HER3 inhibitor for modulating radiosensitivity

Abstract

The present invention relates to the use of an inhibitor of HER-3 for the treatment of a hyperproliferative disease in combination with radiation treatment.

Claims

1. A method of treating lung cancer in a subject comprising: administering to the subject an anti-HER-3 antibody or an antigen-binding fragment thereof in combination with radiation treatment, wherein the radiation treatment comprises X-ray radiation, wherein the anti-HER-3 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence comprising SEQ ID NO:70 and a light chain variable region with an amino acid sequence comprising SEQ ID NO:72, and wherein the anti-HER-3 antibody or antigen-binding fragment thereof is administered before the X-ray radiation and sensitizes the lung cancer to the X-ray radiation.

2. The method of claim 1, wherein the anti-HER-3 antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a multispecific antibody, or an antigen-binding fragment thereof.

3. The method of claim 1, wherein said antigen-binding fragment of the anti-HER-3 antibody is a Fab fragment, a Fab′ fragment, a F(ab′).sub.2 fragment, a Fv fragment, a diabody, or a single chain antibody molecule.

4. The method of claim 1, wherein said anti-HER-3 antibody is coupled to an effector group and/or labelling group.

5. The method of claim 4, wherein said effector group is a radioisotope, a radionuclide, a toxin, a therapeutic group, and/or a chemotherapeutic group.

6. The method of claim 4, wherein said labelling group is a radioisotope or radionuclide, a fluorescent group, an enzymatic group, a chemiluminescent group, a biotinyl group, or a predetermined polypeptide epitope.

7. The method of claim 1, wherein the lung cancer is squamous cell carcinoma of the lung.

8. The method of claim 1, wherein the radiation treatment further comprises brachytherapy, radioisotope therapy, or heat treatment.

9. The method of claim 1, wherein the radiation treatment is based on a single dose or fractioned dosing of radiation.

10. The method of claim 1, wherein the method comprises administering a further active compound.

11. The method of claim 10, wherein the further active compound comprises a chemotherapeutic compound.

12. The method of claim 1 wherein the anti-HER-3 antibody or antigen-binding fragment thereof is administered 4 hours or 48 hours before the X-ray radiation.

13. The method of claim 12 wherein the X-ray radiation is 10 Gy or 16 Gy.

14. The method of claim 13 wherein the X-ray radiation is fractionated.

15. The method of claim 13 wherein the X-ray radiation is a single dose.

16. The method of claim 1 wherein the lung cancer is non-small cell lung cancer.

17. A method of treating lung cancer in a subject, the method comprising: administering to the subject an anti-HER-3 antibody or an antigen-binding fragment thereof, wherein the anti-HER-3 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence comprising SEQ ID NO:70 and a light chain variable region with an amino acid sequence comprising SEQ ID NO:72, and subsequently administering to the subject a radiation treatment, the radiation treatment comprising X-ray radiation, wherein the anti-HER-3 antibody or antigen-binding fragment thereof sensitizes the lung cancer to the X-ray radiation.

18. The method of claim 17 wherein the anti-HER3 antibody or antigen-binding fragment thereof is administered to the subject 4 hours or 48 hours prior to the X-ray radiation.

19. The method of claim 18 wherein the X-ray radiation is 10 Gy or 16 Gy.

20. The method of claim 19 wherein the X-ray radiation is fractionated.

21. The method of claim 19 wherein the X-ray radiation is a single dose.

22. The method of claim 17 wherein the lung cancer is squamous cell carcinoma of the lung.

23. The method of claim 17 wherein the lung cancer is non-small cell lung cancer.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) 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.

(2) FIG. 1.

(3) (A) HER3 profile in multiple carcinoma celllines.

(4) Five human lung carcinoma celllines (NCI-H226, H292, H358, H520 and A549), five colorectal carcinoma celllines (Caco2; SW48, LS180, Lovo and HCT116) and five head and neck carcinoma celllines (SCC1, SCC6, SCC11A, SCC38 and SCC1483) were cultured in relevant mediums. Whole cell lysate was obtained with lysis buffer. Protein was quantitated using the Bradford method, sepreated by SDS gel and followed by immunoblot with the indicated antibodies.

(5) (B) Basal activity of HER3 is blocked by U1-59.

(6) Four head and neck carcinoma celllines (SCC6, SCC11A, SCC38, SCC1483) with high p-HER3 and two lung cancer cellines (H226 and A549) were treated with doses of U1-59 for 24 h. Whole cell lysates were isolated and followed by immumoblot with indicated antibodies. U1-59 inhibited HER3 activity and its downstream signal in a dose-dependent manner in those cells.

(7) FIG. 2:

(8) Radiation-induced activation of HER3 is blocked by U1-59. (A, D) HER3 was transient activated by radiation during the indicated time and blocked by U1-59. SCC6 and H226 cells were incubated with/without U1-59 (20 ug/nnl) for 24 h before radiation. After exposure to 4Gy radiation, whole cell lysates were isolated at the indicated times, followed by immunoblot with p-HER, p-AKT and p-MAPK antibodies. (B, E) U1-59 kept blocking activations of AKT and MAPK 24 h and 48 h after radiation. (C, F) Immunoflurescence showing HER3 were activated by radiation and blocked by U1-59 in SCC6 and H226 cells (30 minutes after radiation).

(9) FIG. 3.

(10) (A, F) U1-59 inhibited cell proliferation in a dose-dependent manner. SCC6 and H226 cells were seeded in 96-well plates and incubated with doses of U1-59 for 72 h. Cell proliferation were detected using CCK8 72 h. (B, G) Sensitizing effect of U1-59 on SCC6 cells in response to radiation. SCC6 and H226 were incubated with U1-59 for 4 h, and radiated with indicated doses. Clonegenic assay were performed as described. Control curves were exposed to radiation without U1-59 treatment.

(11) (C, H) Impact of U1-59 on cell cycle.

(12) SCC6 and H226 cells were incubated with U1-59 (20 ug/ml) for 48 h, followed by exposure to 6Gy radiation. 24 h after radiation, cell cycle (stained with PI) was detected using flow cytometer and analyzed by FlowJo. U1-59 caused G1 arrest and radiation cuased G2 arrest. Combination of U1-59 and radiation reassorted cell within cell cycle, induced accumulation of cells in G1 and G2, and reduced the population of cells in S phase.

(13) (D, I) Impact of U1-59 on cell apoptosis.

(14) Apoptosis (stained with annexin V/PI) was detected using flow cytometer and analyzed by FlowJo. Combination of U1-59 with radiation increased the percentage of apoptotic cells.

(15) (E, J) U1-59 contribute to γ-H2AX focus formation.

(16) SCC6 and H226 were incubated with U1-59 for 24 h before 4 Gy radiation. 4 h after radiation, cells were fixed and performed immunoflurescence staining. Compared with drug or radiation alone, increased number of γ-H2AX foci was detected in the cell treated with XRT combined with U1-59, demonstrated enhanced DNA damage by U1-59

(17) FIG. 4.

(18) Antitumour effect of U1-59 combined with radiation on xenograft tumours in athymic mice. SCC6, SCC1483 and H226 xenograft were performed as described in “Materials and methods”. (A) Mice were treated with single dose of IgG, radiation (10Gy or 16Gy), U1-59 (8 mg/kg) or combination of both. Data points were expressed as mean tumour size±SD. Results were graphed with tumour growth curve and Kaplan Meir survival curve. (B) Mice were treated with fractionated doses of IgG, radiation, U1-59 (100 ug/mouse) or combination of both twice a week as shown. Results were graphed with tumour growth curve and Kaplan Meir survival curve.

(19) FIG. 5.

(20) U1-59 inhibits basal and radiation-induced activation of HER3 in xenograft tumours. SCC6, SCC1483 and H226 xenograft tumours were collected at 24 h post radiation or the last radiation in fractionated treatment groups. Protein was isolated from the tumours and p-HER3 was analyzed by western blot (FIG. 5A and FIG. 5B). Increased p-HER3 was found in radiated tumours and reduced p-HER3 was found in tumours with U1-59 or combined treatment.

(21) FIG. 6.

(22) U1-59 inhibits cell survival signalsand enhances DNA damage in combination with radiation on xenograft tumours in athymic mice. SCC6 xenograft tumours with single dose treatment of radiation (16 Gy), U1-59 (8 mg/kg) or combination of both. tumours were harvested 24 h post treatment. IHC stain were performed as describe in “Materials and methods”. Reduced p-MAPK, p-S6 and PCNA staining, decreased PCNA positive cells and increased γ-H2AX positive cells were detected in tumours with U1-59 or combined treatment.

EXAMPLES

(23) Materials and Methods

(24) Cell Culture and Drug

(25) Five human lung carcinoma celllines (NCI-H226, H292, H358, H520 and A549 and five colorectal carcinoma celllines (Caco2, SW48, LS180, Lovo and HCT116) were purchased from ATCC (Manassas, Va., USA) and maintained in 10% fetal bovine serum (FBS) in RPMl1640 or DMEM (Mediatech Inc., Manassas, Va., USA) with 1% penicillin and streptomycin. Five head and neck carcinoma celllines (UM-SCC1, UM-SCC4, UM-SCC6, UM-SCC11A, and UM-SCC1483 cells) were obtained from University of Michigan and maintained in 10% FBS (Invitrogen, Carlsbad, Calif., USA) in DMED supplemented with 1% hydrocortisone.

(26) The anti-HER-3 antibody U1-59 was used.

(27) Cell Proliferation Assay

(28) Cells were seeded in 96-well plate and exposed to doses of U1-59 for 72 h. Cell proliferation was tested by Cell Counting Kit-8 (Dojindo Molecular Technologies, Gaithersbury, Md., USA).

(29) Clonogenic Assay

(30) A specified number of cells were seeded into each well of six-well tissue culture plates. After allowing cells time to attach (6 hours), U1-59 or the vehicle control (PBS) was added at specified concentrations. The plates were irradiated 4 hours later at the doses of 2, 4, 6, 8Gy. 10 to 14 days after seeding, colonies were stained with crystal violet, the number of colonies containing at least 50 cells was determined and the surviving fractions were calculated. Survival curves were generated after normalizing for cytotoxicity generated by U1-59 alone. Data presented are the mean±SD from at least three independent experiments.

(31) Cell Cycle Analysis

(32) Cells were incubated with U1-59 (20 ug/ml) for 48 h, followed 6Gy radiation. After 24 h, cells were trypsinized and washed with PBS, fixed in 90% ethanol and stored at 4° C. overnight. After remove of ethanol by centrifugation, cells were stained with PI stain buffer (50 ug/ml PI, 100 ug/ml Rnase A, 0.1% Triton X-100). Cells were sorted by FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif., USA). Histogram analysis was performed with FlowJo software (Tree Star Inc., Ashland, Oreg., USA).

(33) Apoptosis

(34) Apoptosis were detected using Annexin V/PI kit. Following treatment, a cell suspension containing 1×10.sup.5 cells in 100 μl staining buffer was incubated with 5 μl Annexin V and PI. Cells were sorted by FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif., USA). Population analysis was performed with FlowJo software.

(35) Immunoblotting Analysis

(36) Following treatment, cells were lysed with buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10 μg/ml of leupeptin and aprotinin). Protein was quantized using a standard Bradford absorbance assay. Equal amounts of protein were fractionated by SDS-PAGE. Thereafter, proteins were transferred to PVDF membrane and analyzed by incubation with the appropriate primary antibody. Proteins were detected via incubation with HRP-conjugated secondary antibodies and ECL chemiluminescence detection system. The NIH ImageJ program was used to measure densitometry of the western bands. The antibodies used in this study were as follows: HERS, AKT, MAPK, horseradish peroxidase-conjugated goat-anti-rabbit IgG and goat-anti-mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif., USA). p-HER3 (Tyr1289), p-AKT and p-MAPK were obtained from Cell Signaling Technology (Beverly, Mass., USA). α-tubulin was from Calbiochem (San Diego, Calif., USA).

(37) Immunofluorescence Assay

(38) Approximately 2×10.sup.3 cells were seeded on a four-well glass chamber slide (Nalgene Nunc, Naperville, Ill., USA). Forty-eight hours later, cells were washed 3 times with PBS and fixed with 2% formaldehyde for 15 min at room temperature. Cells were incubated in ice-cold 100% methanol for 10 min at −20° C. and blocked with 5% normal serum in PBS with 0.3% Triton X100 solution for 1 h at room temperature and incubated with p-HER3 or λ-H2AX antibody overnight at 4° C. Next, cells were incubated with FITC-conjugated appropriate secondary antibody in PBS containing 0.3% Triton X100 and 1% BSA for 2 h. Slides were mounted using ProLong gold with DAPI (Invitrogen). Photographs were captured by confocal microscopy or fluorescence microscopy.

(39) Mouse Xenograft Model

(40) Athymic nude mice (4- to 6-week old; male) were obtained from the Harlan Laboratories (Indianapolis, Ind., USA). All animal procedures and maintenance were conducted in accordance with the institutional guidelines of the University of Wisconsin. Cells were injected bilaterally in the dorsal flanks of the mice at day 0 (2×106 cells). Once tumours reached expected volumes, Mice were single or fractionated dosed-treated with 1) IgG, 2) U1-59, 3) radiation or 4) the combination. Measurements were evaluated by digital calipers and calculated by the formula (π)/6×(large diameter)×(small diameter).

(41) Immunohistochemistry

(42) Xenograft Tumours were Fixed in Neutral Formalin and Embedded in Paraffin.

(43) Immunohistochemical staining was performed for PCNA, p-MAPK, p-AKT, p-S6 and γ-H2AX. In brief, specimen was deparaffinized and rehydratded routinely, following antigene retrieval by citrate buffer for 15 mins at 98° C. in water bath, incubation in 3% hydrogen peroxide for 10 minutes, 3% BSA blocking for 30 min. Staining were performed as below: incubation in primary antibody diluted in recommended antibody diluents at 4° C. overnight, incubation in biotinylated secondary antibody for 30 minutes at room temperature, peroxidase visualization using Dakocytomation Liquid DAB+Substracte Chromogen System, Counterstain in Hematoxylin, routine Dehydrate and Clear, mounting with coverslip.

(44) Statistical Analysis

(45) Xenograft tumour growths were graphed and analyzed using GraphPad Prism 5 (GraphPad, San Diego, Calif.).

(46) Results

(47) HER3 is Expressed in Multiple Solid Tumour Cell Lines

(48) Human epidermal growth factor receptor 3 (HER3) is a key dimerization partner for the HER family and activates oncogenic signaling pathways. Its overexpression in many solid tumours has been linked to poor prognosis. In FIG. 1A we charactered 15 cell lines derived from non-small cell lung carcinoma (H226, H292, H358, H520 and A549), colorectal carcinoma (Caco2, SW48, LS180, Lovo and HCT116) and head and neck squamous cell carcinoma (SCC1, SCC6, SCC11A, SCC38 and SCC1483) for the expression of both HER3 and p-HER3. Both HNSCC and NSCLC cell lines contain activated HER3 suggesting that HER3 may be enhancing their tumourigenic phenotype.

(49) U1-59 can Inhibit Basal Activity of HER3 and Radiation-Induced Activation of HER3

(50) HER3 lacks intrinsic tyrosine kinase activity. However, upon binding to multiple ligands, such as heregulin (neuregulin-1), HER3 can form heterodimers with other HER family member receptors to initiate the activation of multiple signaling pathways that strongly influence cell proliferation and survival. U1-59 is a fully humanized monoclonal antibody that binds to and inactivates HER3 oncogenic signaling pathways. In FIG. 1B, we demonstrate that treatment of multiple HNSCC and NSCLC cell lines with escalating doses of U1-59 can successfully inhibit the activation of both HER3 and AKT (FIG. 1B). In addition, using immunoblotting and immunofluorescence assays, we found that radiation treatment of the HNSCC cell line SCC6 and the NSCLC cell line H226 increased activated forms of HER3 (FIGS. 2A, 2C, 2D, and 2F). The transient activation of HER3 after radiation treatment was also accompanied by an increase in activation of both ERK and AKT in both SCC6 and H226 cell lines (FIGS. 2A and 2D). In FIGS. 2B and 2E we demonstrate that radiation can lead to the continual activation of HER3, AKT, and ERK in both SCC6 and H226 cell lines both 24 and 48 hours post radiation treatment. We subsequently demonstrate that the pre-incubation of either SCC6 or H226 cell lines with U1-59 for 24 hrs prior to radiation treatment could prevent the radiation-induced activation of HER3, ERK, and AKT (FIGS. 2A, 2C, 2D, and 2F). Additionally, U1-59 could lead to the continued inhibition HER3, ERK, and AKT activation at both 24 and 48 hours post radiation treatment (FIGS. 2B and 2E). Collectively, these data suggest that pre-incubation of cells with U1-59 prior to radiation treatment can prevent radiation induced activation of cell survival and proliferation pathways.

(51) U1-59 can Radiosensitize HNSCC and NSCLC Cell Lines In-Vitro

(52) In FIG. 2 we demonstrated that U1-59 could prevent radiation-induced activation of HER3 and subsequent downstream effectors ERK and AKT, therefore we hypothesized that U1-59 could enhance the radiosensitivity of both HNSCC and NSCLC cell lines. First, we demonstrate in FIGS. 3A and 3F that U1-59 can inhibit the growth of both SCC6 and H226 cell lines in a dose-dependent manner. To evaluate the in vitro effect of U1-59 combination with radiation, we conducted clonogenic assays as described in “materials and methods”. We pre-incubated both SCC6 and H226 cells with U1-59 for 4 hr, and subsequently subjected cells to radiation with the indicated doses. As shown in FIGS. 3B and 3G, U1-59 increased the slope of the radiation curves, demonstrating a sensitizing effect of U1-59 on both SCC6 and H226 cells in response to radiation. Control curves were exposed to radiation without U1-59 treatment.

(53) U1-59 can Promote Cell Cycle Arrest and Apoptosis in Combination with Radiation Treatment In-Vitro

(54) We further hypothesized that U1-59 may also enhance HNSCC and NSCLC cell lines to radiation induced cell cycle arrest and apoptosis. To study this we pre-incubated both SCC6 and H226 cells with U1-59 for 48 h followed by exposure to 6Gy radiation. 24 h post radiation we analyzed the cell cycle phase distribution and apoptotic levels by PI and annexin V staining. As shown in FIGS. 3C and 3H U1-59 pre-treated cells demonstrated a G1 phase arrest, while radiation treated cells demonstrated a G2 phase arrest as compared to control cells. We observe a decrease in S-phase with either treatment. It is well known that cells in G1 phase are more sensitive to radiation treatment, while S-phase cells are more resistant. Therefore we demonstrate through Annexin V staining that apoptosis is indeed enhanced in radiation treated cells pre-incubated with U1-59 (FIGS. 3D and 3I), suggesting that the G1 phase arrest induced by U1-59 may have promoted the radiosentitivy of both SCC6 and H226. In addition, we further show an enhanced level of DNA double strand breaks via γ-H2AX staining in SCC6 and H226 cells pre-treated with U1-59 prior to radiation (FIGS. 3E and 3J). Overall, these data demonstrate the enhanced antitumour effect of U1-59 in combination with radiation.

(55) U1-59 in Combination with Radiation can have Antitumour Effects in HNSCC and NSCLC Mouse Tumour Xenografts

(56) To further evaluate the efficacy of radiotherapy combined with U1-59, we inoculated SCC6, SCC1483 and H226 cell lines into athymic mice. Once tumours reached 100-200 mm.sup.3 tumours were divided up for both single and fractionated doses of radiation. In the single dose treated group, xenograft mice were administered with IgG, U1-59 (8 mg/kg), radiation (XRT16Gy or XRT10Gy) or a combination of both (FIG. 4A). In fractionated treated group, xenograft mice were administered with IgG, U1-59 (100 ug/mouse), radiation (twice a week at indicated doses), or combination (FIG. 4B). All three xenografts models demonstrated a marked reduction in tumour volume when treated with both U1-59 in combination with either a single dose or fractionated dose of radiation as compared to single therapy treated mice or IgG control treated mice. Additionally, xenografts treated with both U1-59 and either a single or fractionated dose of radiation demonstrate a clear survival advantage as depicted via Kaplan Meir survival curve analysis.

(57) To validate that U1-59 could effectively inhibit the activation of HER3 in-vivo, we isolated protein from various tumours from each treatment group and performed western blot analysis for both total and activated forms of HER3.

(58) In FIG. 5A we demonstrate that tumours isolated from mice treated with U1-59 alone or U1-59 in combination with a single dose of radiation have lower levels of HER3 activation. In SCC1483 and H226 xenograft models U1-59 treated tumours also contained lower total amounts of HER3 protein (FIG. 5A), something that was not observed in these cell lines treated with U1-59 in-vitro. Additionally, at least one of the two tumours isolated from each xenograft model in FIG. 5A demonstrated a clear activation of HER3 upon a single dose of radiation, which was blocked in the U1-59 treated tumours. In FIG. 5B we further demonstrate that U1-59 treated tumours in combination with fractionated doses of radiation also contained reduced levels of activated and total levels of HER3; activation of HER3 by fractionated doses of radiation was also blocked by U1-59 as well. Collectively, xenograft tumours treated with both U1-59 in combination with either a single dose or fractionated doses of radiation demonstrate a clear growth delay with marked reduction in tumour volumes as compared to controls or either treatment alone, suggesting that the loss of both activated and total levels of HER3 expression may radiosensitize HNSCC and NSCLC tumours.

(59) U1-59 in Combination with Radiation can Prevent the Activation of Cell Survival Signals and Enhance DNA Damage In-Vivo

(60) To further analyze the growth inhibitory effects of U1-59 in combination with radiation treatment, we analyzed the activation of ERK, AKT, S6 kinase, and γ-H2AX in SCC6 treated tumours via immunohistochemistry (FIG. 6). We demonstrate that SCC6 cells treated with both U1-59 and a single dose of radiation can have lower activation levels of the downstream effector molecules ERK, AKT, and S6 kinase. Additionally, U1-59 treated tumours demonstrated a decreased level of PCNA positive cells. Finally, increased levels of γ-H2AX were found in SCC6 tumours treated with both U1-59 and radiation. Overall, these data indicate that U1-59 in combination with radiation can efficiently inhibit cell survival and proliferation pathways, in addition to enhancing DNA damage. These data provide mechanistic evidence for the enhanced antitumour effect of U1-59 combined with radiation in vivo.

(61) The results of these experiments indicate that the combination of U1-59 and radiation had a strong impact on tumour growth in studies using single dose or fractionated dosing of radiation. tumour analysis indicated that radiation treatment activated HER3 in vivo and U1-59 could abrogate this activation. Collectively our findings in vitro and in vivo indicate that U1-59 in combination with radiation has an impact on cell and tumour growth by delaying cell cycle progression, increasing apoptosis and increasing DNA damage. These findings indicate that HER3 may play an important role in response to radiation therapy and blocking its activity may be of strong therapeutic benefit in human tumours.

(62) TABLE-US-00001 Table of CDR Sequences Ab Pat. chain ID: CDR1 CDR2 CDR3 heavy U1-1 GGSINSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 235) (SEQ ID NO: 236) (SEQ ID NO: 237) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 238) (SEQ ID NO: 239) (SEQ ID NO: 240) heavy U1-2 GGSISSGDYYWS YIYYSGSTYYNPSLRS ADYDFWSGYFDY (SEQ ID NO: 241) (SEQ ID NO: 242) (SEQ ID NO: 243) light RASQGIRNDLG AASSLQS LQHNGYPWT (SEQ ID NO: 244) (SEQ ID NO: 245) (SEQ ID NO: 246) heavy U1-3 GGSISSGGYYWS YIYYSGSTYYNPSLKS DGYDSSGYYHGYFDY (SEQ ID NO: 247) (SEQ ID NO: 248) (SEQ ID NO: 249) light KSSQSVLYSSNNKNYLA WASTRES QQYYSTPLT (SEQ ID NO: 250) (SEQ ID NO: 251) (SEQ ID NO: 252) heavy U1-4 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 253) (SEQ ID NO: 254) (SEQ ID NO: 255) light RASQGIRNDLG AASSLQS LQHNNYPWT (SEQ ID NO: 256) (SEQ ID NO: 257) (SEQ ID NO: 258) heavy U1-5 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 259) (SEQ ID NO: 260) (SEQ ID NO: 261) light RASQGIRNDLG AASSLQS LQHNTYPWT (SEQ ID NO: 262) (SEQ ID NO: 263) (SEQ ID NO: 264) heavy U1-6 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWNGYFDY (SEQ ID NO: 265) (SEQ ID NO: 266) (SEQ ID NO: 267) light RASQGIRNDLG AASSLQS LQHNTYPWT (SEQ ID NO: 268) (SEQ ID NO: 269) (SEQ ID NO: 270) heavy U1-7 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 271) (SEQ ID NO: 272) (SEQ ID NO: 273) light RASQDIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 274) (SEQ ID NO: 275) (SEQ ID NO: 276) heavy U1-8 GYTLTELSMY GFDPEDGETIYAQKFQG GWNYVFDY (SEQ ID NO: 277) (SEQ ID NO: 278) (SEQ ID NO: 279) light RSSQSLLHSNGYNYLD LDSHRAS MQALQTPLT (SEQ ID NO: 280) (SEQ ID NO: 281) (SEQ ID NO: 282) heavy U1-9 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWNGYFDY (SEQ ID NO: 283) (SEQ ID NO: 284) (SEQ ID NO: 285) light RASQDIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 286) (SEQ ID NO: 287) (SEQ ID NO: 288) heavy U1-10 GGSISSGDYYWS YIYYSGSTYYVPSLKS ADYDFWSGYFDY (SEQ ID NO: 289) (SEQ ID NO: 290) (SEQ ID NO: 291) light RASQGIRNDLG AASSLQS LQHNNYPWT (SEQ ID NO: 292) (SEQ ID NO: 293) (SEQ ID NO: 294) heavy U1-11 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 295) (SEQ ID NO: 296) (SEQ ID NO: 297) light RASQGIRNDLG AASSLQS LQHNTYPWT (SEQ ID NO: 298) (SEQ ID NO: 299) (SEQ ID NO: 300) heavy U1-12 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 301) (SEQ ID NO: 302) (SEQ ID NO: 303) light RASQGIRNDLG AASSLQS LQHNNYPWT (SEQ ID NO: 304) (SEQ ID NO: 305) (SEQ ID NO: 306) heavy U1-13 GGSISSGGYYWS YIYYSGSTYYNPSLKS EDDGMDV (SEQ ID NO: 307) (SEQ ID NO: 308) (SEQ ID NO: 309) light RSSQSLLHSNGYNYLE LGSNRAS MQALQTPIT (SEQ ID NO: 310) (SEQ ID NO: 311) (SEQ ID NO: 312) heavy U1-14 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 313) (SEQ ID NO: 314) (SEQ ID NO: 315) light RASQGIRNDLG AASSLQS LQHNTYPWT (SEQ ID NO: 316) (SEQ ID NO: 317) (SEQ ID NO: 318) heavy U1-15 GGSVSSGGYYWS YIYYSGSTNYNPSLKS DGDVDTAMVDAFDI (SEQ ID NO: 319) (SEQ ID NO: 320) (SEQ ID NO: 321) light RASQSLSGNYLA GASSRAT QQYDRSPLT (SEQ ID NO: 322) (SEQ ID NO: 323) (SEQ ID NO: 324) heavy U1-16 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 325) (SEQ ID NO: 326) (SEQ ID NO: 327) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 328) (SEQ ID NO: 329) (SEQ ID NO: 330) heavy U1-17 GGSISSGDYYWS YIYYSGSTYYNSSLKS ADYDFWSGYFDY (SEQ ID NO: 331) (SEQ ID NO: 332) (SEQ ID NO: 333) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 334) (SEQ ID NO: 335) (SEQ ID NO: 336) heavy U1-18 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 337) (SEQ ID NO: 338) (SEQ ID NO: 339) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 340) (SEQ ID NO: 341) (SEQ ID NO: 342) heavy U1-19 GGSISSGDYYWS YIYYSGSTYYNPSLKS GDYDFWSGEFDY (SEQ ID NO: 343) (SEQ ID NO: 344) (SEQ ID NO: 345) light sequence not available heavy U1-20 GGSISSGGYYWS YIYDSGSTYYNPSLKS DQGQDGYSYGYGYYYGM (SEQ ID NO: 346) (SEQ ID NO: 347) DV (SEQ ID NO: 348) light QASQDISNYLN VASNLET QQCDNLPLT (SEQ ID NO: 349) (SEQ ID NO: 350) (SEQ ID NO: 351) heavy U1-21 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 352) (SEQ ID NO: 353) (SEQ ID NO: 354) light RASQDIRNDLG AASRLQS LQHNSYPWT (SEQ ID NO: 355) (SEQ ID NO: 356) (SEQ ID NO: 357) heavy U1-22 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 358) (SEQ ID NO: 359) (SEQ ID NO: 360) light RASQGIRNDLG AASSLQN LQHNSYPWT (SEQ ID NO: 361) (SEQ ID NO: 362) (SEQ ID NO: 363) heavy U1-23 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 364) (SEQ ID NO: 365) (SEQ ID NO: 366) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 367) (SEQ ID NO: 368) (SEQ ID NO: 369) heavy U1-24 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWNGYFDY (SEQ ID NO: 370) (SEQ ID NO: 371) (SEQ ID NO: 372) light RASQGIRNDLG AASSLQS LQHNNYPWT (SEQ ID NO: 373) (SEQ ID NO: 374) (SEQ ID NO: 375) heavy U1-25 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 376) (SEQ ID NO: 377) (SEQ ID NO: 378) light RASQGIRNDLG AASSLQN LQHNSYPWT (SEQ ID NO: 379) (SEQ ID NO: 380) (SEQ ID NO: 381) heavy U1-26 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDF (SEQ ID NO: 382) (SEQ ID NO: 383) (SEQ ID NO: 384) light RASQGIRNDLG AASSLQS LQHNGYPWT (SEQ ID NO: 385) (SEQ ID NO: 386) (SEQ ID NO: 387) heavy U1-27 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDP (SEQ ID NO: 388) (SEQ ID NO: 389) (SEQ ID NO: 390) light RASQGIRNDLG AASSLQS LQHNGYPWT (SEQ ID NO: 391) (SEQ ID NO: 392) (SEQ ID NO: 393) heavy U1-28 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDS (SEQ ID NO: 394) (SEQ ID NO: 395) (SEQ ID NO: 396) light RASQGIRNDLG AASSLQS LQHNGYPWT (SEQ ID NO: 397) (SEQ ID NO: 398) (SEQ ID NO: 399) heavy U1-29 GFTFNSYDMH VIWYDGSNKYYADSVKG DRLCTNGVCYEDYGMDV (SEQ ID NO: 400) (SEQ ID NO: 401) (SEQ ID NO: 402) light QASQDISNYLN DASNLET QHYDTLPLT (SEQ ID NO: 403) (SEQ ID NO: 404) (SEQ ID NO: 405) heavy U1-30 GGSISSGDYYWS YIYYSGTTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 406) (SEQ ID NO: 407) (SEQ ID NO: 408) light RAGQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 409) (SEQ ID NO: 410) (SEQ ID NO: 411) heavy U1-31 GYTFTNYGIS WISAYDGYRNYAQKLQG DVQDYGDYDYFDY (SEQ ID NO: 412) (SEQ ID NO: 413) (SEQ ID NO: 414) light RASQSISSYLN AASSLQS QQSYSTPIT (SEQ ID NO: 415) (SEQ ID NO: 416) (SEQ ID NO: 417) heavy U1-32 GGSISSGDYYWS YIYYSGTTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 418) (SEQ ID NO: 419) (SEQ ID NO: 420) light RAGQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 421) (SEQ ID NO: 422) (SEQ ID NO: 423) heavy U1-33 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGHFDC (SEQ ID NO: 424) (SEQ ID NO: 425) (SEQ ID NO: 426) light RASQGIRDDLG AESSLQS LQHHSYPWT (SEQ ID NO: 427) (SEQ ID NO: 428) (SEQ ID NO: 429) heavy U1-34 GYTFTNYGIS WISAYDGYRNYAQKLQG DVQDYGDYDYFDY (SEQ ID NO: 430) (SEQ ID NO: 431) (SEQ ID NO: 432) light RASQSISSYLN AASSLQS QQSYSTPIT (SEQ ID NO: 433) (SEQ ID NO: 434) (SEQ ID NO: 435) heavy U1-35 GFTFSDYYMS YISSSGNNIYHADSVKG ERYSGYDDPDGFDI (SEQ ID NO: 436) (SEQ ID NO: 437) (SEQ ID NO: 438) light QASQDISNYLS DASNLET QQYDNPPCS (SEQ ID NO: 439) (SEQ ID NO: 440) (SEQ ID NO: 441) heavy U1-36 GGSISSGYYYWS YIYYSGTTYYNPSFKS ADYDFWSGHFDY (SEQ ID NO: 442) (SEQ ID NO: 443) (SEQ ID NO: 444) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 445) (SEQ ID NO: 446) (SEQ ID NO: 447) heavy U1-37 GYTFTSYGIS WISAYDGHTNYAQKLQG DPHDYSNYEAFDF (SEQ ID NO: 448) (SEQ ID NO: 449) (SEQ ID NO: 450) light RASQSISSYLN AASSLQS QQSYSTPIT (SEQ ID NO: 451) (SEQ ID NO: 452) (SEQ ID NO: 453) heavy U1-38 GFSLSTSGVGVG LIYWNDDKRYSPSLKS RDEVRGFDY (SEQ ID NO: 454) (SEQ ID NO: 455) (SEQ ID NO: 456) light RSSQSLVYSDGYTYLH KVSNWDS MQGAHWPIT (SEQ ID NO: 457) (SEQ ID NO: 458) (SEQ ID NO: 459) heavy U1-39 GFTVSSNYMS VIYSGGSTYYADSVKG GQWLDV (SEQ ID NO: 460) (SEQ ID NO: 461) (SEQ ID NO: 462) light RSSQSLLHSNGYNYLD LGFHRAS RQALQTPLT (SEQ ID NO: 463) (SEQ ID NO: 464) (SEQ ID NO: 465) heavy U1-40 GGSISSGGYYWS YIYSSGSTYYNPSLKS DRELELYYYYYGMDV (SEQ ID NO: 466) (SEQ ID NO: 467) (SEQ ID NO: 468) light RSSQSLLYSNGYNYLD LGSNRAS MQALQTPLT (SEQ ID NO: 469) (SEQ ID NO: 470) (SEQ ID NO: 471) heavy U1-41 GGSISSGGYYWS YIYYSGSTYYNPSLKS DRELEGYSNYYGVDV (SEQ ID NO: 472) (SEQ ID NO: 473) (SEQ ID NO: 474) light RASQAISNYLN AASSLQS QQNNSLPIT (SEQ ID NO: 475) (SEQ ID NO: 476) (SEQ ID NO: 477) heavy U1-42 GYSFTSYWIG IIYPGDSDTRYSPSFQG HENYGDYNY (SEQ ID NO: 478) (SEQ ID NO: 479) (SEQ ID NO: 480) light RASQSIRSYLN AASSLQS QQSNGSPLT (SEQ ID NO: 481) (SEQ ID NO: 482) (SEQ ID NO: 483) heavy U1-43 GGSISSGGYYWS YIYYSGSTYYNPSLRS DREREWDDYGDPQGMDV (SEQ ID NO: 484) (SEQ ID NO: 485) (SEQ ID NO: 486) light RASQSISSYLH AASSLQS QQSYSNPLT (SEQ ID NO: 487) (SEQ ID NO: 488) (SEQ ID NO: 489) heavy U1-44 GYSFTSYWIG IIWPGDSDTIYSPSFQG HENYGDYNY (SEQ ID NO: 490) (SEQ ID NO: 491) (SEQ ID NO: 492) light RASQSIRSYLN AASSLQS QQSISSPLT (SEQ ID NO: 493) (SEQ ID NO: 494) (SEQ ID NO: 495) heavy U1-45 GYTFTSYDIN WMNPWSGDTGYAQVFQG FGDLPYDYSYYEWFDP (SEQ ID NO: 496) (SEQ ID NO: 497) (SEQ ID NO: 498) light RASQSISSYLN AASSLQS QQSYSTPLT (SEQ ID NO: 499) (SEQ ID NO: 500) (SEQ ID NO: 501) heavy U1-46 GDSVSSNSAAWN RTYYRSKWYNDYAVSVK DLYDFWSGYPYYYGMDV (SEQ ID NO: 502) S (SEQ ID NO: 504) (SEQ ID NO: 503) light sequence not available heavy U1-47 GDSVSSNSAAWN RTYYRSKWYNDYAVSVK DYYGSGSFYYYYGMDV (SEQ ID NO: 505) S (SEQ ID NO: 507) (SEQ ID NO: 506) light RASQSISSYLN AASNLQS QQSYSTPRT (SEQ ID NO: 508) (SEQ ID NO: 509) (SEQ ID NO: 510) heavy U1-48 GGSISSYYWS HIYTSGSTNYNPSLKS EAIFGVGPYYYYGMDV (SEQ ID NO: 511) (SEQ ID NO: 512) (SEQ ID NO: 513) light sequence not available heavy U1-49 GYTFTGYYMH WINPNIGGTNCAQKFQG GGRYSSSWSYYYYGMDV (SEQ ID NO: 514) (SEQ ID NO: 515) (SEQ ID NO: 516) light KSSQSLLLSDGGTYLY EVSNRFS MQSMQLPIT (SEQ ID NO: 517) (SEQ ID NO: 518) (SEQ ID NO: 519) heavy U1-50 GGSVSSGGYYWS YIYYSGSTNYNPSLKS GGDSNYEDYYYYYGMDV (SEQ ID NO: 520) (SEQ ID NO: 521) (SEQ ID NO: 522) light RASQSISIYLH AASSLQS QQSYTSPIT (SEQ ID NO: 523) (SEQ ID NO: 524) (SEQ ID NO: 525) heavy U1-51 GGSISSYYWS YIYYSGSTYYNPSLKS DSSYYDSSGYYLYYYAM (SEQ ID NO: 526) (SEQ ID NO: 527) DV (SEQ ID NO: 528) light KSSQSVLYSSNNKNYLA WASTRES QQYYTTPLT (SEQ ID NO: 529) (SEQ ID NO: 530) (SEQ ID NO: 531) heavy U1-52 GGSISSGGYYWS NIYYSGSTYYNPSLKS GGTGTNYYYYYGMDV (SEQ ID NO: 532) (SEQ ID NO: 533) (SEQ ID NO: 534) light RASQSVSSSYLA GASSWAT QQYGSSPLT (SEQ ID NO: 535) (SEQ ID NO: 536) (SEQ ID NO: 537) heavy U1-53 GFTFSIYSMN YISSSSSTIYYADSVKG DRGDFDAFDI (SEQ ID NO: 538) (SEQ ID NO: 539) (SEQ ID NO: 540) light QASQDITNYLN DASNLET QQCENFPIT (SEQ ID NO: 541) (SEQ ID NO: 542) (SEQ ID NO: 543) heavy U1-55.1 GGSVSSGGYYWN YINYSGSTNYNPSLKS DRELELYYYYYGMDV (SEQ ID NO: 544) (SEQ ID NO: 545) (SEQ ID NO: 546) light will be same as U1-55 heavy U1-55 will be same as U1-55.1 light RSSQSLLYSNGYKYLD LGSNRAS MQALQTPIT (SEQ ID NO: 547) (SEQ ID NO: 548) (SEQ ID NO: 549) heavy U1-57.1 will be same as U1-57 light RSSQSLLYSNGYKYLD LGSNRAS MQALQTPIT (SEQ ID NO: 550) (SEQ ID NO: 551) (SEQ ID NO: 552) heavy U1-57 GGSVSSGGYYWN YINYSGSTNYNPSLKS DRELELYYYYYGMDV (SEQ ID NO: 553) (SEQ ID NO: 554) (SEQ ID NO: 555) light will be same as U1-57.1 heavy U1-58 GFTFSSYGMH VIWYDGSNKYYADSVKG AARLDYYYGMDV (SEQ ID NO: 556) (SEQ ID NO: 557) (SEQ ID NO: 558) light RASQSINSYLN GASGLQS QQSYSSPLT (SEQ ID NO: 559) (SEQ ID NO: 560) (SEQ ID NO: 561) heavy U1-59 GGSFSGYYWS EINHSGSTNYNPSLKS DKWTWYFDL (SEQ ID NO: 562) (SEQ ID NO: 563) (SEQ ID NO: 564) light RSSQSVLYSSSNRNYLA WASTRES QQYYSTPRT (SEQ ID NO: 565) (SEQ ID NO: 566) (SEQ ID NO: 567) heavy U1-61.1 GVSISSGGYYWS YIYYSGSTYYNPSLKS DSESEYSSSSNYGMDV (SEQ ID NO: 568) (SEQ ID NO: 569) (SEQ ID NO: 570) light RASQTISSYLN AASSLQG QQSYSNPLT (SEQ ID NO: 571) (SEQ ID NO: 572) (SEQ ID NO: 573) heavy U1-61 GVSISSGGYYWS YIYYSGSTYYNPSLKS DSESEYSSSSNYGMDV (SEQ ID NO: 574) (SEQ ID NO: 575) (SEQ ID NO: 576) light will be the same U1-61.1 heavy U1-62 GYSFTSYWIG IIYPGDSDTRYSPSFQG QMAGNYYYGMDV (SEQ ID NO: 577) (SEQ ID NO: 578) (SEQ ID NO: 579) light RASQSVISIYLA GASSRAT QQYGSSPCS (SEQ ID NO: 580) (SEQ ID NO: 581) (SEQ ID NO: 582)