATG GAAAGAGCCTTGAGTGGATTGGTCTTGTTTCTCCTTACAATGGTGGTACTTTCTA CAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCAC AGCCTACATGGAACTAAACAGCCTGACTTCTGAGGACTCTGCGGTCTATTACTGT GCAAGAGTGGGTAATAGCTACGTCCATTATGCTATGGACTACTGGGGTCAAGGA ACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCA AGGGCTACTTCCCTGANCA 162 1C01_ NNNNCNNNNNNNNNNNCTTGGTCCCCCCTCCGAACGTGTACGGGTAATTACTCC LC ACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGAG AGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAGC CAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCTG GTACCAGTGCAAGTTGCTGGAACTTATACTTGAGCTGACACTGCAGGTGATGGT GACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGACTGGGTGANNNCAAT GTCANN 163 2C07_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANGGTCCCTGNNNCTCTCC HC TGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGA CTCCAGAGAAGAGGCTGGACTGGGTCGCATACATTAGTAGTGGTGGTGATCACA TCTACTATGCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCA GGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGT ATTACTGTACAAGAGATACCGGTTACTACGTTTCTCGGTACTTCGATGTCTGGGG CACAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTA TCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCNTGGTGACCCTGGGATG CCTGGTCAAGGGCTANTTCCCTGANCAAATA 164 2C07_ NNNNNNNNNNNTNTGNNNNNNNANTTGNTGCCTCCNCCNANCGTCAGAGGATG LC GCTGTTATATTGGTGACAGAAATACTCTGCCAAGTCTTCAGACTGCACATTGCTG ATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCTGTGAAGCGATCAGGGACT CCACTGGACCGGAAGGATGCCGAATAAATCACTACTTTAGGAGATTGCCCTGATT TCTGTTGATACCAGGCAACATTAGTAACCACATTCTGACTGGCCTTGCAGGTGAC GCTGACCCTGTCTCCTACTGATGTGGACATGAATTTTGGAGACTGGGTCATCACA ATGTCA 165 2G04_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANTCTTCACTGAAGATATCCTG HC CAAGGCTTCTGGCTACACCTTCACTGACTACTTTATAAACTGGGTGAAACAGAGG CCTGGACAGGGACTTGACTGGATTGGATGGATTTTTCCTGGAAGTGGTAGTACTT ACTACAATGACAAGTTCAAGGGCAAGGCCACACTTACTGTAGACAAATCCTCCAG CACTGCCTACATGTTGCTCAGCAGCCTGACCTCTGAGGCCTCTGCGGTCTATTTC TGTGCAAGATGGGACTCCGATAGTACCTACTGGTACTTCGATGTCTGGGGCACA GGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCA CTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTG GTCAAGGGCTATTTCCCTGANCA 166 2G04_ NNNNNNCNNNNNTNNNNNCTTGGTNNNCCTCCGAACGTGTACGGGTAAGTACTC LC CACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGA GAGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAATAGGGACTCCAGAAG CCAAATTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTGTGACTTCTGCT GGTACCAGTGCAAGGTGCTGGAACTTATACTTGAATTGACACTGCAGGTGATGG TGACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGTCTGGGTCANNNCAA TGTCA 167 1G01_ NNNNNNNNNCNNNNNNNNNNNNNNANGCCTCAGTGAAGCTTTCCTGCAAGGCT HC ACTGGCTACACATTCACTGGCTACTGGATAGAGTGGTTAAAGCAGAGGCCTGGA CATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGATAATACTAACTACA ATGAGAAGTTCAGGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAG CCTACATGCACCTCAGCAGCCTGACAACTGAGGACTCTGCCATCTATTACTGTGC AAGAGAAGGGGGTTTCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACTGT CTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGC TGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTACTNCCC TGANCAA 168 1G01_ NNNNNNNNNNTGGTGANNGTCACACTCACTTGTCGCTCAAGTATTGGGGCTGTT LC ACAACTAGTAACTACGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTG GTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAG GCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAG GATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTG GAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCC TGTTCCCACCTTCCNCTGAAGAGA 169 2A05_ NNNNNNNNNNNNNNNNNNNNANNCCNGNNNCCTTCNGTGAAGATATCCTGTAAG HO GCTTCTGGATTCACATTCACTGACTACTACATGCACTGGGTGAAGCAGAGCCATG GAAAGAGCCTTGAATGGATTGGACTTGTTTATCCTTACAATGGTGGTACTAGCTA CAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCACCAC AGCCTACATGGAGCTAAACAGCCTGACTTCTGAGGACTCTGCGGTCTATTACTGT GCAAGATTGGGCTCAGGCTACGAGTACTACTTTGACTACTGGGGCCAAGGCACC TCTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCC CTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGG GCTACTNCCCTGANCAN 170 2A05_ NNNNCNNNNNNTTNNNGCTTGGTGCCTCCNCCGAACGTCCACGGGTAAGTTCTC LC CACTGTTGACAGTAATAAGTGGCAGCATCCTCAGCCTCCATGCTGCTGATTGTGA GAGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAG CCAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCT GGTACCAGTGCAAGTTGCTGGAACTTATACTTGAGCTGACACTGCAGGTGATGG TGACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGNCTGGGTNTNN 171 2H05_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNCAGTGANANNTC HC CTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATGCACTGGGTGAAGCAG AGCCATGGAGAGAGCCTTGAGTGGATTGGACTTGTTTCTCCTTACAATGGTGGTA CTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTC CAGCACAGCCTACATGGAACTAAACAGCCTGACTTCTGAGGACTCTGCGGTCTA TTACTGTGCAAGAGTGGGTAGTAGTTACGTCCATTATGCTATGGACTACTGGGGT CAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATC CACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCC TGGTCAAGGGCTACTTCCCTGAGCA 172 2H05_ NNNNNNNNNNNNNNNNNNNNNNTNNNNCTTGGTCNCCCTCCGAACGTGTACGG LC GTACTTACTCCACTGTTGACAGNAATAAGTGGCAGCATCTTCAGCCTCCATGCTG CTGATTGTGAGAGAATAANAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGG ACTCCAGAAGCCAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCT GACTTCTGCTGGTACCAGTGCAAGTTGCTGGAACTTATACTTGAGCTGACACTGC AGGTGATGGTGACCCTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGACTGG GTCATCACAATGTCA 173 2B01_ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANGCTTCAGTGAAGTTGTCCTG HC CANGGCTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGTGCAGCAGAG GCCTGGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTATTGGTGGTGGTACT CACTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCA GCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATT ACTGTGCAAGACTCGGAACTGGTCCGTACTACTTTGACTACTGGGGCCAAGGCA CCACTCTCACAGTCTCCTCACCCAAAACGACACCCCCATCTGTCTATCCACTGGC CCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAA GGGCTATTTCCCTGANNAN 174 2B01_ NGNNNGNTNGATTTCNGCTTGGTGCCTCCACCGAACGTCCACGGGTAACTACTC LC CACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGA GAGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAG CCAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCT GGTACCAGTGCAAGTTGCTGGAACTTATACTTGAGCTGACACTGCAGGTGATGG TGACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGACTGGGTGTN 175 1D01_ NNNNNNNNNNNNNNNNNNNNNNNNNNNTNCTGTGAANATTTCCTGCAAGGCTTC HC TGGCTACACCTTCACTGACTACTATATAAACTGGGTGAATCAGAGGCCTGGACAG GGACTTGATTGGATTGGATGGATTTTTCCTGGAANTGNTTTTATTTATTACAATGA NAAGTTCAAGGACAAGGTCACTTCTACTGTATACAAATCCTCCANCACATTCTACT TGTTGCTCANCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAANATG GAAGGATTACGGGTGGTACTTCTATGTCTGGGGCACAGGGACCACGGTCACCGT CTCCTCATCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCT GCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCT TANCAN 176 1D01_ NNNNNNNNNNNNNNNCTTGGTCCCCCCTCCGAACGTGTAAGGGTAAGTCCTCCA LC CTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGAGA GAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAGCC AGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCTGG TACCAGTGCAAGTTGCTGGAACTTAGACTTGAGCTGACATTGCAGGTGAGGGTG ACCTTCTCCCCTGGAGATGCAGCCATGAGTGCTGGAGACTGGGTGANNNCAATG TCA 177 2E02_ NNNNNNNNNNNNNNNNNNNNNNNNNNNAGNANCCTTCAGTGAAGATATCCTGTA HC AGGCTTCTGGATTCACATTCACTGACTACTACATTCACTGGGTGAAGCGGAGCCA TGGAAAGAGCCTTGAGTGGATTGGACTTGTTTCTCCTTACAATGGTGGTACTTTC TACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGATACATCCTCCAATA CAGCCTACATGGAGCTAACCAGCCTGACTTCTGAGGACTCTGCGGTCTATTACT GTGCAAGATTGGGGGTTAACTGGTACTTCGATGTCTGGGGCACAGGGACCACG GTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCT GGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGC TATTTCCCTGANNN 178 2E02_ NNNNNNNNNNNNNNNTNTTCNNCTTGTTTCNCCCCTCCTANCAAGTGGNNNTNN LC AAACTCCTCTGNTGGANAACAATANNNTANNCATCNNNTNCCNCCTCCCTGCNNA TGAAGNGNNATAANANNTCCNNCNNNNNCTGCGCTNNTGNANACAAGNNCTACT CANNAANNGNNAGNNGTGCCNTAAATCNACNGTTTGGNGGAGGTTTCTGACTTC TGCTGGTACCANNGCNANGTGCTGGAACTTATACTNCTGNTGACACTGCGGNNN AGNAGGACCTTTTCCCCCCCNGGATGCANCCATGAGTGCTNTGNANTGTGTCAT CACAATATCANCA 179 1A05_ NNNNNNNNCTGNNTNNNNNNNGNANCNTTCAATGAAGATATCCTGTAAGGCTTC HC TGGGTTCACTTTCACTGACTACTTCATACACTGGGTGAGACAGAGCCATGGAAAG AGCCTTGAGTGGATTGGACTTGTTTCTCCTTATAATGGTGGTACTTACTACAACCA GAAGTTCAAGGGCAAGGCCTCATTGACTGTAGACACATCCTCCAGCACAGCCTA CATGGAACTAAGCAGCCTGACTTCTGAGGACTCTGCGGTCTATTACTGTGCAAGA TTGGGCTACTATGGTAACTGGTACTTCTTTGACTACTGGGGCCAAGGCACCCCTC TCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTG GATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCT ATTNCCCTGAGCAN 180 1A05_ NNNNCNNNNNNNNNNNCTTGGTCCCCCCTCCGAACGTGTATGGGTAACTACTCC LC ACTGTTGACAGTAATAAGTGGCAGCATCTTCAGCCTCCATGCTGCTGATTGTGAG AGAATAAGAGGTCCCAGATCCACTGCCACTGAAGCGAACAGGGACTCCAGAAGC CAGGTTGGATGTGCCATAAATCCAGGGTTTGGGGGAGGTTTCTGACTTCTGCTG GTACCAGTTCAAGTTGCTGGAACTTATACTTGAGCTGACACTACAGGTGATGGTG ACCTTTTCCCCTGGAGATGTAGCCATGAGTGCTGGAGACTGGGTGANNNCAATG TCANNA

TABLE-US-00004 TABLE4 Heavyandlightchainproteinvariableregionis encodedbyaplasmidnucleotidesequence SEQ IDNO Label Sequence 181 1E04_ NNNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG HC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAG GTCCAGCTGCAACAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAA GTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGGTGCACTGGGT GAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAATGATTCATCCTAATAG TGGTAGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGAC AAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCT GCGGTCTATTACTGTGCAAGTTATTACTACGGTAGTAGCTATTACTTTGACTACTG GGGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTG GGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGG ACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGNN NNNNGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACC TGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC CCTCATGATCTCCCGGANCCCTGAGGTCACATGCGTGGNGGTGGACGTGAGCN 182 1E04_ NNNNNNNNNNNNNNNTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA CATTGTGATGACCCAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGT CACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAAC TACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACT GGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTG GAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTA TTACTGTCAGAATGATTATAGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTG GAGCTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACT CCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG AGAGTGTTAGAAGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGA CTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATG CCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAA TCCTGGTTGCTGTCTCTTTATGAG 183 1C08_ NNNNNNNNNNNNNNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGG HC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA GGTCCAGCTGCAACAGTCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGA AGCTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAACTACTGGATGCACTGGGT GAAGCAGAGGCCTGGGCAAGGCCTTGAGTGGATTGGAATGATTCATCCTAATAG TGGTACTTCTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGAC AAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCT GCGGTCTTTTACTGTACAAGATCTGACTGGGCCTTTGACTACTGGGGCCAAGGT ACCTCTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTG GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT CAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCT CAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCA AATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAN 184 1C08_ NNNNNNNNNNNNNNNNNNNCTGCACCTCGGTTCTATCGATTGAATTCCACCATG LC GGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCG ACATTGTGATGACCCAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGG TCACTATGAGCTGCAAGTCCAGTCAGAGCCTGTTAAACAGTGGAAATCAAAAGAA CTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTA CGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGAT CTGGAACCGATTTCACTCTTACCATCAGTAGTGTGCAGGCTGAAGACCTGGCAG TTTATTACTGTCAGAATGATCATAGTTATCCGCTCACGTTCGGTGCTGGGACCAA GCTGGAGCTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATC TGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTC TATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGT AACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCT CAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGTTAGAAGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGA TTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTT AATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGT ATAAATCCTGGNTGCTGN 185 1B02_ NNNNNNNNNCNGNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGAT HC GGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGGT CCAACTGCAGCAGTCTGGAGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGA TGTCCTGCAAGACTTCTGGATATACATTCACATTCTACGGTATAAACTGGGTGAA GCAGAGGCCTGGACAGGGCCTGGAATGGATTGGATATATTTATGTTGGAAATGG TTATTCTGAGTACAATGAGAAGTTCAAGGTCAAGGCCACACTGACTTCAGACACA TCCTCCAGCACAGCCTACATGCAGCTCAGCGGCCTGACATCTGAGGACTCTGCA ATCTATTTCTGTGCAAGATCAGATGGTTACAACTACTTTGACTACTGGGGCCAAG GCACCACTCTCACAGTCTCCTCAACGTCGACCAAGGGCCCATCGGTCTTCCCCC TGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG GTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTG ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC TCCCGGACCCCTGAGGNCN 186 1B02_ NNNNNNNNNNNNNNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCA AATTGTTCTCACCCAGTCTCCAGCAATCCTATCTGCATCTCCAGGGGAGAAAGTC ACAATGACTTGCAGGGCCAGCTCAAGTTTACATTACATGCACTGGTACCAGCAGA AGACAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTG GAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAA TCAGCAGAGTGGAGGCTGAAGATACTGCCACTTATTACTGCCAGCAGTGGAGTA GTAACCCACCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTG GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA GCAGGACAGCAAGGACAGCACCTACAGCNNNNNNNNCACCCTGACGCTGAGCA AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTGATC CTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGC TTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTT ATGAGGAGTTGTGGNCCGTTGTCAGGNAACGTGGCGTGGTGTGCACTG 187 1A09_ NNNNNNNNNNNGNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGAT HC GGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGGT CCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGT TGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGTGA AGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTAATAGTG GTAGTACTTACTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAA ATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGC GGTCTATTACTGTGCAAGATGGGATGATTCCTACTGGTACTTCAAAGTCTGGGGC ACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTT CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGNNNCACATGCGTGGTGGTGGA 188 1A09 NNNNNNNNNNNNNNNNGNNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCA LC TGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTC CCAAATTGTTCTCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAG GTCACAATGACTTGCAGGGCCAGTTCAAGTGTAAGTTACATGCACTGGTACCAG CAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCT TCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTC ACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGG AGTAGCAACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACG GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC AGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTG ATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTAT GTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTAT TGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGCTN 189 2A03_ NNNNNNNNNNNNNNGNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGG HC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA GGTCCAACTGCAGCAGTCTGGAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGA AGCTGTCCTGCAAGGCTTCTGGCTACACTTTCACTGACTACTATATAAACTGGGT GAAGCAGAGGCCTGGACAGGGACTCGAGTGGATTGCAAGGATTTATCCTGGAAG TGGTAATACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGAA AAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCT GCTGTCTATTTCTGTGCAAGACGGCTAACTGCGGGATACTTCGATGTCTGGGGC ACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTT CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGANTCACATGCGTGGTGGTGGACNNN 190 2A03_ NNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGAT LC GGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGACAT CAAGATGACCCAGTCTCCATCTTCCATGTTTGCATCTCTAGGAGAGAGAGTCACT ATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTATCCTGGTTCCAGCAGA AACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGG GGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATC AGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGT TTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTGGCT GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTGATCCTC AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCT CCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTC CCGTATGGCTTTCATTTTCTCCTCNNTGTATAAATCCTGGTTGCTGTCTCTTTATG AGGAGTTGTGGN 191 2C09_ NNNNNNNNNNNNNNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGG HC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA AGTGATGCTGGTGGAGTCTGGGGAAGACTTAGTGAAACCTGGAGGGTCCCTGAA ACTCTCCTGTATAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTT CGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATATATTAGTAGTGGTGGT GATTACATCTACTATACAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACA ATGCCAGGAACACCCTGTTCCTACAAATGAGCAGTCTGAAGTCTGAGGACACAG CCATGTATTACTGTACAAGAGATACCGGTTACTACGTTTCTCGGTACTTCGATGT CTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCAT CGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAAC TCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGANGN 192 2C09_ NNNNNNNNNNNNNNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA CATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTTGGAGACAGGGTC AGTGTCACCTGCAAGGCCAGTCAGAATGTGGGAACTAATGTAGCCTGGTATCAA CAGAAACCAGGACAATCTCCTAAAGCACTGATTTACTCGGCATCCTTCCGGAACA GTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCATCAATATAA CAACTATCCTCTGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGT GGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAG AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTGAT CCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTG CTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTT TATGAGGAGTTGTGGCCCNNN 193 2G02_ NNNNNNNNNNNGNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGAT HC GGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGGT CCAACTGCAGCAGTCTGGACCTGTGCTGGTGAAGCCTGGGCCTTCAGTGAAGAT ATCCTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATGCACTGGGTGAAG CAGAGCCATGGAAAGAGCCTTGAATGGATTGGACTTGTTTATCCTTACAATGGTG GTACTTATTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATC CTCCAGCACAGCCTACATGGAGCTAAACAGCCTGACTTCTGAGGACTCTGCGGT CTATTACTGTGTACGATTAGGGAACGGTAGTAGCAACGAGTGGTACTTCGATGTC TGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCNN 194 2G02_ NNNNNNNNNNNNNNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCA AATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGGT CACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCTACAATTTGCACTGGTAC CAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTG GCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTC TCACAATCAGCAGTATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTG GAGAACCTACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTAC GGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGC TTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAAN 195 1D09_ NNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGATG HC GTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGGTC CAACTGCAGCAGCCTGGAGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGCT GTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATACACTGGATGAAG CAGAGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGAGCCTAATAGCGG TGATACTAAATACAATGAGAAGATCAAGAGCAGGGCCACACTGACTGTAGACAAA CCGTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCG GTCTATTATTGTGCAAGATCTGGGTATGATTACCCTGAGGCCTGGGGCCAAGGC ACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTG GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT CAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCT CAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCA AATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG GGGGANCGTCAGTCTTCCTCTTCCCCCCAAAACCCAANNA 196 1D09_ NNNNNNNNNNNNNGTNACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG LC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAC ATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCCAGGGCAGAGAGCC ACCATCTCCTGCAGAGCCAGTGAAGGTGTTGAATATTATGGCACAACTTTAATGC AGTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCAT CCAACGTAGAATCTGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACA GACTTCAGCCTCAACATCCATCCTGTGGAGGAGGATGATATTGCAATGTATTTCT GTCAGCAAAGTAGGAAGGTTTCTTGGACGTTCGGTGGAGGCACCAAGCTGGAAA TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAGAAGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGT ATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTT GTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCT GGNTGCTGTCTCTTTATGAGNGTTGTGGGCCCGTTGTCNGGCAACNTGGCGNG GNGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATNNNNACTCNN CTGTCAGCNNCT 197 2B06_ NNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGA HC TGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGG TCCAGCTGCAACAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGT TGTCCTGCAAGGCTTCTGGCTATACCTTCACCAACTACTGGATGCACTGGGTGAA ACACAGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTGG TGGTACTAAGTACAATGAGAAGTTCAAGAATAAGGCCACACTGACTGTAGACAAT CCCTCCAGCACAGGCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCG GTCTATTATTGTACAAGATCTGGGTATGATTACCCTGACTACTGGGGCCAAGGCA CCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGG CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTC AAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACC AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTG CAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAA ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGG GGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT 198 2B06_ NNNNNNNNNNNNNNNNGNNNNNTGNACCTCGGTTCTATCGATTGAATTCCACCA LC TGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTC CGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGA GCCACCATCTCCTGCAGAGCCAGTGAAAGTGTTGAATATTATGGCACAAGTTTAA TGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAAGTCCTCATCTATGCTG CATCCAACGTAGATTCTGGGGTCCCTCCCAGGTTTAGTGGCAGTGGGTCTGGGA CAGACTTCAGCCTCAACTTCCATCCTGTGGAGGAGGATGATATTGCAATGTATTT CTGTCAGCAAAGTAGGAAGGTTCCTTGGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCA GAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGTTAGAAGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG GTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCT TTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCC TGGTTGCTGTCTCTTTATGANGAGTTGTGGCCCGTTGTCAGGNACGTGGCGTGG TGTGCACTGTGNTTGCTGACNCAACCCCCCACTGGTTGGGGCATTGCCACCNAC CTGN 199 2A02_ NNNNNNNNCCNNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGA HC TGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGG TCCAACTGCAACAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGC TGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGA AGCAGAGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTG GTGGTACTAAGTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACA AACCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTG CGGTCTATTATTGTGCAAGATCTGGGTATGATTACCCTGACTACTGGGGCCAAGG CACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCT GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCA AATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGANGTGAGCCACGAAGANCCT GAGGTN 200 2A02_ NNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGAT LC GGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGACATT GTGCTGACCCAATCTCCAGCTCCTTTGGCTGTGTCTCTAGGGCAGAGAGCCACC ATCTCCTGCAGAGCCAGTGAAGGTGTTGAATATTATGACACAAGTTTAATGCAGT GGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCA ACGTAGAACCTGGGGTTCCTGCCAGGTTTGGTGGCAGTGGGTCTGGGACAGAC TTCAGCCTCAACATCCATCCTGTGGAGGAGGATGATATTGCAATGTATTTCTGTC AGCAAAGTAGGAAGGTTCCTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCA AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAG GCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA GAAGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTC TTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTAT CATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTT GCTGTCTCTTTATGAGNAGTTGTGGCCCGTTGTCNNNAACGTGGCGTGGTGTGN NACTGTGTTTGCTG 201 1B07_ NNNNNCNNNNNNNNNGTNACTGCACCTCGGTTCTATCGATTGAATTCCACCATG HC GGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCG AGGTCCAACTGCAACAGCCTGGGGCTGAGCTGGTAAAGCCTGGGACTTCAGTGA AGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGT GAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAATGATTCATCCTAATAG TGGTAGTACTAACTACAATGAGATGTTCGAGAGCAAGGCCTCACTGACTGTAGAC AAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCT GCGGTCTATTACTGTGCAAGAATCGGATACTCCGGCTGGGGTCAAGGAACCTCA GTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCC TCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA CTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCG GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACG 202 1B07_ NNNNNNNNNNNNNNGTNACTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA CATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGT CACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAATGGTGGAAATCAAAAGAAC TACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAAGTGTTGATCAAC GGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAATGGATCT GGAACCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTT TATTACTGTCAGAATGATCATACTTATCCGCTCACGTTCGGTGCTGGGACCAAGC TGGAGCTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTG ATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGG GGAGAGTGTTAGAAGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGAT TGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTA ATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTA T 203 2D04_ NNNNNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATG HC GGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCG AGGTCCAACTGCAGCAGCCTGGAGGTGAGCTGGTGAGGCCTGGGTCCTCAGTG AAGATGTCCTGCAAGACTTCTGGATATACATTCACATTCTACGGTATAAACTGGG TGAAGCAGAGGCCTGGACAGGGCCTGGAATGGATTGGATATATTTATGTTGGAA ATGGTTATACTGAGTACAATGAGAAGTTCAAGGTCAAGGCCACACTGACTTCAGA CACATCCTCCAGCACAGCCTACATGCAGCTCAGCGGCCTGACATCTGAGGACTC TGCAATCTATTTCTGTGCAAGATCCGATGGTTACAACTACTTCGACTACTGGGGC CAAGGCACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGC CCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCT ACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTG AGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAC TCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA TGATCTC 204 2D04_ NNNNNNNNNNNNNNNNNNNCACTGCACCTCGGTTCTATCGATTGAATTCCACCA LC TGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTC CCAAATTGTTCTCACCCAGTCTCCAGCAATCCTGACTGCATCCCCAGGGGAGAA AGTCACAATGACTTGCAGGGCCAGTTCAAGTGTACATTACATGCACTGGTACCAG CAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCT TCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTC ACAATCAGCAGAGTGGAGGCTGAAGATACTGCCACTTATTACTGCCATCAGTGG AGTAGTAACCCACCCACGTTCGGTGCTGGGACAAAGTTGGAAATAAAACGTACG GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC AGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTG ATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTAT GTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTAT TGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGCTGTCTC TTTATGAGGAGTTGTGGGCCCGTTGTCNNNAACGTGGCGTGGTGTGCACTGTGT TTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTT CCGGGACTTTCGC 205 1E07_ NNNNNNNNNNNGNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGAT HC GGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGGT CCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGT TGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGTGA AGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTAATAGTG GTAGTACTTACTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAA ATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGC GGTCTATTACTGTGCAAGATGGGATGATTCCTACTGGTACTTCAAAGTCTGGGGC ACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTT CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAAN 206 1E07_ NNNNNNNNNNNNNNGTCACTGCNCCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCA AATTGTTCTCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTC ACAATGACTTGCAGGGCCAGTTCAAGTGTAAGTTACATGCACTGGTACCAGCAG AAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTG GAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAA TCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTA GCAACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTG GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCA AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTGATC CTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGC TTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGCTGTCTCTTT ATGAGGAGTTGTGGCCCGTTGTCNNNAACGTGGGCGTGGNGTGCACTGTGTTTG CTGA 207 2D07_ NNNNNNNNNNNNNNGTNACTGCACCTCGGTTCTATCGATTGAATTCCACCATGG HC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA GGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAGGCCTGGGGCTTCAGTGA AGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACTACTATATACACTGGCT GAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATTGATTTTTCCTGGAAG TGGTAGTATTTACTGTAATGAGAAGTTCAAGGGCAAGGCTACACTTACTGTAGAC AAATCCTCCACCACAGCCTACATGTTGCTCAGCAGCCTGACCTCTGAGGACTCT GCGGTCTACTTCTGTGCAAGATGGGAGACTACGGCGTGGTACTTCGATGTCTGG GGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGG GCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAG AGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACC TGAACTCCTGGGGGGANCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC CCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA 208 2D07_ NNNNNNNNNNNNNNNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG LC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCAAA TTGTTCTCACCCAGTCTCCCCAAATTGTTCTCACCCAGTCTCCAGCAATCCTGTC TGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGTTTAAGTGTAAG TTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTAT GCCACATCCAACCTGGCTTCTGGAGTCCCTGCCCGCTTCAGTGGCAGTGGGTCT GGGACCTCTTACTCTCTCACAGTCAGCAGAGTGGAGGCCGAAGATGCTGCCACT TATTACTGCCAGCAGTGGAGGAGTTACCCGTACACGTTCGGAGGGGGCACCAAG CTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTG ATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGG GGAGAGTGTTAGAAGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGAT TGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTA ATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTA TAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGGCCCGTTGTCAGNAACGT GGNGTGGNGTGCACTGTGTTTGCTGACGCAA 209 1A02_ NNNNNNNNNNNNNGTNACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG HC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAG GTCCAACTGCAACAGTCTGGACCTGTGCTGGTGAAGCCTGGGCCTTCAATGAAG ATATCCTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATACACTGGGTGA GGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACTTGTTTCTCCTTACAATG GTGGTACTTACTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACTC ATCCTCCAGCACAGCCTACATGGAGCTAAGCAGCCTGACTTCTGAGGACTCTGC GGTCTATTACTGTGCAAGATTGGGCTACTATGGTGACTGGTACTACTTTGACTAC TGGGGCCAAGGCACCCCTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGNNTCACATGCGTGGTGGTGGACGTGAGC CACGAAGANCCTGAGNNNAGTTCAACTGGTACG 210 1A02_ NNNNNNNNNNNNNNNNTNAACTGCACCTCGGTTCTATCGATTGAATTCCACCAT LC GGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCC CAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTACATCTCCAGGGGAAAAGG TCACCATCACCTGTAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGAACTGGTA CCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCT GGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCT CTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGT GGAGTAGTTACCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTA CGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGC TTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGANTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGT CTCTTTATGAAGGAGTTGTGGCCCGTTGTCAGGNAACGTGGGCGNGGTNNGCAC TGN 211 2D10_ NNNNNNNNNNNNNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGAT HC GGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGGT CCAGCTGCAACAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGT TGTCCTGCAAGGCTTCTGGCTACACTTTCACCACCTACTGGATGCACTGGATGAA GCAGTGGCCTGGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTAATAGTGG TAGTACTTACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAA TCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCG GTCTATTACTGTGCAAGATTTGAGGAGCTATGGGGAGGTTACTGGTACTTCGATG TCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCA TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAA CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT CANGACTCTACTCCCTCAGCANCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCA CCCAGACCTACATCTGCNACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGANAGTTGAGCCCAAATCTTGTGACAANNCTCACACATGCCCACCGTGCCCAN CACCTGAACTCCTGGGGGGACCGNCAGTCTTCCTCTTCCCCCCAAAACCCANGA CNNCCNN 212 2D10_ NNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGA LC TGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCAAAT TGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGGTCAC CATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCACTGGTACCAG CAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTGGCTT CTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCAC AATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAG TAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGT GGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAG AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTGAT CCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTG CTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGCTGTCTCTT TATGAGGAGTTGTGGCCCGTTGTCAGGNAACGTGGNGTG 213 2E08_ NNNNNNNNNNNNCNGGTNACTGCACCTCGGTTCTATCGATTGAATTCCACCATG HC GGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCG AGGTCCAGCTGCAGCAGCCTGGGGCTGAGCTGGCAAAGCCTGGGGCTTCAGTG AAGTTGTCCTGCAAGGCTTCTGGCTACAATTTCACCAGCTACTGGATGCACTGGG TGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTAATA GTGGTGGTACTTACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTTTAGA CAGATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTC TGCGGTCTATTATTGTGCAAGATTTGAGGAGGAATGGGGAGGGTATTGGTACTTC GATGTCTGGGGCACGGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGG CCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAG CGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG GACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC GTGAGCCACGAAGANCCTGAGGNCAAGTTCAACTGGTACGNNN 214 2E08_ NNNNNNNNNNNNNNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGA LC TGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCAAAT TGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAAAAGGTCAC CATCACCTGCAGTGTCAGTTCAAGTATAAGTTCCAGCAACTTGCACTGGTACCAA CAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACTTCCAACCTGGCTT CTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCAC AATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAG TAGTTACCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAACGTACGGT GGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAG AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTGAT CCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTG CTTCCCGTATGGCTTTCATTTTCTCCTCNTT 215 2H04_ NNNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG HC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAG GTCCAACTGCAACAGTCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAG TTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGTGA AGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGACTGATTCATCCTAATAGTA GTAGTACTTACTACAATGAGAAGTTCAAGACCAGGGCCACACTGACTGTAGACAA GTCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGC GGTCTATTACTGTGCAAGATTGGGCTATGGTAACTCCTACTGGTACTTCGATGTC TGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGNC ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG CCNNN 216 2H04_ NNNNNNNNNNNCNGGTNNACTGCACCTCGGTTCTATCGATTGAATTCCACCATG LC GGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCC AAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGG TCACCATCACCTGCAGTGTCAGCTCAAGTATTAGTTCCAGCAACTTGCACTGGTA CCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCT GGCTTCTGGAGTCCCTATTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCT CTCACAATCAGCAGTTTGGAGGCTGAGGATGCTGCCACTTATTACTGTCAACAGT GGAGAAGTTACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTA CGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGC TTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGCTGT CTCTTTATGAGGAGTTGTGGGCCCGTTGTCANGNACGNNGNNGTGGTGTGCACT GTGTTTGCTGACGCANNNCCNNNNNGGN 217 1B03_ NNNNNNNNNNCNGGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG HC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAG GTCCAACTGCAGCAGTCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAA GTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGT GCAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTATTGG TGGTGGTACTCACTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGAC AAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCT GCGGTCTATTACTGTGCAAGACTCGGAACTGGTCCGTACTACTTTGACTACTGGG GCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCT TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC TGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGC GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGAC CTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCT CATGATCTCCCGGACCCCTGANTCACATGCGTGGTGGTGGACGTGAGCCACGAA GANCCTGA 218 1B03_ NNNNNNNNNNNNNNNNNNNNCTGCACCTCGGTTCTATCGATTGAATTCCACCAT LC GGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCC CAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAG GTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCACCTTGGACTGGT ACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGGTTTATGGCACATCCAACC TGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTC TCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAG TGGAGTAGTTACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGT ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAG CTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAA CTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATG CTATTGCTTCCCGTATGGNTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGCTG TCTCTTTNATGAGGAGTTGTGGCCCGTNGNCAGGNAACGNGGCGTGGTGTGCA CTGNNTTTGCT 219 2H07_ NNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGA HC TGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGG TCCAGCTGCAGCAGTCTGGACCTGTGCTGGTGAAGCCTGGGCCTTCAGTGAATA TATCCTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATTCACTGGGTGAAA CAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACTTGTTTATCCTTACAGTGGTG GTACTTACTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACGCATC CTCCAGCACAGCCTACATGGAGCTAGGCAGCCTGACTTCTGAGGACTCTGCGGT CTATTACTGTGCAAGATTGGGCGACAACTTCTACTACTTTGACTACTGGGGCCAA GGCACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCT GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGC CCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCN 220 2H07_ NNNNNNNNNNNNNNNNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCA AATTGTTCTCACCCAGTCTCCAACACTCATGGCTGCATCTCCAGGGGAGAAGGT CACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCACTGGTAC CAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTG GCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACATCTTATTCTC TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTG GAGAAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTAC GGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGC TTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNNGCTGT CTCTTTATGAGGAGTTGTGGGCCCGTTGTCAGGNAACGTGGCGTGGNGTGNN 221 1C01_ NNNNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGG HC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA GGTCCAACTGCAACAGCCTGGACCTGTGCTGGTGAAGCCTGGGCCTTCAGTGAA GATATCCTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATGCACTGGGTG AAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGTCTTGTTTCTCCTTACAATG GTGGTACTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAC ATCCTCCAGCACAGCCTACATGGAACTAAACAGCCTGACTTCTGAGGACTCTGC GGTCTATTACTGTGCAAGAGTGGGTAATAGCTACGTCCATTATGCTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGANGTCACATGCGTGGNN 222 1C01_ NNNNNNNNNNNNNCCNGGTNACTGCACCTCGGTTCTATCGATTGAATTCCACCA LC TGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTC CCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAA GGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCACTGG TACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACC TGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTC TCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAG TGGAGTAATTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGT ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAG CTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAA CTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATG CTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGNATAAATCCTGGNTGCTG TCTCTTTATGANGAGTTGTGGNCCCGTTGTCNGNNAACGTGGCGTGGTGTGCAC TGTGNTTGCTGN 223 2C07_ NNNNNNNNNNNCNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG HC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAA GTGAAGCTGGTGGAGTCTGGGGAAGGCTTAGTGAAGCCTGGAGGGTCCCTGAA ACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTT CGCCAGACTCCAGAGAAGAGGCTGGACTGGGTCGCATACATTAGTAGTGGTGGT GATCACATCTACTATGCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACA ATGCCAGGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAG CCATGTATTACTGTACAAGAGATACCGGTTACTACGTCTCTCGGTACTTCGATGT CTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCAT CGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAAC TCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGANCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCA 224 2C07_ NNNNNNNNNNNNNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG LC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAC ATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCA GCGTCACCTGCAAGGCCAGTCAGAATGTGGTTACTAATGTTGCCTGGTATCAACA GAAATCAGGGCAATCTCCTAAAGTAGTGATTTATTCGGCATCCTTCCGGTCCAGT GGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACC ATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCACCAATATAACA GCCATCCTCTGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGG CTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAC TGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAG CAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGCTTGATCC TCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTG CTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCT TCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGCTGNCTCTTNA NTNNNNGANNTGTGGCCCGTTGTCAGGNAAC 225 2G04_ NNNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG HC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAG GTCCAGCTGCAGCAGCCTGGACCTGAATTGGTGAAGCCCGGGTCCTCACTGAA GATATCCTGCAAGGCTTCTGGTTACACCTTCACTGACTACTTTATAAACTGGGTG AAACAGAGGCCTGGACAGGGACTTGACTGGATTGGATGGATTTTTCCTGGAAGT GGTAGTACTTACTACAATGACAAGTTCAAGGGCAAGGCCACACTTACTGTAGACA AATCCTCCAGCACTGCCTACATGTTGCTCAGCAGCCTGACCTCTGAGGCCTCTG CGGTCTATTTCTGTGCAAGATGGGACTCCGATAGTACCTACTGGTACTTCGATGT CTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCAT CGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAAC TCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCAC 226 2G04_ NNNNNNNNNNNNCNNNNNNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCA LC TGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTC CCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAA GGTCACCATCACCTGCAGTGTCAATTCAAGTATAAGTTCCAGCACCTTGCACTGG TACCAGCAGAAGTCACAAACCTCCCCCAAACCCTGGATTTATGGTACATCCAATT TGGCTTCTGGAGTCCCTATTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTC TCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAG TGGAGTACTTACCCGTACACGTTCGGAGGTGGCACCAAGCTGGAAATCAAACGT ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAG CTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAA CTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATG CTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTG TCTCTTTATGAGGAGTTGTGGGCCCGTTGTCAGGCAACGTGGNGNGGNGTGCAC TGTGTTTGCTGACGCAACCCCNCTGGTNGGGGCATTGCCACCACCTGTCAGCTC CTTTCCGGGACTTTCGNTTNNNNNN 227 1G01_ NNNNNNNNNNNNNNGGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATG HC GGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCG AGGTCCAACTGCAACAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGA AGCTTTCCTGCAAGGCTACTGGCTACACATTCACTGGCTACTGGATAGAGTGGTT AAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAG TGATAATACTAACTACAATGAGAAGTTCAGGGGCAAGGCCACATTCACTGCAGAT ACATCCTCCAACACAGCCTACATGCACCTCAGCAGCCTGACAACTGAGGACTCT GCCATCTATTACTGTGCAAGAGAAGGGGGTTTCTACTTTGACTACTGGGGCCAA GGCACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCT GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGC CCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT GGGGGGANCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGANCN 228 1G01_ NNNNNNNNNNNNNNNNNNNNNNNCCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCA GGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCAC ACTCACTTGTCGCTCAAGTATTGGGGCTGTTACAACTAGTAACTACGCCAACTGG GTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACC GAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTG CCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTC TATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAG GCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCGCCCTCGAGTGAGGAGC TTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAG CCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAG ACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTG AGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCAC GCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAGAA GCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTA ACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCAT GCTATTGCTNCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCT GTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGNAACGTGGCGTGGNGTGCAC TGNGTTTGCTGACGCAACCCCCACTGGTNGGGGCATTGCNNCCACCTGTCANCT NCTTTCCGGGACTTTCGCTTN 229 2A05_ NNNNNNNNNNNNNCCNNGNNNACTGCACCTCGGTTCTATCGATTGAATTCCACC HC ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATT CCGAGGTCCAGCTGCAACAGTCTGGACCTGTGCTGGTGAAGCCTGGGCCTTCA GTGAAGATATCCTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATGCACT GGGTGAAGCAGAGCCATGGAAAGAGCCTTGAATGGATTGGACTTGTTTATCCTTA CAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGT AGACACATCCTCCACCACAGCCTACATGGAGCTAAACAGCCTGACTTCCGAGGA CTCTGCGGTCTATTACTGTGCAAGATTGGGCTCAGGCTACGAGTACTACTTTGAC TACTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCA TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAA CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGANCCCTGAGGTCACATGCGTGGTG 230 2A05_ NNNNNNNNNNNNNNGTNACTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCA AATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGGT CACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCACTGGTAC CAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTG GCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTC TCACAATCAGCAGCATGGAGGCTGAGGATGCTGCCACTTATTACTGTCAACAGT GGAGAACTTACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTA CGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGC TTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGNTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGN CTCTTTATNNNGNAGTTGNNGGCCCGTNNNNNNNANCGTNNN 231 2H05_ NNNNNNNNNNNNNGNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG HC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAG GTCCAACTGCAGCAGCCTGGACCTGTGCTGGTGAAGCCTGGGCCTTCAGTGAA GATATCCTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATGCACTGGGTG AAGCAGAGCCATGGAGAGAGCCTTGAGTGGATTGGACTTGTTTCTCCTTACAATG GTGGTACTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAC ATCCTCCAGCACAGCCTACATGGAACTAAACAGCCTGACTTCTGAGGACTCTGC GGTCTATTACTGTGCAAGAGTGGGTAGTAGTTACGTCCATTATGCTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGANNG AGAGTTGAGCCCAAATCTTGTGACAAACTCACACATGCCCACCGTGCCCAGCAC CTGAACTCCTGGGGGGANCGTCNGTCTTCCTCTTCCCCCAAAACCCNANGANAC CCTCATGATCTCCCGGACCCCTGAGGTCNCNTGCGTGGTGGTGGANGTGANNC CNNNNAANNNCNNTNAN 232 2H05_ NNNNNNNNNNNCNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCA AATTGTTCTCACCCAGTCTCCAGCACTCACGGCTGCATCTCCAGGGGAGAGGGT CACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCACTGGTAC CAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTG GCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTC TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTG GAGTAAGTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTAC GGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGC TTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGT CTCTTTATGA 233 2B01_ NNNNNNNNNNNNNNGNCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGG HC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA GGTCCAACTGCAACAGTCTGGGGCTGGGCTGGTAAAGCCTGGGGCTTCAGTGA AGTTGCCTTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGATGCACTGGGT GCAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGATTGATTCATCCTATTGG TGGTGGTACTCACTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTGGA CAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTC TGCGGTCTATTACTGTGCAAGACTCGGAACTGGTCCGTACTACTTTGACTACTGG GGCCAAGGCACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGG GCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAG AGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACC TGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC CCTCATGATCTCCCGGAN 234 2B01_ NNNNNNNNNNNNNNNNGNNNACTGCACCTCGGTTCTATCGATTGAATTCCACCA LC TGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTC CCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAA GGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCACTGG TACCAGCAGAAGTCAGAGACCTCCCCCAAACCCTGGATTTATGGCACATCCAAC CTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATT CTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACA GTGGAGTAGTTACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACG TACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT CAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAA GCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTA ACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGNCTTTGTATCAT GCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGNNNNNATCCTGGNTGC TGTCTCTTTATGAGGAGTTGTGGGCCCGTTGTCAGGCAACNNNGCGTGGNGTGC ACTGTGTTTGCTGACGCAACCCCCNACTGGNNNNNNNATTGCCACCNNNCNGTC AGCNNCTTTNCGGGACTTTCGCTTTNCCCNTNCNTATTNNNNNNNN 235 1D01_ NNNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGG HC ATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAG GTCCAGCTGCAGCAGCCTGGACCTGACGTGGTGAAGCCTGGGGCTTCAGTGAA GATTTCCTGCAAGGCTTCTGGCTACACCTTCACTGACTACTATATAAACTGGGTG AAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAAGT GGTAGTAGTTATTACAATGAGAAGTTCAAGGACAAGGCCACATCTACTGTAGACA AATCCTCCAGCACAGCCTACATGTTGCTCAGCAGCCTGACCTCTGAGGACTCTG CGGTCTATTTCTGTGCAAAATGGAAGGATTACGGGTGGTACTTCGATGTCTGGG GCACAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTC TTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG CTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGG CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAG TTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC TCATGATCTCCCGGANCCCTGNNTCACATGCGTGGTGGTGGACGTGAGCCACGA AGACCTNA 236 1D01_ NNNNNNNNNNNNNNNNNGNNNNCTGNACCTCGGTTCTATCGATTGAATTCCACC LC ATGGGATGGTCATGTATCATCCTNNTTTCTAGTAGCAACTGCAACCGGTGTACAT TCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAG AAGGTCACCCTCACCTGCAATGTCAGCTCAAGTCTAAGTTCCAGCAACTTGCACT GGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCA ACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTT ATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCA ACAGTGGAGGACTTACCCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGA GTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG AAGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGANTGACTGGTATTCT TAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATC ATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAATCCTGGTTGC TGTCTCTTTATGAGGAGTTGTGGNCCGTTGTCAGGCAACGTGGCGTGGTGTGCA CTGTGTTTGCNNGACNNCACCCCCNNCTGGTTGGGGNATTGCCACCNNCCTGTC AGCTNNTTTCCGGGANNNTN 237 2E02_ NNNNNNNNNNNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCATGGGA HC TGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGG TCCAACTGCAGCAGTCTGGACCTGTGCTGGTGAAGCCTGGGCCTTCAGTGAAGA TATCCTGTAAGGCTTCTGGATTCACATTCACTGACTACTACATTCACTGGGTGAA GCGGAGCCATGGAAAGAGCCTTGAGTGGATTGGACTTGTTTCTCCTTACAATGG TGGTACTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGATACA TCCTCCAATACAGCCTACATGGAGCTAACCAGCCTGACTTCTGAGGACTCTGCG GTCTATTACTGTGCAAGATTGGGGGTTAACTGGTACTTCGATGTCTGGGGCACA GGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCC CCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTAC ATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAG CCCAAATCTTGTGACAAAACTCACNNNTGCCCACCGTGCCCAGCACCTGAACTC CTGGGGGGANCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG ATCTC 238 2E02_ NNNNNNNNNNNNNNCNNGTCACTGCACCTCGGTTCTATCGATTGAATTCCACCA LC TGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTC CCAAATTGTTCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAAAA GGTCACCCTCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCACCTTGCACTG GTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAA CCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTA TTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAA CAGTGGAGTACTTACCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGC CAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCC ATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGA AGCTTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTT AACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCA TGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGNTGC TGTCTCTTTATGAAGGAGTTGTGGGCCCGTTGTCNNNAACGTGGGCGTGGTGTG 239 1A05_ NNNNNNNNNNNNNNGNNNNTGCACCTCGGTTCTATCGATTGAATTCCACCATGG HC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGA GGTCCAACTGCAACAGCCTGGACCTGTACTGGTGAAGCCTGGGCCTTCAATGAA GATATCCTGTAAGGCTTCTGGGTACGCTTTCACTGACTACTTCATACACTGGGTG AGACAGAGCCATGGAAGGAGCCTTGAGTGGATTGGACTTGTTTCTCCTTATAATG GTGGTACTTACTACAACCAGAAGTTCAAGGGCAAGGCCTCATTGACTGTAGACAC ATCCTCCAGCACAGCCTACATGGAACTAAGCAGCCTGACTTCTGAGGACTCTGC GGTCTATTACTGTGCAAGATTGGGCTACTATGGTAACTGGTACTTCTTTGACTACT GGGGCCAAGGCACCCCTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATCG GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCT GGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTC AGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAG GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACC CAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAG AGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCA CCTGAACTCCTGGGGGGANCGTCAGTCTTCCTCTTCCCCCAAAACCCAAGGACA CCCTCATGATCTCCCGGACCCCTGAGTCACATGCGTGGTGGTGGACGTGAGCCA CGANNA 240 1A05_ NNNNNNNNNNNNNNNNNNCTGCACCTCGGTTCTATCGATTGAATTCCACCATGG LC GATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCA AATTGTTCTCACCCAGTCTCCAGCACTCATGGCTACATCTCCAGGGGAAAAGGTC ACCATCACCTGTAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGAACTGGTACC AGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTGG CTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCT CACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTG GAGTAGTTACCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTAC GGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAAGC TTGATCCTCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCNTGTATAAATCCTGNNGCTGNC TCTTTATGAGGANTTGNGGCCCGTTGTCAGGNAACGTGGCGNGGTGTGCACTGT GNTTGCNNGACNNNNNNCN

[0104] In various other aspects, the monoclonal antibody to FimH comprises a heavy chain protein variable region and a light chain protein variable region configured to bind to and deactivate the binding of the FimH lectin domain protein of E. coli to exposed mannose residues on the surface of epithelial cells. SEQ ID Nos of the amino acid and nucleotide sequences of heavy and light chains of the E. coli-derived monoclonal antibody sequences re summarized in Table 5 below.

[0105] In some aspects, the heavy chain protein variable region comprises the amino acid sequence selected from SEQ ID NOS: 241, 243, 245, 247, 249, 251, 253, 255, and 257 and the light chain protein variable region comprises the amino acid sequence selected from SEQ ID NOS: 242, 244, 246, 248, 250, 252, 254, 256, and 258 as listed in Table 5 below. In other aspects, the heavy chain protein variable region is encoded by a nucleotide sequence selected from SEQ ID NOS: 259, 261, 263, 265, 267, 269, 271, 273, and 275, and the light chain protein variable region is encoded by a nucleotide sequence selected from SEQ ID NOS: 260, 262, 264, 266, 268, 270, 272, 274, and 276, shown listed in Table 5 below.

TABLE-US-00005 TABLE5 E.coli-DerivedAntibodySequences E.coli SEQID Sequence NO Label Sequence 241 A2HC TGVHSEVQLQQSGAEQVRPGASVKMSCKASGYTFTTYNMNWMKQT PGQGLEWIGVIYPGNGDISYNQKFKGKATLTVDKSSSTAYMQLSSLTSE DSAVYFCAREGDYGPWFAYWGQGTLVTVSAAS(SEQ_ID_NO:241) 242 A2LC TGVHSQIVLTQSPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSG ASPKFWIYSTSKLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQH YSRYPLTFGGGTKLEIKRT(SEQ_ID_NO:242) 243 A11HC TGVHSEVQLQQSGPVLVKPGPSVKISCKASGFTFTDYYIHWVKQSHGK SPEWIGLVSPYNGGTYYNQKFMGRATLTEDTSSNTAYMELNSLTSEDS AVYYCARLLRGYWYFDVWGTGTTVTVSSAS(SEQ_ID_NO:243) 244 A11LC TGVHSQIVLTQSPALMAASPGEMVTITCSVSSSISSSNLHWYQQKSET SPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQ WRTYPYTFGGGTKLEIKRT(SEQ_ID_NO:244) 245 B7HC TGVHSEVQLQQPGAELVRPGASVKMSCKASGYTFTIYNLHWVKQTPR QGLEWIGTIYPGDGDTSYNQKFKGKATLTVDKSSSTAYMQLSNLTSED SAVYFCAREGDYGPWFAYWGQGTLVTVSAAS(SEQ_ID_NO:245) 246 B7LC TGVHSQIVLTQSPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSG ASPKLWIYSSSNLASGVPARFSGSGSGTSYSLTINSVEAEDAATYYCQH YGSYPLTFGGGTKLEIKRT(SEQ_ID_NO:246) 247 C7HC TGVHSEVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRP GKGLKWIGRIYPGDGDTNYNGKFKGKATLTADKSSXTAYMQLSSLTSE DSAVYFCAREGNLXFDYWGQGTTLTVSSAS(SEQ_ID_NO:247) 248 C7LC TGVHSDIVLTQSPASLDVSLGQRATISCRASQSVSTSTYSYMHWYQQK PGQSPKVLIKYASNLEAGVPARVSGSGSGTDFTLNIHPVEEEDTATYYC QHSWEIPPTFGGGTKLEIKRT(SEQ_ID_NO:248) 249 C12HC TGVHSQVQLKQSGPGLVAPSQSLSITCTVSGFSLTNYAISWVRQPPGK GLEWLGIIWTGGGTLYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTA RYYCARAYYSNYDWYFDVWDTGTTVTVSSAS(SEQ_ID_NO:249) 250 C12LC TGVHSDIQMTQSPSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGT VKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYS QVPYTFGGGTKLEIKRT(SEQ_ID_NO:250) 251 E7HC TGVHSEVQLQQSGAELVKPGASVKVSCKASGYSFTNYWVHWVKQRP GQGLEWIGRIYSSDGDSNYNQKFKGKATLTVDKSSSTAYMQLSSLTSE DSAVYYCAIEGNLYFDYWGQGTTLTVSSAS(SEQ_ID_NO:251) 252 E7LC TGVHSDIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQK PGQPPKFLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYC QHSWEIPLTFGAGTKLKLKRT(SEQ_ID_NO:252) 253 F7HC TGVHSEVQLQQPGPVLVKPGPSVKISCKASGFTFTDYFIHWVKQSHGK SLEWIGLVSPYNGGTYYNQQFKGKATLTVDTSSSTAYMDLNSLTSEDS AVYYCTRLLRGYWYFDVWGTGTTVTVSSAS(SEQ_ID_NO:253) 254 F7LC TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSIGSSNLHWYQQKSETS PKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQ WSTYPYTFGGGTKLEIKRT(SEQ_ID_NO:254) 255 F11HC TGVHSQVQLKESGPGLVAPSQSLSITCTVSGFSLTNYAISWVRQPPGK GLEWLGIIWTGGGTLYNSALKSRPSISKDNSKSQVFLKMNSLQTDDTA RYYCARAYYSNYDWYFDVWDTGTTVTVSSAS(SEQ_ID_NO:255) 256 F11LC TGVHSDIQMTQSPSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGT VKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYS QVPYTFGGGTKLEIKRT(SEQ_ID_NO:256) 257 H9HC TGVHSEVQLQQSGPVLVKPGPSVKISCKASGFTFTDYFVHWVKQSHG KSLEWIGLVSPYNGGTYYNQQFKGKATLTVDTSSSTAYMDLNSLTSED SAVYYCARLLRGYWYFDVWGTGTTVTVSSAS(SEQ_ID_NO:257) 258 H9LC TGVHSQIVLTQSPALMAASPGEKVTITCSVSSSISSSTLHWYQQKSETS PKPWIYGTSNLASGVPVRFGGSGSGTSYSLTISSMEAEDAATYYCQQ WSTYPYTFGGGTKLEIKRT(SEQ_ID_NO:258) 259 A2HC ACCGGTGTACATTCCGAGGTCCAACTGCAACAGTCTGGGGCTGAG CAGGTGAGGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCT GGCTACACATTTACCACTTACAATATGAACTGGATGAAGCAGACACC TGGACAGGGCCTGGAATGGATTGGAGTTATTTATCCAGGAAATGGT GATATTTCCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGT AGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACA TCTGAAGACTCTGCGGTCTATTTCTGTGCAAGAGAGGGAGATTATG GGCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCT CTGCAGCGTCGAC(SEQ_ID_NO:259) 260 A2LC ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCAATCAT GTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAG CTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCA GGTGCCTCCCCCAAATTCTGGATTTATAGTACATCCAAGTTGGCTTC TGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTA CTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATT ACTGCCAGCACTACAGTCGTTACCCACTCACGTTCGGAGGGGGGA CCAAGCTGGAAATAAAACGTACG(SEQ_ID_NO:260) 261 A11HC ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGTCTGGACCTGTGC TGGTGAAGCCTGGGCCTTCAGTGAAGATATCCTGTAAGGCTTCTGG ATTCACATTCACTGACTACTACATACACTGGGTGAAGCAGAGCCATG GAAAGAGCCCTGAGTGGATTGGACTTGTTTCTCCTTACAATGGTGG TACTTACTACAACCAGAAGTTCATGGGCAGGGCCACATTGACTGAA GACACATCCTCCAACACAGCCTACATGGAGCTAAACAGCCTGACTT CTGAGGACTCTGCGGTCTATTACTGTGCGAGGTTACTACGGGGCTA CTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTC CTCAGCGTCGAC(SEQ_ID_NO:261) 262 A11LC ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCAT GGCTGCATCTCCAGGGGAGATGGTCACCATCACCTGCAGTGTCAG CTCAAGTATAAGTTCCAGCAACTTGCACTGGTACCAGCAGAAGTCA GAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTGGCTT CTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTA TTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATT ACTGTCAACAGTGGCGTACTTACCCGTACACGTTCGGAGGGGGGA CCAAGCTGGAAATAAAACGTACG(SEQ_ID_NO:262) 263 B7HC ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGCCTGGGGCTGAG CTGGTGAGGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCT GGCTACACATTTACCATTTACAATTTGCACTGGGTAAAGCAGACACC TAGACAGGGCCTGGAATGGATTGGAACTATTTATCCAGGAGATGGTG ATACTTCCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGTA GACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAACCTGACAT CTGAAGACTCTGCGGTCTATTTCTGTGCAAGAGAGGGAGATTATGG GCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT GCAGCGTCGAC(SEQ_ID_NO:263) 264 B7LC ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCAATCAT GTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAG CTCAAGTGTAAGTTCCAGTTATTTGCACTGGTACCAGCAGAAGTCAG GTGCCTCCCCCAAACTCTGGATCTATAGCTCATCCAACTTGGCTTCT GGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTAC TCTCTCACAATCAACAGTGTGGAGGCTGAAGATGCAGCCACTTATTA CTGCCAGCACTACGGTAGTTACCCACTCACGTTCGGAGGGGGGAC CAAGCTGGAAATAAAACGTACG(SEQ_ID_NO:264) 265 C7HC ACCGGTGTACATTCCGAGGTCCAACTGCAACAGTCTGGACCTGAGC TGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAGGCTTCTG GCTACGCATTCAGTAGCTCCTGGATGAACTGGGTGAAGCAGAGGCC TGGAAAGGGTCTTAAGTGGATTGGACGGATTTATCCTGGAGATGGA GATACTAATTACAATGGGAAGTTCAAGGGCAAGGCCACACTGACTG CAGACAAATCCTCCNGCACAGCCTACATGCAACTCAGCAGCCTGAC ATCTGAGGACTCTGCGGTCTACTTCTGTGCAAGAGAGGGTAACTTG NATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG CGTCGAC(SEQ_ID_NO:265) 266 C7LC ACCGGTGTACATTCCGACATTGTGCTGACCCAATCTCCTGCTTCCTT AGATGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAG CCAAAGTGTCAGTACATCTACCTATAGTTATATGCACTGGTACCAACA GAAACCAGGACAGTCACCCAAAGTCCTCATCAAGTATGCATCCAAC CTAGAAGCTGGGGTCCCTGCCAGGGTCAGTGGCAGTGGGTCTGGG ACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTG CAACATATTACTGTCAGCACAGTTGGGAGATTCCTCCGACGTTCGGT GGAGGCACCAAGCTGGAAATCAAACGTACG(SEQ_ID_NO:266) 267 C12HC ACCGGTGTACATTCCCAGGTGCAGCTGAAGCAGTCAGGACCTGGC CTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCTG GGTTCTCATTAACCAACTATGCTATAAGCTGGGTTCGCCAGCCACCA GGAAAGGGTCTGGAGTGGCTTGGAATAATATGGACTGGTGGAGGCA CACTTTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAAGAC AACTCCAAGAGTCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGA TGACACAGCCAGGTACTACTGTGCCAGAGCCTACTATAGTAACTACG ACTGGTACTTCGATGTCTGGGACACAGGGACCACGGTCACCGTCTC CTCAGCGTCGAC(SEQ_ID_NO:267) 268 C12LC ACCGGTGTACATTCCGACATCCAGATGACTCAGTCTCCATCCTCCCT GTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGT CAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGG AACTGTTAAACTCCTGATCTATTACACATCAAGTTTACACTCAGGAGT CCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTC ACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCA GCAATATAGTCAGGTTCCGTACACGTTCGGAGGGGGGACCAAGCTG GAAATAAAACGTACG(SEQ_ID_NO:268) 269 E7HC ACCGGTGTACATTCCGAGGTCCAACTGCAGCAGTCTGGGGCTGAA CTGGTGAAGCCTGGGGCTTCAGTGAAGGTGTCCTGCAAGGCTTCT GGCTACTCCTTCACCAACTACTGGGTGCACTGGGTGAAGCAGAGG CCTGGCCAAGGCCTTGAGTGGATTGGAAGGATTTATTCTTCTGATG GTGATTCTAATTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACT GTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGA CATCTGAGGACTCTGCGGTCTATTACTGTGCAATAGAGGGAAACTTG TACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG CGTCGAC(SEQ_ID_NO:269) 270 E7LC ACCGGTGTACATTCCGACATTGTGCTGACCCAATCTCCTGCTTCCTT AGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAG CCAAAGTGTCAGTACATCTAGCTATAGTTATATGCACTGGTACCAACA GAAACCAGGACAGCCGCCCAAATTCCTCATCAAGTATGCATCCAAC CTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGG ACAGACTTCACCCTCAATATCCATCCTGTGGAGGAGGAGGATACTG CAACATATTACTGTCAGCACAGTTGGGAGATTCCTCTCACGTTCGGT GCTGGGACCAAGCTGAAGCTGAAACGTACG(SEQ_ID_NO:270) 271 F7HC ACCGGTGTACATTCCGAGGTCCAACTGCAACAGCCTGGACCTGTGC TGGTGAAGCCTGGGCCTTCAGTGAAGATATCCTGTAAGGCTTCTGG ATTTACATTCACTGACTACTTCATACACTGGGTGAAGCAGAGCCATG GAAAGAGCCTTGAGTGGATTGGACTTGTTTCTCCTTACAATGGTGGT ACTTACTACAACCAGCAGTTCAAGGGCAAGGCCACATTGACTGTAG ACACATCCTCCAGCACAGCCTACATGGACCTAAACAGCCTGACTTCT GAGGACTCTGCGGTCTATTACTGTACGAGGTTACTACGGGGCTACT GGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCT CAGCGTCGAC(SEQ_ID_NO:271) 272 F7LC ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCGCTCAT GGCTGCATCTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAG CTCGAGTATAGGTTCCAGCAACTTGCATTGGTACCAGCAGAAGTCA GAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTGGCTT CTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTA TTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATT ACTGTCAACAGTGGAGTACTTACCCGTACACGTTCGGAGGGGGGA CCAAGCTGGAAATAAAACGTACG(SEQ_ID_NO:272) 273 F11HC ACCGGTGTACATTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGC CTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCTG GGTTCTCATTAACCAACTATGCTATAAGCTGGGTTCGCCAGCCACCA GGAAAGGGTCTGGAGTGGCTTGGAATAATATGGACTGGTGGAGGCA CACTTTATAATTCAGCTCTCAAATCCAGACCGAGCATCAGTAAAGAC AACTCCAAGAGTCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGA TGACACAGCCAGGTACTACTGTGCCAGAGCCTACTATAGTAACTACG ACTGGTACTTCGATGTCTGGGACACAGGGACCACGGTCACCGTCTC CTCAGCGTCGAC(SEQ_ID_NO:273) 274 F11LC ACCGGTGTACATTCCGACATCCAGATGACTCAGTCTCCATCCTCCCT GTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGT CAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGG AACTGTTAAACTCCTGATCTATTACACATCAAGTTTACACTCAGGAGT CCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTOTTA CCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAG CAATATAGTCAGGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAACGTAC(SEQ_ID_NO:274) 275 H9HC ACCGGTGTACATTCCGAGGTCCAGCTGCAACAGTCTGGACCTGTGC TGGTGAAGCCTGGGCCTTCAGTGAAGATATCCTGTAAGGCTTCTGG ATTCACATTCACTGACTACTTCGTACACTGGGTGAAGCAGAGCCATG GAAAGAGCCTTGAGTGGATTGGACTTGTTTCTCCTTACAATGGTGGT ACTTACTACAACCAGCAGTTCAAGGGCAAGGCCACATTGACTGTAG ACACATCCTCCAGCACAGCCTACATGGACCTAAACAGCCTGACTTCT GAGGACTCTGCGGTCTATTACTGTGCGAGGTTACTACGGGGCTACT GGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCT CAGCGTCGAC(SEQ_ID_NO:275) 276 H9LC ACCGGTGTACATTCCCAAATTGTTCTCACCCAGTCTCCAGCGCTCAT GGCTGCATCTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAG CTCGAGTATTAGTTCCAGCACCTTGCACTGGTACCAGCAGAAGTCA GAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTGGCTT CTGGAGTCCCTGTTCGCTTCGGTGGCAGTGGATCTGGGACCTCTTA TTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATT ACTGTCAACAGTGGAGTACTTACCCGTACACGTTCGGAGGGGGGA CCAAGCTGGAAATAAAACGTACG(SEQ_ID_NO:276)

[0106] A FimH modulating agent can be an agent that induces or inhibits progenitor cell differentiation into FimH expressing cells (e.g., by blocking FimH). For example, anti-FimH antibodies can be used to block FimH.

FimH Signal Reduction, Elimination, or Inhibition by Small Molecule Inhibitors, shRNA, siRNA, or ASOs

[0107] As described herein, a FimH modulation agent can be used for use in UTI and other bacterial infection therapy. A FimH modulation agent can be used to reduce/eliminate or enhance/increase FimH signals. For example, a FimH modulation agent can be a small molecule inhibitor of FimH. As another example, a FimH modulation agent can be a short hairpin RNA (shRNA). As another example, a FimH modulation agent can be a short interfering RNA (siRNA).

[0108] As another example, RNA (e.g., long noncoding RNA (lncRNA)) can be targeted with antisense oligonucleotides (ASOs) as a therapeutic. Processes for making ASOs targeted to RNAs are well known; see e.g. Zhou et al. 2016 Methods Mol Biol. 1402:199-213. Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

FimH Inhibiting Agent

[0109] One aspect of the present disclosure provides for targeting of FimH, its receptor, or its downstream signaling. The present disclosure provides methods of treating or preventing UTIs and other bacterial infections based on the discovery that treatment with FimH inhibiting antibodies protects against UTI in vivo.

[0110] As described herein, inhibitors of FimH (e.g., antibodies, fusion proteins, small molecules) can reduce or prevent UTIs and other bacterial infections. A FimH inhibiting agent can be any agent that can inhibit FimH, downregulate FimH, or knockdown FimH.

[0111] As an example, a FimH inhibiting agent can inhibit FimH signaling.

[0112] For example, the FimH inhibiting agent can be an anti-FimH antibody. As an example, the anti-FimH antibody can be any anti-FimH antibody identified by ELISA binding assays that bind with high affinity to antigenic FimH. Furthermore, the anti-FimH antibody can be a murine antibody, a humanized murine antibody, or a human antibody.

[0113] As another example, the FimH inhibiting agent can be an anti-FimH antibody, wherein the anti-FimH antibody prevents binding of FimH to mannose or prevents activation of FimH and downstream signaling.

[0114] As another example, the FimH inhibiting agent can be a fusion protein. For example, the fusion protein can be a decoy receptor for FimH. Furthermore, the fusion protein can comprise a mouse or human Fc antibody domain fused to the ectodomain of FimH.

[0115] As another example, a FimH inhibiting agent can be the anti-FimH antibodies identified in the present disclosure, which has been shown to be a potent and specific inhibitor of FimH signaling.

[0116] As another example, a FimH inhibiting agent can be an inhibitory protein that antagonizes FimH. For example, the FimH inhibiting agent can be a viral protein, which has been shown to antagonize FimH.

[0117] As another example, a FimH inhibiting agent can be a short hairpin RNA (shRNA) or a short interfering RNA (siRNA) targeting FimH or associated biological machinery.

[0118] As another example, a FimH inhibiting agent can be an sgRNA targeting FimH of associated machinery.

[0119] Methods for preparing a FimH inhibiting agent (e.g., an agent capable of inhibiting FimH signaling) can comprise the construction of a protein/Ab scaffold containing the natural FimH receptor as a FimH neutralizing agent; developing inhibitors of the FimH receptor down-stream; or developing inhibitors of the FimH production up-stream.

[0120] Inhibiting FimH can be performed by genetically modifying FimH in a subject or genetically modifying a subject to reduce or prevent expression of the FimH gene, such as through the use of CRISPR-Cas9 or analogous technologies, wherein, such modification reduces or prevents UTIs and other bacterial infections.

Chemical Agent:

[0121] Examples of FimH inhibiting agents are described herein. FimH inhibiting agents can be of a formula that binds to the mannose-binding domain of the FimH adhesin proteins.

[0122] R groups can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C.sub.1-10alkyl hydroxyl; amine; C.sub.1-10carboxylic acid; C.sub.1-10carboxyl; straight chain or branched C.sub.1-10alkyl, optionally containing unsaturation; a C.sub.2-10cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C.sub.1-10alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C.sub.1-10alkyl hydroxyl; amine; C.sub.1-10carboxylic acid; C.sub.1-10carboxyl; straight chain or branched C.sub.1-10alkyl, optionally containing unsaturation; straight chain or branched C.sub.1-10alkyl amine, optionally containing unsaturation; a C.sub.2-10cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C.sub.1-10alkyl amine; heterocyclyl; heterocyclic amine; aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms; and the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C.sub.1-10alkyl hydroxyl; amine; C.sub.1-10carboxylic acid; C.sub.1-10carboxyl; straight chain or branched C.sub.1-10alkyl, optionally containing unsaturation; straight chain or branched C.sub.1-10alkyl amine, optionally containing unsaturation; a C.sub.2-10cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; heterocyclyl; straight chain or branched C.sub.1-10alkyl amine; heterocyclic amine; and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms. Any of the above can be further optionally substituted.

[0123] The term imine or imino, as used herein, unless otherwise indicated, can include a functional group or chemical compound containing a carbon-nitrogen double bond. The expression imino compound, as used herein, unless otherwise indicated, refers to a compound that includes an imine or an imino group as defined herein. The imine or imino group can be optionally substituted.

[0124] The term hydroxyl, as used herein, unless otherwise indicated, can include OH. The hydroxyl can be optionally substituted.

[0125] The terms halogen and halo, as used herein, unless otherwise indicated, include chlorine, chloro, Cl; fluorine, fluoro, F; bromine, bromo, Br; or iodine, iodo, or I.

[0126] The term acetamide, as used herein, is an organic compound with the formula CH.sub.3CONH.sub.Z. The acetamide can be optionally substituted.

[0127] The term aryl, as used herein, unless otherwise indicated, includes a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, or anthracenyl. The aryl can be optionally substituted.

[0128] The terms amine and amino, as used herein, unless otherwise indicated, include a functional group that contains a nitrogen atom with a lone pair of electrons and wherein one or more hydrogen atoms have been replaced by a substituent such as, but not limited to, an alkyl group or an aryl group. The amine or amino group can be optionally substituted.

[0129] The term alkyl, as used herein, unless otherwise indicated, can include saturated monovalent hydrocarbon radicals having straight or branched moieties, such as but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl groups, etc. Representative straight-chain lower alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; while branched lower alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, unsaturated C.sub.1-10 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, or -3-methyl-1 butynyl. An alkyl can be saturated, partially saturated, or unsaturated. The alkyl can be optionally substituted.

[0130] The term carboxyl, as used herein, unless otherwise indicated, can include a functional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group (COOH). The carboxyl can be optionally substituted.

[0131] The term alkenyl, as used herein, unless otherwise indicated, can include alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety. An alkenyl can be partially saturated or unsaturated. The alkenyl can be optionally substituted.

[0132] The term alkynyl, as used herein, unless otherwise indicated, can include alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above. An alkynyl can be partially saturated or unsaturated. The alkynyl can be optionally substituted.

[0133] The term acyl, as used herein, unless otherwise indicated, can include a functional group derived from an aliphatic carboxylic acid, by removal of the hydroxyl (OH) group. The acyl can be optionally substituted.

[0134] The term alkoxyl, as used herein, unless otherwise indicated, can include O-alkyl groups wherein alkyl is as defined above and O represents oxygen. Representative alkoxyl groups include, but are not limited to, O-methyl, O-ethyl, O-n-propyl, O-n-butyl, O-n-pentyl, O-n-hexyl, O-n-heptyl, O-n-octyl, O-isopropyl, O-sec-butyl, O-isobutyl, O-tert-butyl, O-isopentyl, O-2-methylbutyl, O-2-methylpentyl, O-3-methylpentyl, O-2,2-dimethylbutyl, O-2,3-dimethylbutyl, O-2,2-dimethylpentyl, O-2,3-dimethylpentyl, O-3,3-dimethylpentyl, O-2,3,4-trimethylpentyl, O-3-methylhexyl, O-2,2-dimethylhexyl, O-2,4-dimethylhexyl, O-2,5-dimethylhexyl, O-3,5-dimethylhexyl, O-2,4dimethylpentyl, O-2-methylheptyl, O-3-methylheptyl, O-vinyl, O-allyl, O-1-butenyl, O-2-butenyl, O-isobutylenyl, O-1-pentenyl, O-2-pentenyl, O-3-methyl-1-butenyl, O-2-methyl-2-butenyl, O-2,3-dimethyl-2-butenyl, O-1-hexyl, O-2-hexyl, O-3-hexyl, O-acetylenyl, O-propynyl, O-1-butynyl, O-2-butynyl, O-1-pentynyl, O-2-pentynyl and O-3-methyl-1-butynyl, O-cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl, O-cycloheptyl, O-cyclooctyl, O-cyclononyl and O-cyclodecyl, OCH2-cyclopropyl, OCH2-cyclobutyl, OCH2-cyclopentyl, OCH2-cyclohexyl, OCH2-cycloheptyl, OCH2-cyclooctyl, O CH2-cyclononyl, OCH.sub.2-cyclodecyl, O(CH.sub.2).sub.2-cyclopropyl, O(CH.sub.2).sub.2-cyclobutyl, O(CH.sub.2).sub.2-cyclopentyl, O(CH.sub.2).sub.2-cyclohexyl, O(CH.sub.2).sub.2-cycloheptyl, O(CH.sub.2).sub.2-cyclooctyl, O(CH.sub.2).sub.2-cyclononyl, or O(CH.sub.2).sub.2-cyclodecyl. An alkoxyl can be saturated, partially saturated, or unsaturated. The alkoxyl can be optionally substituted.

[0135] The term cycloalkyl, as used herein, unless otherwise indicated, can include an aromatic, non-aromatic, saturated, partially saturated, or unsaturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 1 to 10 carbon atoms (e.g., 1 or 2 carbon atoms if there are other heteroatoms in the ring), preferably 3 to 8 ring carbon atoms. Examples of cycloalkyls include, but are not limited to, C3-10 cycloalkyl groups include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. The term cycloalkyl also can include -lower alkyl-cycloalkyl, wherein lower alkyl and cycloalkyl are as defined herein. Examples of -lower alkyl-cycloalkyl groups include, but are not limited to, CH2-cyclopropyl, CH2-cyclobutyl, CH2-cyclopentyl, CH2-cyclopentadienyl, CH2-cyclohexyl, CH2-cycloheptyl, or CH2-cyclooctyl. The cycloalkyl can be optionally substituted. A cycloheteroalkyl, as used herein, unless otherwise indicated, can include any of the above with a carbon substituted with a heteroatom (e.g., O, S, N).

[0136] The term heterocyclic or heteroaryl, as used herein, unless otherwise indicated, can include an aromatic or non-aromatic cycloalkyl in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. Representative examples of a heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1,4)-dioxane, (1,3)-dioxolane, 4,5-dihydro-1H-imidazolyl, or tetrazolyl. Heterocycles can be substituted or unsubstituted. Heterocycles can also be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heterocyclic ring). A heterocyclic can be saturated, partially saturated, or unsaturated. The hetreocyclic can be optionally substituted.

[0137] The term indole, as used herein, is an aromatic heterocyclic organic compound with the formula C.sub.8H.sub.7N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. The indole can be optionally substituted.

[0138] The term cyano, as used herein, unless otherwise indicated, can include a CN group. The cyano can be optionally substituted.

[0139] The term alcohol, as used herein, unless otherwise indicated, can include a compound in which the hydroxyl functional group (OH) is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms. The alcohol can be optionally substituted.

[0140] The term solvate is intended to mean a solvate form of a specified compound that retains the effectiveness of such compound. Examples of solvates include compounds of the invention in combination with, for example: water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.

[0141] The term mmol, as used herein, is intended to mean millimole. The term equiv, as used herein, is intended to mean equivalent. The term mL, as used herein, is intended to mean milliliter. The term g, as used herein, is intended to mean gram. The term kg, as used herein, is intended to mean kilogram. The term g, as used herein, is intended to mean micrograms. The term h, as used herein, is intended to mean hour. The term min, as used herein, is intended to mean minute. The term M, as used herein, is intended to mean molar. The term L, as used herein, is intended to mean microliter. The term M, as used herein, is intended to mean micromolar. The term nM, as used herein, is intended to mean nanomolar. The term N, as used herein, is intended to mean normal. The term amu, as used herein, is intended to mean atomic mass unit. The term C., as used herein, is intended to mean degree Celsius. The term wt/wt, as used herein, is intended to mean weight/weight. The term v/v, as used herein, is intended to mean volume/volume. The term MS, as used herein, is intended to mean mass spectroscopy. The term HPLC, as used herein, is intended to mean high performance liquid chromatograph. The term RT, as used herein, is intended to mean room temperature. The term e.g., as used herein, is intended to mean example. The term N/A, as used herein, is intended to mean not tested.

[0142] As used herein, the expression pharmaceutically acceptable salt refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1,1-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion. As used herein, the expression pharmaceutically acceptable solvate refers to an association of one or more solvent molecules and a compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. As used herein, the expression pharmaceutically acceptable hydrate refers to a compound of the invention, or a salt thereof, that further can include a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

Molecular Engineering

[0143] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0144] The terms heterologous DNA sequence, exogenous DNA segment or heterologous nucleic acid, as used herein, each refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A homologous DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0145] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0146] A promoter is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0147] A transcribable nucleic acid molecule as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0148] The transcription start site or initiation site is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3 direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5 direction) are denominated negative.

[0149] Operably-linked or functionally linked refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be operably linked to or associated with a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0150] A construct is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

[0151] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3 transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3-untranslated region (3 UTR). Constructs can include but are not limited to the 5 untranslated regions (5 UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0152] The term transformation refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as transgenic cells, and organisms comprising transgenic cells are referred to as transgenic organisms.

[0153] Transformed, transgenic, and recombinant refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term untransformed refers to normal cells that have not been through the transformation process.

[0154] Wild-type refers to a virus or organism found in nature without any known mutation.

[0155] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0156] Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0157] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid that is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in vitro using the specific codon-usage of the desired host cell.

[0158] Highly stringent hybridization conditions are defined as hybridization at 65 C. in a 6SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (T.sub.m) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65 C. in the salt conditions of a 6SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 C. in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: T.sub.m=81.5 C.+16.6(log.sub.10[Na.sup.+])+0.41 (fraction G/C content)0.63(% formamide)(600/l). Furthermore, the T.sub.m of a DNA:DNA hybrid is decreased by 1-1.5 C. for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0159] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated into the host cell genome.

TABLE-US-00006 Conservative Substitutions I Side Chain Characteristic Amino Acid Aliphatic Non-polar G A P I L V Polar-uncharged C S T M N Q Polar-charged D E K R Aromatic H F W Y Other N Q D E

TABLE-US-00007 Conservative Substitutions II Side Chain Characteristic Amino Acid Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C. Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T Y B. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged (Basic): K R H Negatively Charged (Acidic): D E

TABLE-US-00008 Conservative Substitutions III Original Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met(M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp(W) Tyr, Phe Tyr (Y) Trp, Phe, Tur, Ser Val (V) Ile, Leu, Met, Phe, Ala

[0160] Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term exogenous is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term exogenous gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0161] Host strains developed according to the approaches described herein can be evaluated by any number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0162] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3 overhangs.

Genome Editing

[0163] As described herein, FimH signals can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing. Processes for genome editing are well known; see e.g. Aldi 2018 Nature Communications 9(1911). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0164] For example, genome editing can comprise CRISPR/Cas9, CRISPR-Cpf1, TALEN, or ZNFs. Adequate blockage of FimH by genome editing can result in protection from autoimmune or inflammatory diseases.

[0165] As an example, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR/Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9 (thus, a (N).sub.20NGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double strand break instigates either non-homologous end-joining, which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology-directed repair, which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Thus, genomic editing, for example, using CRISPR/Cas systems could be useful tools for therapeutic applications for UTIs and other bacterial infections to target cells by the removal of FimH signals.

[0166] For example, the methods as described herein can comprise a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein.

Formulation

[0167] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0168] The term formulation refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a formulation can include pharmaceutically acceptable excipients, including diluents or carriers.

[0169] The term pharmaceutically acceptable as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 (USP/NF), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc. may also be used.

[0170] The term pharmaceutically acceptable excipient, as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0171] A stable formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 C. and about 60 C., for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0172] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0173] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of the agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0174] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition.

Therapeutic Methods

[0175] Also provided is a process of treating, preventing, or reversing UTIs and other bacterial infections in a subject in need of administration of a therapeutically effective amount of a FimH inhibiting agent, so as to protect against UTIs and other bacterial infections.

[0176] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a UTI and other bacterial infection. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0177] Generally, a safe and effective amount of a FimH inhibiting agent is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a FimH inhibiting agent described herein can substantially inhibit UTIs and other bacterial infections, slow the progress of UTIs and other bacterial infections, or limit the development of UTIs and other bacterial infections.

[0178] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0179] When used in the treatments described herein, a therapeutically effective amount of a FimH inhibiting agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to protect against a UTI and other bacterial infections.

[0180] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0181] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0182] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single-dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0183] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

[0184] Administration of a FimH inhibiting agent can occur as a single event or over a time course of treatment. For example, a FimH inhibiting agent can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0185] Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for UTIs and other bacterial infections.

[0186] A FimH inhibiting agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a FimH inhibiting agent can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through the administration of separate compositions, each containing one or more of a FimH inhibiting agent, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through the administration of one composition containing two or more of a FimH inhibiting agent, an antibiotic, an anti-inflammatory, or another agent. A FimH inhibiting agent can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a FimH inhibiting agent can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.

Administration

[0187] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0188] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0189] Agents and compositions described herein can be administered in a variety of methods well-known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 m), nanospheres (e.g., less than 1 m), microspheres (e.g., 1-100 m), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0190] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0191] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve the taste of the product; or improve the shelf life of the product.

Screening

[0192] Also provided are methods for screening.

[0193] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.

[0194] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0195] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals etc.).

[0196] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character x log P of about 2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character x log P of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0197] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being drug-like. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0198] Several of these drug-like characteristics have been summarized into the four rules of Lipinski (generally known as the rules of fives because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of compounds during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0199] The four rules of five state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8 to about 15 .

Kits

[0200] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to, a FimH inhibiting antibody, antibiotics, solubilizing agents, and salts. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing the activity of the components.

[0201] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0202] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.

[0203] A control sample or a reference sample as described herein can be a sample from a healthy subject. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0204] The methods and algorithms of the invention may be enclosed in a controller or processor. Furthermore, methods and algorithms of the present invention can be embodied as a computer-implemented method or methods for performing such computer-implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer-readable storage medium containing a computer program or other machine-readable instructions (herein computer program), wherein when the computer program is loaded into a computer or other processor (herein computer) and/or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer programs include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, thumb drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and/or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general-purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.

[0205] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0206] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0207] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term about. In some embodiments, the term about is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0208] In some embodiments, the terms a and an and the and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term or as used herein, including the claims, is used to mean and/or unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0209] The terms comprise, have and include are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as comprises, comprising, has, having, includes and including, are also open-ended. For example, any method that comprises, has or includes one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that comprises, has or includes one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0210] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0211] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0212] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0213] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

[0214] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Example 1Monoclonal Antibodies Targeting FimH Adhesin Protect Against Uropathogenic E. coli UTI

[0215] In this example, monoclonal antibodies targeting the FimH adhesin protein are developed and identified. Their use in the treatment of UTI in vitro and in vivo is described, which showed protection against UTI in a murine model.

Introduction

[0216] Urinary tract infections (UTIs) affect over 400 million individuals worldwide yearly, leading to $2.8 billion in healthcare and productivity-related costs annually in the US alone. Around 25% of individuals will suffer from recurrent UTIs which severely impairs their quality of life. Further, 27% of all sepsis cases can be traced to urinary origin. This is aggravated by the increased prevalence of multidrug-resistant uropathogens, such as uropathogenic Escherichia coli (UPEC), responsible for 70-90% of UTIs, and Klebsiella pneumoniae, one of the most prevalent non-UPEC uropathogens. UTIs represent the fourth leading cause of death attributed to or associated with antibiotic resistance. Thus, developing antibiotic-sparing strategies to prevent UTIs caused by these difficult-to-treat uropathogens is crucial. One promising approach for new antibacterial treatments is to neutralize key extracellular adhesins to prevent bacterial colonization and invasion into tissue and biofilm formation.

[0217] UPEC and K. pneumoniae express chaperone-usher pathway (CUP) type 1 pili that are tipped with the mannose-binding FimH adhesin essential in i) bladder colonization, ii) ascension to cause pyelonephritis, iii) invasion into terminally differentiated umbrella cells of the bladder, iv) the formation of intracellular biofilms in luminal bladder cells, and v) causing an epigenetic imprint in the bladder that predisposes to recurrent UTI. FimH is a two-domain protein with an N-terminal lectin domain containing a deep binding pocket that recognizes mannose with stereochemical specificity and a C-terminal pilin domain linking the adhesin to the pilus. At the tip of the assembled type 1 pilus, FimH exists in a conformational equilibrium between a high-affinity relaxed state and a low-affinity tense state controlled by structural interactions between the FimH lectin and pilin domains. In the high-affinity relaxed state, the FimH lectin domain is highly mobile with respect to the pilin domain. In contrast, in the low-affinity tense state, the pilin domain constrains the lectin domain and allosterically deforms the mannose-binding pocket. UPEC FimH occupies both tense and relaxed conformations whereas the equilibrium in the highly invariant and conserved K. pneumoniae FimH is primarily shifted towards the tense low-binding conformation, explaining its poor mannose binding properties despite an identical mannose binding site.

[0218] A vaccine against UPEC FimCH has revealed an 73% reduction in recurrent UTIs caused by UPEC or Klebsiella spp. in a phase 1A/1B clinical trial, showing potential to prevent the two most common UTI pathogens with a FimH targeted therapeutic. The effectiveness of FimH vaccination is associated with antibody responses that inhibit FimH binding. Here, we characterized monoclonal antibodies (mAb) from mice immunized with E. coli and K. pneumoniae FimH lectin domains and discovered cross-reactive antibodies that bind to four distinct FimH structural epitopes (Class 1-4), which block FimH binding in vitro. Using cryo-EM, we discovered that the mAbs are selectively bound to the epitopes displayed in the high-affinity relaxed conformation of FimH. Using binding studies and mouse UTI models, we identified Class 1 mAbs that blocked FimH binding to mannose through steric interference leading to protection against UPEC in mouse UTI models. From structure to an antibiotic alternative therapeutic, these results guide future optimization of FimH mAbs and vaccination strategies to treat E. coli and K. pneumoniae UTIs, two of the most prominent uropathogens with increasing antibiotic resistance.

Results

FimH.sub.LD mAbs can Cross-React with E. coli and K. pneumoniae FimH

[0219] Female C57BL/6J mice were immunized with the lectin domain truncates of FimH (FimH.sub.LD) from either E. coli (UT189) or K. pneumoniae (TOP52). Heavy chain V-D-J and light chain V-J fragments from sorted plasmablasts isolated from draining lymph nodes were cloned into human IgG expression vectors to create chimeric murine/human mAbs as previously described. In total, 33 clonally distinct mAbs were generated from E. coli (8 mAbs) and K. pneumoniae (25 mAbs) that bound the respective FimH.sub.LD (referred to as Ec or Kp mAbs respectively). The FimH.sub.LD structures of E. coli and K. pneumoniae are highly homologous (RMSD=0.420) with an 86% amino acid sequence identity, including an identical binding pocket (FIG. 1A, FIG. 1B, FIG. 6A, FIG. 6B). By ELISA, most mAbs bound to their respective FimH.sub.LD antigen with half-maximal effective binding concentrations (EC.sub.50) ranging from 8 to 50 ng/mL, while some bound more weakly with EC.sub.50 values from 50 to 751 ng/mL (FIG. 1D). Half (4 of 8) of the Ec mAbs also reacted with K. pneumoniae FimH.sub.LD and 80% (20 of 25) of Kp mAbs reacted with E. coli FimH.sub.LD. Further, eight mAbs (2 Ec and 6 Kp mAbs) that reacted with both FimH antigens also bound with high affinity to a third structurally similar (RMSD=0.699 to Ec FimH) E. coli adhesin, FmIH lectin domain (FmIH.sub.LD), an adhesin that binds to exposed galactose residues on bladder tissue during chronic cystitis infections (FIG. 1C, FIG. 1D, FIG. FIG. 6C).

FimH.sub.LD mAbs Bind Diverse Epitopes

[0220] To determine the structural regions of FimH.sub.LD that were recognized by each mAb, we generated a mutant library consisting of 45 surface-exposed mutations to bulky charged residues across the lectin domain of full-length E. coli FimH. Binding of the mAbs to the stable mutant FimH proteins was determined by ELISA, which revealed four binding site classes (FIG. 1E, structural regions of FimH are defined in FIG. 6D and FIG. 26). Class 1 mAbs (4 Ec and 13 Kp mAbs), bind with high affinity to both E. coli and K. pneumoniae FimH.sub.LD and FmIH.sub.LD. Class 1 mAbs were unable to bind E. coli FimH Class 1 epitope residue mutants V27D, N152K, and V155D located in the insertion and swing loops, and in the part of the linker between the pilin and receptor binding domains, suggesting they bind to the base of the lectin domain. Class 2 mAbs (4 Kp mAbs) bound to E. coli and K. pneumoniae FimH.sub.LD, but did not react with FmIH.sub.LD, consistent with the sequence differences between E. coli FimH.sub.LD and E. coli FmIH.sub.LD (FIG. 6D). Class 2 mAbs shared the inability to bind to the Class 2 epitope residue mutants N23K and T40K in E. coli FimH suggesting that they bind to the side of the FimH.sub.LD body between the base of the binding clamp loop and basal swing loop. A majority of Class 3 mAbs (2 Ec mAbs and 3 Kp mAbs), bound to both E. coli and K. pneumoniae FimH.sub.LD, but not to FmIH.sub.LD. Class 3 mAbs were unable to bind to Class 3 epitope residue mutants S62K, Y64D, V67D, E89K, K121 D, V128D, V145D, and V155D in E. coli FimH, suggesting that they bind below the binding pocket to the opposite lateral side of the FimH.sub.LD body from the Class 2 epitope, covering regions on -sheet B below the clamp loop and peripheral alpha-helix. Class 4 mAbs (2 Ec mAbs) were only able to bind E. coli FimH.sub.LD, and not to K. pneumoniae FimH.sub.LD or E. coli FmIH.sub.LD and were unable to bind to Class 4 epitope residue mutants Y55D, S80K, R92D, and K101 D in E. coli FimH, suggesting that they bound at the base of binding loop two and backside of FimH.sub.LD. Five Kp mAbs were not mapped as they did not bind E. coli FimH. Thus, immunization of E. coli and K. pneumoniae FimH.sub.LD antigens generated mAbs bound to four unique surfaces (Class 1-4 epitopes) on E. coli FimH. Below, mAb nomenclature denotes the epitope class recognized by the mAb after the Ec or Kp designation. Kp FimH.sub.LD mAbs whose epitopes could not be determined are left unnumbered.

Structural Basis of FimH mAb Recognition

[0221] The structural basis of Class 1-3 mAbs binding to FimH.sub.LD was determined by cryo-electron microscopy (cryo-EM) of FimCH complexed with fragment antigen-binding regions (Fabs) of Kp1 2H04, Ec1 F7, Kp2 2C07, and Ec3 B7 (FIG. 2A, FIG. 2B, FIG. 2D, FIG. 2E, FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 8, FIG. 9C). In each case, the observed interaction between FimH.sub.LD and Fabs confirmed and extended the epitope mapping described above. The structural basis of Kp1 2H04 Fab and Ec1 F7 Fab binding to Class 1 epitope of FimH revealed that they both interacted with the FimH.sub.LD swing loop and linker regions (Class 1 epitope residues A24 to N29 and N151 to T158) and coordinated multiple aromatic residues around FimH.sub.LD Class 1 residue P26 (FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2F, FIG. 2G). However, Kp1 2H04 and Ec1 F7 Fabs bound to the E. coli FimH.sub.LD at differing angles. Ec1 F7 Fab was rotated 20 degrees relative to Kp1 2H04 Fab bound to FimH.sub.LD (FIG. 2C, FIG. 9A, FIG. 9B, FIG. 9C). The structure of the Class 2 Kp2 2C07 Fab-FimH.sub.LD complex revealed strong binding to the Class 2 epitope residues Y21 to A27 and N151 to D153 (FIG. 2D, FIG. 2H). Class 3 Ec3 B7 Fab bound to -sheet B of E. coli FimH including Class 3 residue Y64 (FIG. 2I, FIG. 2F), which has been identified as a toggle switch between tense and relaxed FimH conformation, with the residue undergoing a major solvent-accessible surface area change in the conformational transition. This binding mode suggests a role of our Class 3 mAbs specifically binding and stabilizing the relaxed FimH conformation, as mutagenesis of Class 3 residue Y64 abolished Class 3 mAb binding in the epitope mapping (FIG. 1E). Altogether, the high-resolution cryo-EM structures identified critical FimH interactions of the mAb epitope classes.

FimH.sub.LD mAbs Bind Preferentially to the Relaxed Conformation

[0222] When FimH is incorporated at the tip of type 1 pili, the receptor binding domain samples a conformational equilibrium between low-affinity tense and high-affinity relaxed conformations (FIG. 6A, FIG. 6B). The identified mAb binding epitopes of FimH.sub.LD, particularly Class 1-3, are in regions that vary extensively between tense and relaxed conformational states (FIG. 6A, FIG. 6B, FIG. 26). Thus, we investigated whether the conformational dynamics influence the epitopes recognized by Class 1-4 mAbs by measuring binding to FimH-tipped piliated E. coli bacteria using ELISA. Class 1 mAbs displayed the highest reactivity, while Class 2-4 mAbs displayed greatly diminished reactivity to FimH tipped type 1 pili of E. coli (FIG. 3A). Thus, we tested Class 1-4 mAbs against E. coli expressing conformational FimH variants. A majority of mAbs from Classes 1-4 had increased binding to the relax-shifted A27V/V163A E. coli FimH mutant tipping type 1 pili and very weak binding to the tense-shifted A62S E. coli FimH mutant, despite similar levels of type 1 pili expression as measured by western blot (FIG. 3B, FIG. 10). To investigate binding of the highest affinity Class 1-2 mAbs to FimH in a tip-like state, we used Bio-Layer Interferometry (BLI) to measure binding to recombinant FimH. We prepared tip-like recombinant full-length FimH by incubating E. coli FimCH with the N-terminal extension (Nte) peptide of FimG resulting in a donor strand exchange reaction where the FimG Nte displaces the FimC chaperone to produce E. coli FimG.sub.nteH complex that samples dynamic conformations like that of FimH tipping type 1 pili. Class 1-2 mAbs had varied binding affinity to E. coli FimG.sub.nteH, with K.sub.on rates lower than binding to E. coli FimH.sub.LD (K.sub.on rates between 210.sup.4 to 110.sup.5 M.sup.1 s.sup.1) (FIG. 3C, FIG. 3D, FIG. 11A, FIG. 11B, FIG. 12A, FIG. 12B). This finding indicated that Class 1-2 mAbs had a binding preference for the relaxed FimH conformation likely due to FimH.sub.LD being the immunizing antigen.

FimH.sub.LD mAbs Inhibit FimH Mannosylated Protein Binding

[0223] The structural analysis showed mAbs recognizing epitopes near the mannose binding pocket suggesting mAbs may interfere with FimH binding. Thus, FimH.sub.LD mAbs were tested for their ability to block E. coli and K. pneumoniae FimH.sub.LD binding to highly mannosylated glycoprotein bovine submaxillary mucin (BSM). At a 5:1 molar ratio of mAb to FimH.sub.LD, assays measuring mAbs that inhibit binding to BSM in ELISA assays ranged from no inhibition to 85% inhibition compared to untreated control (FIG. 4A). Only eight mAbs (representing epitope Classes 1-3) inhibited both FimH.sub.LD proteins at greater than 50% at a 5:1 molar ratio. We selected mAbs from Classes 1-3 from both E. coli and K. pneumoniae antigens with the highest inhibition to FimH.sub.LD and the strongest binding to FimH tipping type 1 pili on the surface of bacteria to test for the ability to inhibit mannose-dependent E. coli bacterial hemagglutination of guinea pig erythrocytes. When present in high concentrations (17 uM), we found all mAbs tested can inhibit hemagglutination with more potency than -D-mannopyranoside. However, the FimH mAbs displayed greatly variable inhibition potency with 50% inhibition concentration ranging from 700 nM to above 17 uM (FIG. 4B). We selected Kp1 2H04 to further test for ability to block FimH.sub.LD binding to mouse bladder tissue, as Kp1 2H04 had high inhibition in the FimH BSM ELISA and E. coli hemagglutination. At a 10:1 molar ratio of mAb to FimH.sub.LD protein, Ec FimH.sub.LD and Kp FimH.sub.LD mixed with control IgG bound strongly to the bladder epithelial cells. However, Kp1 2H04 mAb treatment completely blocked Ec FimH.sub.LD and Kp FimH.sub.LD binding to mouse bladder tissue (FIG. 4C).

FimH.sub.LD mAbs Protect Against UTI

[0224] To determine the ability of the mAbs to prevent UTI, we screened eight mAbs in a prophylactic model. We chose Class 1-3 mAbs Ec1 F7, Kp1 2H04, Kp1 1A02, Kp1 2E02, Kp1 1B03, Kp1 2E08 mAbs, Kp2 2C07, and Ec3 B7 mAb for these assays since they bind with high-affinity to tip-like FimH, inhibit bacterial E. coli FimH binding, and represent three epitope classes from E. coli and K. pneumoniae FimH antigens. Each mAb was administered via intraperitoneal injection at 0.5 mg per mouse 24 hours before infection with E. coli UT189. Bladder and kidney titers were enumerated 24 hours post-infection (FIG. 5A). There was no detectable IgG in urine before infection (24 hours after IP injection of mAbs) but mAbs were detected in the bladder approximately 3 to 6 hours after infection, consistent with bladder damage being necessary for antibodies to reach the urine as previously suggested (FIG. 13). Three of the mAbs from epitope Class 1, (Ec1 F7, Kp1 2H04, and Kp1 1A02), resulted in significantly decreased bladder titers (1 log; P<0.05) and two Class 1 mAbs (Ec1 F7 and Kp1 2H04) also significantly decreased kidney titers (0.5 log; P<0.05) (FIG. 5B, FIG. 5C). The effect of prophylactic administration of Kp1 2H04, one of the strongest inhibitors of bladder titers at 24 hpi, on formation of intracellular bacterial communities (IBCs) at 6 hpi was assessed. Kp1 2H04 significantly decreased the amount of bladder IBCs compared to the control IgG at 6 hpi (FIG. 5D, FIG. 5E, FIG. 5F).

[0225] To test if immune system signaling functions associated with the fragment crystallizable (Fc) region of mAbs were needed for protection, we created a LALAPG Fc variant of Kp1 2H04 (2H04.sub.LALAPG) which inhibits the ability of the mAb to bind Fc receptors or fix complement. 2H04.sub.LALAPG had a comparable binding affinity to FimH.sub.LD as 2H04 when assayed by ELISA (FIG. 14). When tested in the prophylactic model, 2H04.sub.LALAPG inhibited UT189 infection to the same degree as Kp1 2H04 in the bladder and kidney (FIG. 5G, FIG. 5H), suggesting that Kp1 2H04 prevents infection primarily by directly inhibiting FimH rather than through Fc receptor functions. To further test this, we repeated the prophylactic model but monitored the infection over 14 days. Kp1 2H04 treated mice resulted in lower urine and bladder titers over the 14 days. Still, this difference did not increase over time despite detectable amounts of mAb in the serum and bladder homogenates up to 14 days post-infection (FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D) suggesting that the primary mechanism of the mAb is blocking initial FimH attachment to the bladder epithelium.

Discussion

[0226] Carbapenem-resistant Enterobacteriaceae (CRE) and extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae are listed as urgent and serious threats by the Centers for Diseases Control and Prevention (CDC) as they are resistant to numerous antibiotics needed to treat common infections, including UTIs. New antibiotic-sparing strategies are needed to treat these and other Enterobacteriaceae infections. mAbs have been exceptional drugs in treating cancer and viral infections but few mAb therapies have been notably developed for treating or preventing bacterial infections. Here, we generated mAbs to neutralize FimH, the critical adhesin used by UPEC and K. pneumoniae to mediate UTI pathogenesis. We: i) generated mAbs that define four classes of anti-FimH mAbs that bind to distinct FimH epitopes; ii) identified cross-reactive mAbs with high affinity to E. coli and K. pneumoniae FimH; and iii) characterized the most potent FimH blocking antibodies in a structural and functional analysis. When we tested these mAbs in vivo, we found significant quantities of antibody could be detected in the urine of mice after (but not prior to) infection, suggesting that mAbs penetrate the urinary tract in the context of infection. Two Class 1 Kp and Ec mAbs (Kp1 2H04 and Ec1 F7) significantly reduced bacterial titers in the bladder and kidneys when administered before UPEC infection in a robust mouse model of UTI through direct inhibition of FimH function. This is in contrast to previous studies suggesting that mAbs to the relaxed FimH conformation may stabilize a high-affinity conformation thereby increasing affinity to the bladder. These data suggest that some mAbs, which bind outside the binding pocket to a high-affinity relaxed-conformation by our structural analysis, inhibit FimH binding through steric hindrance preventing access to the binding pocket.

[0227] In the bladder, FimH binds uroplakin Ia, which forms an oligomeric complex with other uroplakin proteins in a dense crystalline lipid superstructure burying the mannose glycan deep in the complex. The structure of uroplakin plaques requires FimH to reach into a tight spatial pocket to bind mannose which may be sterically hindered by the binding of mAbs (FIG. 16A, FIG. 16B, FIG. 16C). However, this proposed mechanism does not preclude the additional possibility of stabilizing the high-affinity conformation as shifting the conformational equilibrium towards the high-affinity state via mutations can also result in virulence attenuation. Bacteria with low-affinity tense-shifted FimH alleles may be able to evade vaccination or mAb therapy targeting the relaxed FimH conformation; however, bacteria with tense-shifted FimH alleles are attenuated in infection due to decreased ability to bind to the bladder epithelium. Nevertheless, the highly variable ability of antibodies to bind to tense or relaxed states of FimH suggests that the conformational equilibrium may provide an advantage in vivo by helping UPEC avoid antibodies that effectively bind to only one of the two states. Notably, we did not identify a mAb that directly binds to the FimH mannose-binding pocket, suggesting that vaccination of FimH in the relaxed state may function by providing antibodies that sterically inhibit FimH rather than directly blocking the mannose-binding pocket. Future studies are needed to determine the antigenicity of a tense state FimH and if tense state-specific anti-FimH mAbs are effective at protecting from UTI.

[0228] Our results show that FimH-inhibiting mAbs have promising antibiotic-sparing therapeutic potential to treat both UPEC and K. pneumoniae UTIs, lay the groundwork for identifying FimH mAbs with increased efficacy, and provide a roadmap to leverage mAbs to inhibit other bacterial uropathogenic CUP adhesins. While there have been few mAbs developed to treat bacterial infection, mAbs directed at treating UTI could offer unique advantages over antibiotics since they both avoid selection of antibiotic resistance and would have a sustained period of effectiveness. These mAbs may be deployed effectively in patients with highly recurrent UTI, who are often administered prophylactic antibiotics, or in hospital settings with high-risk UTI patients, such as patient populations requiring catheterization, in which UPEC and K. pneumoniae can repeatedly colonize catheters over many months.

Materials and Methods

Protein Generation and Purification

[0229] FimCH, FimHLD and FmIH lectin domain truncated proteins were purified. Briefly, proteins were expressed and isolated from crude periplasmic preparations using affinity chromatography. In vitro DSE using FimGNTE peptide was performed and purified. FimHLD labeled with EZ-Link NHS-PEG4 Biotinylation Kit (ThermoFisher) or Alexa Fluor 647 with NHS Ester conjugation (Invitrogen) was generated according to the manufacturer's instructions. Surface topology diagram of FimH was generated using PDBsum.

Mouse Immunization for Monoclonal Antibody (mAb) Generation

[0230] All procedures involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Washington University in Saint Louis. Female C57BL/6J mice (Jackson Laboratories) were immunized intramuscularly with 30 g E. coli FimHLD or 25 g K. pneumoniae FimHLD emulsified with AddaVax (InvivoGen). Four weeks later, mice were boosted with a second dose of FimHLD emulsified with AddaVax. One control mouse received PBS emulsified with AddaVax according to the same schedule. Draining iliac and inguinal lymph nodes were harvested 5 days after the boost for plasmablast sorting.

Cell Sorting for mAb Generation

[0231] Staining for sorting was performed using fresh lymph node single cell suspensions in PBS supplemented with 2% FBS and 1 mM EDTA (P2). Cells were stained for 30 min on ice with CD138-BV421 (281-2, 1:200), CD4-PerCP (GK1.5, 1:100), CD19-PE (6D5, 1:200), B220-PE-D594 (RA3-6B2, 1:200), CD38-PE-Cy7 (90, 1:200), Fas-APC (SA367H8, 1:400), IgD-APC-Cy7 (11-26c.2a, 1:100), and Zombie Aqua (all Biolegend) diluted in P2. Cells were washed twice and single plasmablasts (B220lo CD138+ IgDlo CD19+ CD4 live singlet lymphocytes) were sorted using a FACSAria II into 96-well plates containing 2 L Lysis Buffer (Clontech) supplemented with 1 U/L RNase inhibitor (NEB) and immediately frozen on dry ice.

Monoclonal Antibody (mAb) and Fragment Antigen Binding (Fab) Generation

[0232] Antibodies were cloned. In brief, VH, V, and VA genes were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR from singly-sorted plasmablasts using cocktails of primers specific for IgG, IgM/A, IgK, and IgA using first round and nested primer sets (Table 6) and then sequenced. Clonally related cells were identified by the same length and composition of IGHV, IGHJ and heavy-chain CDR3 and shared somatic hypermutation at the nucleotide level. To generate recombinant antibodies, heavy chain V-D-J and light chain V-J fragments were PCR-amplified from 1st round PCR products with mouse variable gene forward primers and joining gene reverse primers having 5 extensions for cloning by Gibson assembly as previously described (Table 6), and were cloned into pABVec6W antibody expression vectors in frame with either human IgG, IgK, or IgL constant domain. Plasmids were co-transfected at a 1:2 heavy to light chain ratio into Expi293F cells using the Expifectamine 293 Expression Kit (Thermo Fisher), and antibodies were purified with protein A agarose (Invitrogen). For monovalent Fab generation, the VH segment of selected antibodies were cloned into a Fab expression vector with a thrombin cleavage site preceding a 6His tag by GenScript. Fab and light chain plasmids were co-transfected into Expi293F cells for expression and purified with HisPur Ni-NTA resin (Thermo Scientific). For controls in experiments, IgG mAb 2B04, specific for SARS-CoV-2 receptor binding domain was used.

TABLE-US-00009 TABLE6 PrimersusedformAbgeneration 1stRoundPCRPrimers IgG,IgK,IgLprimers IgM/A Forward VH/Outer:GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG(SEQ_ID_NO:280) Reverse 3Couter:AGGGGGCTCTCGCAGGAGACGAGG(SEQ_ID_NO:281) 3Couter:GAAAGTTCACGGTGGTTATATCC(SEQ_ID_NO:282) NestedPCRprimers IgG,IgK,IgLprimers IgM/A Forward VH/Outer:GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG(SEQ_ID_NO:283) Reverse 3Cinner:AGGGGGAAGACATTTGGGAAGGAC(SEQ_ID_NO:284) 3Cinner:TGCCGAAAGGGAAGTAATCGTGAAT(SEQ_ID_NO:285) Gibsoncloningprimers IgH Forward VH01: ATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGGTCCARCTGCARC AGYCTGG(SEQ_ID_NO:286) VH02: CCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCCAGGTGCAGCTGAAGSAG TC(SEQ_ID_NO:287) VH06: CCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAAGTGAAGCTTGARGWG TCTG(SEQ_ID_NO:288) VH14: CCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCCGAGGTTCAGCTGCAGCAG (SEQ_ID_NO:289) Reverse JH01:GAAGACCGATGGGCCCTTGGTCGACGCTGAGGAGACGGTGACCGTG (SEQ_ID_NO:290) JH02:GAAGACCGATGGGCCCTTGGTCGACGCTGAGGAGACTGTGAGA (SEQ_ID_NO:291) JH03:GAAGACCGATGGGCCCTTGGTCGACGCTGCAGAGACAGTGACCAGAG (SEQ_ID_NO:292) JH04:GAAGACCGATGGGCCCTTGGTCGACGCTGAGGAGACGGTGACTGAG (SEQ_ID_NO:293) IgK

ELISAs

[0233] To generate ELISA binding curves to FimHLD and FmIHLD adhesin truncates, plates were first coated with the antigen (0.1 ug/mL) overnight. Plates were washed once with PBS supplemented with Tween-20 (PBS-T, 0.05%) and blocked for 2 h with PBS-T with 10% FBS. mAbs were serially diluted starting at 30 ug/mL and added to the plate to bind for 1 h. Plates were washed 3 in PBS-T. mAb binding was detected with anti-human IgG (Jackson ImmunoResearch, 1:2,500 dilution) for 1 h before washing 3 times in PBS-T and developed with O-phenylenediamine dihydrochloride in citrate buffer (Sigma). Reactions were quenched with 1 M HCl and absorbance was read at 490 nm.

[0234] To measure inhibition of FimHLD binding, plates were coated with BSM (10 ug/mL) overnight. Plates were blocked for 2 h with 1PBS with 1% BSA. FimHLD (2.5 ug/mL) was mixed with a serial curve of mAb (to ensure dose-dependent inhibition) for 1 h. FimHLD mAb mixtures were then added to the plate to let bind for 1 h at RT. Plates were washed 3 times with PBS-T and incubated with anti-streptavidin-HRP (BD Pharmagen) for 1 h before washing 3 times with PBS-T and development with TMB substrate (BD Pharmagen). Reactions were quenched with 1 M H2SO4 and absorbance at 450 nm was recorded.

[0235] For measurements of the reactivity of mAbs to bacteria, bacteria were grown statically 224 (grown for 24 h and subcultured 1:1000 for another 24 h growth period) in LB and normalized to OD600=1.0 in 1PBS. Bacteria (100 uL) were added to the plate, spun down 5 min at 3000 rcf, and allowed to bind for 1 h. The supernatant was decanted, and formalin (10%) was added to the wells to fix bacteria for 10 min. Plates were washed 3 in PBS-T and then assayed and developed with the same protocol as the ELISA binding curves to adhesin truncates. To measure levels of humanized IgG (mAbs) in serum, urines and bladder homogenates, plates were coated with diluted 1:100 serum, 1:10 mouse urine, or 1:2 bladder homogenates along with a standard curve of hIgG isotype overnight at 4 C. Plates were then blocked for 2 hours at room temperature with 1PBS with 1% BSA. Then, plates were washed three times with PBS-T. To measure hIgG levels, plates were then incubated with 1:10,000 dilution of goat anti-Human IgG H&L (HRP) preabsorbed IgG (Abcam, ab97175) for 1 hr at room temperature. For plate development, TMB substrate reagent was added and incubated for approximately 5 minutes at room temperature. Reactions were quenched with 3M HCl and absorbance at 450 nm was recorded.

Generation of FimH Surface Mutants

[0236] Surface FimH (J96) mutants were generated via one-step mutagenesis (19055817) using a pBAD33.1 vector plasmid encoding for FimH (J96 strain) template, Pfu Ultra HF polymerase (Agilent, NC9666083), and the primers listed in Table 6. After PCR amplification, reactions were digested with DpnI (NEB Biolabs, R0176S) to remove the original template from the reaction products. PCR reaction products were transformed into E. coli DH5a for ligation. Plasmids confirmed by Sanger sequencing were then transformed into Escherichia coli C600 fim expression strain.

Epitope Mapping

[0237] Epitope mapping of FimH mAbs to E. coli FimChisH was performed using a modified ELISA technique. E. coli strain C600 fim carrying pBAD33 plasmids encoding FimH mutants and a ptrc99a plasmid encoding FimChis, were grown in LB to an OD of 0.6-0.8 and then induced with 0.1 mM IPTG and 0.05% arabinose for 1 h. Cells were harvested and periplasm extracts containing FimChisH variants were obtained. FimChisH periplasmic extracts were titrated using anti-FimH sera to normalize the amount of FimH. Normalized FimChisH periplasm was used to coat plates for 1 h at room temperature. Plates were blocked for 2 h with 1PBS with 1% BSA and mAbs (0.1 ug/mL, except for low-binding mAb 2A02 1 ug/ml was used) were allowed to bind for 1 h. Plates were washed in 1PBS-T before detection with anti-human IgG (Jackson ImmunoResearch, 1:1000 dilution) and developed with TMB substrate (BD Pharmagen) and H2SO4 as outlined above. mAb binding to each FimChisH mutant was normalized to WT FimChisH binding. Variants that decreased binding below 10% of WT binding and clustered together (3 or more residues) were considered an epitope. Mapping data was visualized and clustered (one-minus Pearson correlation) in Morpheus.

Western Blotting

[0238] Western blotting to detect FimA in bacterial lysates was performed. Bacteria grown 224 statically was normalized to optical density at 600 nm of 1.0 and were acid treated with HCl and boiled to disrupt FimA DSE interactions. To measure FimA, rabbit antitype 1 pili (1:2000) was used. A secondary antibody of goat anti-rabbit-HRP (1:10,000, KPL) was used to detect followed by development with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher). Images were obtained on a BioRad ChemiDoc system. A colorimetric image (to view protein size ladder) was overlaid on the chemiluminescent image (detection signal) to create the figure reported in this study.

Biolayer Interferometry (BLI) Studies

[0239] Kinetic binding studies were performed on an Octet Red instrument (ForteBio). Avi-tag biotinylated antibody fragments (Fab) were loaded up to 2.5 nm onto Streptavidin sensor tips (Sartorius) that were pre-equilibrated in HEPES Buffered Saline (HBS) with 0.05% Tween-20 and 1% BSA (kinetic buffer A). Diluted antigens (Ec FimHLD, Ec FimGnteH) were monitored for 200 s of association and 600 s of dissociation in kinetic buffer A. Loaded sensor tips dipping in kinetic buffer A were used as reference sensors. Reference subtracted kinetic traces were used to calculate kinetic rate constants (kon, koff) and equilibrium dissociation rate constant (KD) using a Langmuir 1:1 binding model. Resulting binding traces and fits were plotted with GraphPad Prism v10.

Hemagglutination Inhibition (HAI) Assay

[0240] E. coli guinea pig erythrocyte hemagglutination inhibition assays were performed. Briefly, mAbs were serially diluted in microtiter plates, and 25 uL of bacterial suspension (serially diluted from OD600=10.0 in 1PBS) was added to each well. After incubation for 10 min at room temperature, 25 ul (OD640=2.0) guinea pig erythrocytes in 1PBS were added for a final volume of 50 ul. The plates were incubated at 4 C. overnight. For each mAb concentration, the HA titer was defined as the greatest dilution of bacteria that caused hemagglutination.

Cryo-EM Data Collection and Analysis

[0241] Fab and FimCH protein were mixed at a ratio of 1.2:1 and dialyzed into 20 mM HEPES pH 7.5 with 50 mM NaCl. Complexes were flash frozen on EM grids in liquid ethane using an FEI Vitrobot (ThermoFisher) and imaged on Titan Krios (2H04 complex) or Glacios (F7, B7 and 2C07 complexes) microscopes using a Falcon 4 electron detector (Thermo Fisher). Movies were processed in Cryosparc v4.4.1 and particles were picked using Topaz. Densities were post-processed using DeepEMhancer for model building. The collection parameters and workflow are described in more detail in FIG. 8. Initial Fab models were built using homology models in SwissModel. FimHLD model was generated by trimming and threading the UT189 FimH sequence on PDB 1KLF. Rough models were initially docked in ChimeraX before multiple rounds of real space refinement in Phenix v1.20.1 with manual editing in COOT v0.9.6. Refinement statistics are shown in Table 7.

TABLE-US-00010 TABLE 7 CryoEM model refinement and validation statistics Kp1 2H04-FimH Ec1 F7-FimH Ec3 B7-FimH Kp2 2C07-FimH Composition (#) Chains 3 3 3 3 Atoms 4532 (Hydrogens: 0) 4501 (Hydrogens: 0) 4491 (Hydrogens: 0) 4558 (Hydrogens: 0) Residues Protein: 597 Protein: 595 Protein: 595 Protein: 601 Nucleotide: 0 Nucleotide: 0 Nucleotide: 0 Nucleotide: 0 Water 0 0 0 0 Ligands 0 0 0 0 Bonds (RMSD) Length () (# > 0.005 (0).sup. 0.005 (0).sup. 0.004 (0).sup. 0.004 (0).sup. 4) Angles () (# > 0.852 (1).sup. 0.911 (2).sup. 0.707 (3).sup. 0.809 (4).sup. 4) MolProbity score 2.26 2.5 2.08 2.19 Clash score 15.01 15.67 11.64 12.25 Ramachandran plot (%) Outliers 0.34 0.34 0.34 0.34 Allowed 10.83 16.81 8.15 11.43 Favored 88.83 82.85 91.51 88.24 Rama-Z (Ramachandran plot Z-score, RMSD) whole (N = 591) 3.25 (0.32) 3.22 (0.33) 1.79 (0.35) 3.03 (0.32) helix (N = 22) 2.39 (0.70) 3.52 (0.92) 1.60 (1.00) 1.32 (1.92) sheet (N = 220) 2.27 (0.30) 1.22 (0.35) 0.51 (0.35) 1.41 (0.36) loop (N = 349) 2.07 (0.33) 2.78 (0.32) 1.72 (0.34) 2.47 (0.29) Rotamer outliers 0.78 1.38 0 0 (%) C outliers (%) 0 0 0 0 Peptide plane (%) Cis 9.1/0.0 8.6/0.0 8.8/0.0 10.3/0.0 proline/general Twisted 3.0/0.2 0.0/0.0 0.0/0.0 3.4/0.0 proline/general CaBLAM outliers 5.13 8.92 5.49 7.3 (%) ADP (B-factors) Iso/Aniso (#) 4532/0 4501/0 4491/0 4558/0 min/max/mean Protein 18.79/129.80/74.18 61.50/146.72/91.65 25.70/128.99/62.80 49.62/132.53/75.87 Nucleotide Ligand Water Occupancy Mean 1 1 1 1 occ = 1 (%) 100 100 100 100 0 < occ < 1 (%) 0 0 0 0 occ > 1 (%) 0 0 0 0 CC (mask) 0.65 0.61 0.77 0.58 CC (box) 0.67 0.66 0.78 0.66 CC (peaks) 0.64 0.61 0.77 0.59 CC (volume) 0.65 0.61 0.78 0.58 Resolution () 3.2 3.8 3.5 3.7

Immunofluorescence Studies

[0242] C3H/HeN 7-8 week old female mice (Envigo) bladders were fixed in formalin, embedded in paraffin, and sectioned. Tissue sections were heat deparaffinized and rehydrated in xylene, followed by stepwise hydration in 100% ethanol, 90% ethanol, 75% ethanol, 50% ethanol to 30% ethanol (each step having a 5 min incubation in fresh solution). Slides were rinsed in 1PBS followed by blocking solution (1PBS with 5% fetal bovine serum). Primary mouse antibody to uroplakin IIIa (Progen, 1:50) was allowed to bind overnight at 4 C. Slides were washed in 1PBS and a secondary anti-mouse Alexa fluor 488 antibody (Invitrogen, 1:1000) was allowed to bind for 2 h, and then washed again. FimH.sub.LD-Alexa Fluor 647 (588 nM) was mixed with mAb (6 uM) for 20 min at room temperature in 1PBS. FimH.sub.LD mAb mixtures were applied to the section along with Hoechst DNA dye (8 uM) for 20 min at room temperature. The slides were washed again in 1PBS and allowed to dry. ProLong gold antifade mountant (Invitrogen) was added and slides were imaged using the confocal function of a Zeiss Cell Observer Spinning Disk Confocal Microscope with a 10 air objective lens.

[0243] Splayed bladders were analyzed with a Zeiss Axio Observer D1 inverted fluorescence microscope equipped with an X-Cite120 mini LED light source (Excelitis Technologies) and DAPI, GFP, DsRed, and Cy5 filter sets. EC Plan-Neofluar (NA 0.075) 2.5 and EC Plan-Neofluar (NA 0.15) 5 objectives (Zeiss), an Axiocam 503 color camera (Zeiss), and ZEN 2 (blue version) software were used for image acquisition.

Mouse Infection Experiments

[0244] For acute and 2-week infection models, 7-8 week old female C3H/HeN mice (Envigo) were infected with 210.sup.8 CFUs of UT189. Intraperitoneal injections of mAb were given in 1PBS buffer 24 h before infection. Urines were taken by clean catch at specified time points. For obtaining serum, 3 to 4 mice per treatment group were bled at specific time points during the length of the experiment via submental bleeding method. At the conclusion of the experimental time points, mice were humanely sacrificed, and bladder and kidney organs were homogenized and tittered. For screening mAbs in the prophylactic infection model with UT189, if a phenotype was observed after 1 replicate, the experiment was repeated an additional 1-2 times.

TABLE-US-00011 TABLE8 PrimersforFimHMutagenesis Mutant Primer AA Codon Codon Name PrimerSequence pBAD N/A N/A pBAD33F ATGCCATAGCATTTTTTATCC (SEQ_ID_NO:294) pBAD33R GATTTAATCTGTATCAGG(SEQ_ID_NO:295) 4K AAA GAT(D) J96_FimH GCCTGTGATACCGCCAATGGTACC (K) 4KF (SEQ_ID_NO:296) J96_FimH GGCGGTATCACAGGCGAATGACC 4KR (SEQ_ID_NO:297) 7N AAT AAA(K) J96_FimH GCCTGTAAAACCGCCAAAGGTACC (N) 7NF (SEQ_ID_NO:298) J96_FimH GATAGCGGTACCTTTGGCGGTTTT 7NR (SEQ_ID_NO:299) 10A GCT GAT(D) J96_FimH GGTACCGATATCCCTATTGGCGGTG (A) 10AF (SEQ_ID_NO:300) J96_FimH GCCAATAGGGATATCGGTACCATTGGC 10AR (SEQ_ID_NO:301) 13I ATT AAA(K) J96_FimH GCTATCCCTAAAGGCGGTGGCAGC (I) 13I_F (SEQ_ID_NO:302) J96FimH CCACCGCCTTTAGGGATAGCGG 13I_R (SEQ_ID_NO:303) 17S AGC AAA(K) J96_FimH GGTGGCAAAGCCAATGTTTATGTAAACCT (S) 17SF TGCG(SEQID_NO:304) J96_FimH GTTTACATAAACATTGGCTTTGCCACCGC 17SR CAATAGG(SEQ_ID_NO:305) 19N AAT AAA(K) J96_FimH GCAGCGCCAAAGTTTATGTAAACCTTGC (N) 19NF (SEQ_ID_NO:306) J96_FimH GTTTACATAAACTTTGGCGCTGCCACC 19NR (SEQ_ID_NO:307) 21Y TAT GAT(D) J96_FimH GCCAATGTTGATGTAAACCTTGCGCCC (Y) 21YF (SEQ_ID_NO:308) J96_FimH GTTTACATCAACATTGGCGCTGCCACC 21YR (SEQ_ID_NO:309) 23N AAC AAA(K) J96_FimH GTTTATGTAAAACTTGCGCCCGTCGTG (N) 23NF (SEQ_ID_NO:310) J96_FimH GACGGGCGCAAGTTTTACATAAACATTGGC 23NR (SEQ_ID_NO:311) 25A GCG GAT(D) J96_FimH GTAAACCTTGATCCCGTCGTGAATGTGGGG (A) 25A-2F (SEQ_ID_NO:312) J96FimH CACGACGGGATCAAGGTTTACATAAACAT 25A-2R TGGC(SEQ_ID_NO:313) 27V GTC GAC(D) J96_FimH CCTTGCGCCCGACGTGAATG (V) 27VF (SEQ_ID_NO:314) J96_FimH CCACATTCACGTCGGGCGC 27VR (SEQ_ID_NO:315) 29N AAT AAA(K) J96_FimH GCCCGTCGTGAAAGTGGGG (N) 29NF (SEQ_ID_NO:316) J96_FimH GGTTTTGCCCCACTTTCACGAC 29NR (SEQ_ID_NO:317) 30V GTG GAT(D) J96_FimH CCCGTCGTGAATGATGGGCAAAAC (V) 30VF (SEQ_ID_NO:318) J96_FimH CGACCAGGTTTTGCCCATCATTCAC 30VR (SEQ_ID_NO:319) 33N AAC AAA(K) J96_FimH CGTGAATGTGGGGCAAAAACTGGTCG (N) 33NF (SEQ_ID_NO:320) J96_FimH CGAAAGATCCACGACCAGTTTTTGCCC 33NR (SEQ_ID_NO:321) 37D GAT AAA(K) J96_FimH CCTGGTCGTGAAACTTTCG (D) 37DF (SEQ_ID_NO:322) J96_FimH GCGTCGAAAGTTTCACGAC 37DR (SEQ_ID_NO:323) 40T ACG AAG(K) J96_FimH CGTGGATCTTTCGAAGCAAATC (T) 40TF (SEQ_ID_NO:324) J96_FimH GGCAAAAGATTTGCTTCGAAAG 40TR (SEQ_ID_NO:325) 43F TTT GAT(D) J96_FimH CGCAAATCGATTGCCATAACGATT (F) 43FF (SEQ_ID_NO:326) J96_FimH GGCAATCGATTTGCGTCGAAAGAT 43FR (SEQ_ID_NO:327) 48Y TAT GAT(D) J96_FimH GCCATAACGATGATCCGGAAACC (Y) 48YF (SEQ_ID_NO:328) J96_FimH CTGTAATGGTTTCCGGATCATCGTTATGG 48YR (SEQ_ID_NO:329) 50E GAA AAA(K) J96_FimH GCCATAACGATTATCCGAAAACCATTAC (E) 50EF (SEQ_ID_NO:330) J96_FimH GTCTGTAATGGTTTTCGGATAATCG 50ER (SEQ_ID_NO:331) 55Y TAT GAT(D) J96_FimH CCATTACAGACGATGTCACACTGC (Y) 55YF (SEQ_ID_NO:332) J96_FimH GTGACATCGTCTGTAATGGTTTCC 55YR (SEQ_ID_NO:333) 60R CGA AAA(K) J96_FimH CACTGCAAAAAGGCTCGGCTTATGGCGG (R) 60RF (SEQ_ID_NO:334) J96_FimH GCCGAGCCTTTTTGCAGTGTGACATAGTC 60RR (SEQ_ID_NO:335) 62S TCG AAG(K) J96_FimH GCAACGAGGCAAGGCTTATGGC (S) 62SF (SEQ_ID_NO:336) J96_FimH GCCGTTCCTCGTTGCAGTGTGACA 62SR (SEQ_ID_NO:337) 64Y TAT GAT(D) J96_FimH CTCGGCTGATGGCGGCGTGTTAT (Y) 64YF (SEQ_ID_NO:338) J96_FimH CCATCAGCCGAGCCTCGTTGCA 64YR (SEQ_ID_NO:339) 67V GTG GAT(D) J96_FimH GGCGGCGATTTATCTAATTTTTCCGGG (V) 67VF (SEQ_ID_NO:340) J96_FimH TTAGATAAATCGCCGCCATAAGCCGAG 67VR (SEQ_ID_NO:341) 70N AAT AAA(K) J96_FimH GTTATCTAAATTTTCCGGGACCG (N) 70NF (SEQ_ID_NO:342) J96_FimH GGAAAATTTAGATAACACGCCGC 70NR (SEQ_ID_NO:343) 74T ACC AAA(K) J96_FimH CCGGGAAAGTAAAATATAGTGGC (T) 74TF (SEQ_ID_NO:344) J96_FimH CTATATTTTACTTTCCCGGAAAAATTAG 74TR (SEQ_ID_NO:345) 76K AAA GAT(D) J96_FimH CCGGGACCGTAGATTATAGTGGCAGTAGC (K) 76KF (SEQ_ID_NO:346) J96_FimH GCCACTATAATCTACGGTCCCGGAAAAAT 76KR TAG(SEQ_ID_NO:347) 78N AGT AAA(K) J96_FimH CGTAAAATATAAAGGCAGTAGCTATCC (S) 78NF (SEQ_ID_NO:348) J96_FimH CTGCCTTTATATTTTACGGTCCC 78NR (SEQ_ID_NO:349) 80S AGT AAA(K) J96_FimH GTGGCAAAAGCTATCCATTTCC (S) 80SF (SEQ_ID_NO:350) J96_FimH GGATAGCTTTTGCCACTATATTTTACGG 80SR (SEQ_ID_NO:351) 87T ACC AAA(K) J96_FimH CCTACCAAAAGCGAAACGCCG (T) 87TF (SEQ_ID_NO:352) J96_FimH CGTTTCGCTTTTGGTAGGAAATGG 87TR (SEQ_ID_NO:353) 89E GAA AAA(K) J96_FimH CCTACCAAAAGCGAAACGCCGC (E) 89EF (SEQ_ID_NO:354) J96_FimH CGGCGTTTTGCTGGTGGTAGGA 89ER (SEQ_ID_NO:355) 92R CGC GAC(D) J96_FimH CGCCGGACGTTGTTTATAATTCG (C) 92RF (SEQ_ID_NO:356) J96_FimH TAAACAACGTCCGGCGTTTCGC 92RR (SEQ_ID_NO:357) 96N AAT AAA(K) J96_FimH GCGTTGTTTATAAATCGAGAACGG (N) 96NF (SEQ_ID_NO:358) J96_FimH CCGTTCTCGATTTATAAACAACGCG 96NR (SEQ_ID_NO:359) 98R AGA AAA(K) J96_FimH GTTTATAATTCGAAAACGGATAAGCCG (R) 98RF (SEQ_ID_NO:360) J96_FimH GCTTATCCGTTTTCGAATTATAAACAACG 98RR (SEQ_ID_NO:361) 99T ACG AAG(K) J96_FimH GTTTATAATTCGAGAAAGGATAAGCCG (T) 99TF (SEQ_ID_NO:362) J96_FimH CGGCTTATCCTTTCTCGAATTATAAAC 99TR (SEQ_ID_NO:363) 101K AAG GAT(D) J96_FimH GAGAACGGATGATCCGTGGCCGGTG (K) 101KF (SEQ_ID_NO:364) J96_FimH CCGGCCACGGATCATCCGTTCTCGAA 101KR (SEQ_ID_NO:365) 110T ACG AAG(K) J96_FimH GGTGGCGCTTTATTTGAAGCCTGTGAGC (T) 110TF (SEQ_ID_NO:366) J96_FimH CGCACTGCTCACAGGCTTCAAATAAAGC 110TR (SEQ_ID_NO:367) 121K AAA GAT(D) J96_FimH GGCGATTGATGCTGGCTCATTAATTGC (K) 121KF (SEQ_ID_NO:368) J96_FimH GAGCCAGCATCAATCGCCACC 121KR (SEQ_ID_NO:369) 128V GTG GAT(D) J96_FimH CATTAATTGCCGATCTTATTTTGCGAC (V) 128VF (SEQ_ID_NO:370) J96_FimH CAAAATAAGATCGGCAATTAATGAGCC 128VR (SEQ_ID_NO:371) 132R CGA AAA(K) J96_FimH GCTTATTTTGAAACAGACCAACAAC (R) 132RF (SEQ_ID_NO:372) J96_FimH GGTCTGTTTCAAAATAAGCACGGC 132RR (SEQ_ID_NO:373) 139S AGC AAA(K) J96_FimH CAACTATAACAAAGATGATTTCCAG (S) 139SF (SEQ_ID_NO:374) J96_FimH GGAAATCATCTTTGTTATAGTTGTTGG 139SR (SEQ_ID_NO:375) 142F TTC GAC(D) J96_FimH CTATAACAGCGATGATGACCAGTTTGTGTGG (F) 142FF (SEQ_ID_NO:376) J96_FimH CACAAACTGGTCATCATCGCTGTTATAGTTG 142FR (SEQ_ID_NO:377) 145V GTG GAT(D) J96_FimH TTTCCAGTTTGATTGGAATATTTACG (V) 145VF (SEQ_ID_NO:378) J96_FimH GTAAATATTCCAATCAAACTGGAAATC 145VR (SEQ_ID_NO:379) 152N AAT AAA(K) J96_FimH CGCCAATAAAGATGTGGTGGTG (N) 152NF (SEQ_ID_NO:380) J96_FimH CACCACATCTTTATTGGCGTAA 152NR (SEQ_ID_NO:381) 155V GTG GAT(D) J96_FimH CCAATAATGATGTGGATGTGCCTACTGGC (V) 155VF (SEQ_ID_NO:382) J96_FimH GTAGGCACATCCACATCATTATTGGCG 155VR (SEQ_ID_NO:383)

Example 2Monoclonal Antibodies Targeting FimH Adhesin Protect Against UTI in a Murine Model

[0245] The K. pneumoniae FimH amino acid sequence is 86% similar to that of uropathogenic Escherichia coli (UPEC) and is highly conserved among K. pneumoniae isolates. FimH consists of an N-terminal lectin mannose binding domain and a pilin domain that connects it to the pilus. We generated monoclonal antibodies (mAbs) to UPEC and K. pneumoniae FimH lectin domains to identify mAbs that inhibit FimH binding and prevent infection in vivo. Using ELISA binding assays, we identified mAbs that bind with high affinity to the antigenic FimH (FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D). In addition, a subset of mAbs cross-reacted to both UPEC and K. pneumoniae FimH proteins and related chaperone usher pili galactose binding adhesin FmIH, which contributes to UPEC attachment in chronic UTI. Further, we identified monoclonal antibodies that inhibited K. pneumoniae and UPEC FimH lectin domain binding in vitro by performing binding inhibition ELISAs (FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D).

[0246] On the tip of a pilus, FimH is a two-domain protein that samples a conformational equilibrium between a high-affinity relaxed state and low-affinity tense state. The FimH lectin domain antigen is exclusively in the relaxed state. When tested for binding to UT189 bacteria in an ELISA assay, we found that mAbs differentially bind to the bacteria and not all mAbs bind with high affinity (FIG. 3A; LON designates fim locked on an overexpression strain as a positive control). In UPEC, naturally occurring FimH variants can strongly shift the conformational equilibrium towards either state. In tip-like two-domain FimH, the mutations A27V/V163 strongly shift FimH towards the relaxed conformation and A62S strongly shifts FimH towards tense. mAbs were tested for binding to these FimH conformational variants overexpressed on the surface of UT189 (FIG. 3B). We found that there were two subsets of mAbs: 1) a set that binds with slight preference to the relaxed conformational mutant and II) a set that binds with a strong preference to the relaxed state. 2H04 mAb was also tested for binding to K. pneumoniae isolate TOP52 by ELISA and immunogold and we found that it bound more strongly to UT189 corroborating previous work that shows K. pneumoniae FimH to be strongly skewed towards a low-affinity conformation (FIG. 20, FIG. 21A, FIG. 21B).

[0247] Using the subset of mAbs that bound with high-affinity to UT189 wild type bacteria, we tested 7 different representative mAbs from this subset for ability to block type 1 pili red blood cell hemagglutination. We found that most mAbs inhibited type 1 pili binding slightly at the highest concentration, but two mAbs (2H04 and 2C07) had a strong titratable inhibitory effect (FIG. 22)

[0248] We first tested our highest inhibiting mAbs from our FimH lectin domain protein assays for ability to protect in an acute murine model of UPEC UTI with an infection of 107 CFUs of UT189 (FIG. 23A, FIG. 23B). Two out of four mAbs (2H04 and F7) tested significantly reduced bacterial titers in the urine and bladders of infected mice. The two mAbs that did not affect infection were 2G04 and 2E08 which do not bind to UT189 bacteria as well as 2H04 and F7. These results showed that the ability to bind to native FimH on the pilus of bacteria is important for protection.

[0249] We then tested 7 different representative mAbs that bound well to UT189 bacteria and were tested in the hemagglutination assay in an acute model of infection. mAbs were given before infection to test preventative ability. We found that again 2H04 and F7 significantly protected mice from infection along with one other mAb 1A02 (FIG. 24A, FIG. 24B, FIG. 24C). Interestingly, 2C07 with the highest inhibitory power did not appear to significantly protect against infection suggesting that the ability to recognize and bind to native tip-like FimH drives protection.

[0250] Using cryo-EM, we obtained the structure of 2H04 fAb to FimH lectin domain (FIG. 25). We found that 2H04 bound to the base of the lectin domain, in an area that does not change dramatically between conformational states. Further, this suggests that the mechanism of inhibiting FimH binding is by steric hindrance and preventing FimH from reaching the mannose ligands, as opposed to the mAb binding directly to the mannose-binding pocket.

[0251] These results demonstrate that monoclonal antibodies inhibiting FimH function are an antibiotic-sparing therapeutic strategy for K. pneumoniae and UPEC UTIs.

Methods

Monoclonal Antibody Generation

[0252] Monoclonal antibodies against TOP52 and E. coli FimH truncates were generated. Briefly, C57BL/6 mice were immunized with 25 ug of purified lectin mixed 1:1 with the squalene oil-in-water adjuvant Addavax (Invivogen). 3 weeks post-immunization, mice were boosted with a second dose of the same protein. Mice were sacrificed at 5 days post-boost and B cells from draining lymph nodes were stained and sorted into 96 well plates. B cell RNA was converted to cDNA and the VDJ regions amplified and cloned into human IgG1 plasmid expression vectors.

Mab Expression and Purification

[0253] mAbs were expressed using the Expi293 protein expression system and purified on a Protein A column.