Antibodies having specificity for BTN2 and uses thereof

Abstract

The present invention relates to antibodies having specificity for BTN2 and uses thereof, in particular for the treatment of cancer.

Claims

1. A method of treating an autoimmune or inflammatory disorder or transplant rejection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-BTN2 antibody, wherein the anti-BTN2 antibody comprises a. the H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb 4.15 of SEQ ID NOs:3-8 respectively; b. the H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb 5.28 of SEQ ID NOs:11-16 respectively; c. the H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb 7.28 of SEQ ID NOs:19-24 respectively; d. the H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb 7.48 of SEQ ID NOs:27-32 respectively; e. the H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb 8.15 of SEQ ID NOs:35-40 respectively; or f. the H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb 8.16 of SEQ ID NOs:43-48 respectively.

2. The method of claim 1, wherein the anti-BTN2 antibody comprises a. a heavy chain wherein the VH region has the sequence SEQ ID NO:9 and a light chain wherein the VL region has the sequence SEQ ID NO:10; b. a heavy chain wherein the VH region has the sequence SEQ ID NO:17 and a light chain wherein the VL region has the sequence SEQ ID NO:18; c. a heavy chain wherein the VH region has the sequence SEQ ID NO:25 and a light chain wherein the VL region has the sequence SEQ ID NO:26; d. a heavy chain wherein the VH region has the sequence SEQ ID NO:33 and a light chain wherein the VL region has the sequence SEQ ID NO:34; e. a heavy chain wherein the VH region has the sequence SEQ ID NO:41 and a light chain wherein the VL region has the sequence SEQ ID NO:42; or f. a heavy chain wherein the VH region has the sequence SEQ ID NO:49 and a light chain wherein the VL region has the sequence SEQ ID NO:50.

3. The method of claim 1, wherein the autoimmune or inflammatory disorder is selected from the group consisting of: rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (Type 1 diabetes), multiple sclerosis (MS), Crohn's disease, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, pemphigus vulgaris, pemphigoid, Addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, coeliac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic leucopenia, idiopathic thrombocytopenic purpura, male infertility, mixed connective tissue disease, myasthenia gravis, pernicious anemia, phacogenic uveitis, primary biliary cirrhosis, primary myxoedema, Reiter's syndrome, stiff man syndrome, thyrotoxicosis, ulcerative colitis, and Wegener's granulomatosis.

4. The method of claim 1, wherein the anti-BTN2 antibody has specificity for both human butyrophilin-2A1 (BTN2A1) and human butyrophilin-2A2 (BTN2A2).

5. The method of claim 1, wherein the anti-BTN2 antibody does not cross-react with human CD277.

6. The method of claim 1, wherein the anti-BTN2 antibody is a human, chimeric or humanized antibody.

Description

LEGENDS OF THE FIGURES

(1) FIG. 1: Binding of BTN2 mAbs on HEK293F cells. HEK293F cells were transfected with BTN2A2-Flag plasmid. At day 1 after transfection, BTN2A2 transfected HEK293F cells (A) and non-transfected HEK293F cells (B) were stained with purified mouse anti-human BTN2 mAbs for 20 min at 4° C. Monoclonal ANTI-FLAG™ antibody serves as positive control for transfection. After 2 washes, cells were incubated with a secondary goat anti-mouse IgG PE for 20 min at 4° C. and a marker for cell viability. Cells were acquired on a LSRFortessa™ (BD) and analyzed with FlowJo™ (TreeStar).

(2) FIG. 2: BTN2 is expressed on cancer cell lines. Cancer cell lines derivated from solid tumors (A), or hematopoietic malignancies (B) were stained with purified BTN2 (8.16) mAb (10 μg/ml) for 20 min at 4° C. After 2 washes, cells were incubated with a secondary goat anti-mouse IgG PE for 20 min at 4° C. and a marker for cell viability. Cells were acquired on a LSRFortessa™ (BD) and analyzed with FlowJo™ (TreeStar). Cell lines were derived from: prostate cancer (PC3, DU145, LNCaP), melanoma (Gerlach), pancreatic cancer (L-IPC, Panc-1, Mia-PACA-2), colorectal cancer (Caco-2), Hela (cervical cancer), renal cancer (A498), lung cancer (A549), breast cancer (MDA-MB-134, MDA-MB-231, SKBR3), Burkitt lymphoma (Daudi, Raji), Non-Hodgkin's lymphoma (RL), chronic myelogenous leukemia (K562), acute myeloid leukemia (U937).

(3) FIG. 3: BTN2 mAbs inhibit the cytolytic function of Vγ9Vδ2 T cells. γδ-T cells were expanded from PBMCs of 6 healthy donors (see Material and Methods). Purified γδ-T cells were simulated overnight in IL-2 (200 UI/ml). Then, γδ-T cells were co-cultured at 37° C. with Daudi target cell line (at effector: target (E:T) ratio of 1:1) with anti-CD107a and anti-CD1072 antibodies and with or without anti-BTN2 (4.15, 5.28, 7.28, 7.48, 8.15, 8.16, 8.33) mAbs (10 μg/ml). After 4 h, cells were collected, fixed and permeabilized, then stained with intracellular mAb (IFN-γ, TNF-α) and analyzed by flow cytometry. The figure shows (A) the degranulation of γδ-T cells, and the production of inflammatory cytokines (B) TNF-α, (C) IFN-γ. Lower dashed line represents the basal cytolytic function of γδ-T cells against Daudi cell line.

(4) FIG. 4: BTN2 mAbs inhibit the agonist effect of anti-CD277 (20.1) mAb on the cytolytic function of Vγ9Vδ2 T cells. Purified γδ-T cells were simulated overnight in IL-2 (200 UI/ml). Then, γδ-T cells were co-cultured at 37° C. with Daudi target cell line (at effector: target (E:T) ratio of 1:1) with anti-CD107a and anti-CD1072 antibodies and Golgi stop, with or without anti-BTN2 (4.15, 5.28, 7.28, 7.48, 8.15, 8.16, 8.33) mAbs in combination or not with anti-CD277 20.1 mAb. After 4 h, cells were collected and analyzed by flow cytometry. The figure shows the percentage of CD107 (degranulation marker) positive cells among γδ-T cells. Lower dashed line represents the basal degranulation of γδ-T cells against Daudi cell line. Upper dashed line represents the median degranulation observed with anti-CD277 20.1 antibody.

(5) FIG. 5: BTN2 mAbs does not cross-react with HEK293F cells transfected with CD277 isoforms (BTN3A1, BTN3A2 or BTN3A3). HEK293F cells transfected with BTN3A1, BTN3A2, BTN3A3 and non-transfected HEK293F cells were stained with purified mouse anti-human BTN2 mAbs, or with mouse anti-human CD277 20.1 mAb, or with isotypic control IgG1 for 20 min at 4° C. ANTI-FLAG™ serves as positive control for transfection. After 2 washes, cells were incubated with a secondary goat anti-mouse IgG PE for 20 min at 4° C. and a marker for cell viability. Cells were acquired on a LSRFortessa™ (BD) and analyzed with FlowJo™ (TreeStar).

EXAMPLES

(6) Material and Methods

(7) Cell Culture

(8) Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteer donors (HV) provided by the local Blood Bank (EFS-Marseille-France) and isolated by density gradient (Eurobio).

(9) The Burkitt lymphoma cell line, Daudi, was obtained from the American Type Culture Collection and cultured (0.5×10.sup.6/mL) in RPMI 1640 medium with 10% FCS.

(10) Expansion of γδ-T Cells

(11) Effector γδ-T cells were established as previously described. PBMCs from HV were stimulated with Zoledronate (Sigma, 1 μM) and rhIL-2 (Proleukin, 200 IU/mL) at Day 0. From Day 5, rhIL-2 was renewed every two days and cells were kept at 1.15×10.sup.6/mL for 15 days. The last day, the purity of γδ T cells was evaluated by flow cytometry. Only cells cultures that reached more than 80% of γδ T Cells, were selected to be used in functional tests. Purified γδ-T cells were thawed until use.

(12) Generation of Monoclonal Antibodies (mAbs)

(13) The mouse anti-human BTN2 antibodies (clones 4.15, 5.28, 7.28, 7.48, 8.15, 8.16, 8.33 with IgG1 isotype) and mouse anti-human CD277 (also known as BTN3A; clone 20.1 with IgG1 isotype) were purified from cell culture supernatants.

(14) Flow Cytometry

(15) PBMCs, purified γδ-T cells or tumor cell lines were incubated with specified mAbs before analysis on LSRFortessa™ (Becton Dickinson) using DIVA software (BD bioscience). Antibodies used for γδ-T cell degranulation assay were: anti-CD107a-FITC (BD Biosciences), anti-CD107b-FITC (BD Biosciences), anti-CD3-PeVio700 (Miltenyi), anti-Tgd-PE (Miltenyi), live/dead near IR (Thermofisher). Antibodies used for screening of BTN2 expression on tumor cell lines were: purified anti-BTN2 (clone 8.16, 10 μg/ml), FcR Block reagent (Miltenyi), goat anti-mouse-PE (Jackson immunoresearch), live/dead near IR (Thermofisher).

(16) Functional assay on γδ-T cells

(17) Purified γδ-T cells from HV were cultured overnight in IL-2 (200 UI/ml). Then, γδ-T cells were co-cultured at 37° C. with Daudi target cell line (at effector: target (E:T) ratio of 1:1), and cytotoxic tests were performed in 4-hours assays in the presence of GolgiStop™ and soluble CD107 (a&b)-FITC, with or without anti-BTN2 (4.15, 5.28, 7.28, 7.48, 8.15, 8.16, 8.33) mAbs and/or anti-CD277 20.1 mAb (10 μg/ml), with or without activation by phosphoagonists (Pag). After 4 hours, cells were collected, fixed and permeabilized then stained with intracellular mAb (IFN-γ, TNF-α). Cells were finally re-suspended in PBS 2% paraformaldehyde and extemporaneously analyzed on a BD LSRFortessa™ (BD Biosciences, San Jose, Calif.). The degree of cytolytic function of γδ-T cells was measured based on the percentage of cells positive for CD107a and CD107b (degranulation) and/or the production of inflammatory cytokines (IFN-γ, TNF-α).

(18) Binding Test of Anti-BTN2 mAbs on for BTN2A1 and BTN2A2 Expressing Cells

(19) HEK-293F cells bearing CRISPR-Cas9-mediated deletions of all isoforms of BTN3 and BTN2 (293F BTN3/BTN2 KO) were generated (data not shown), cultured in DMEM (Life Technologies) 10% fetal bovine serum (FBS, Gibco) 1 mM sodium pyruvate (Thermofisher scientific), and transfected independently with pcDNA3-Zeo-BTN2A1-CFP or pcDNA3-Zeo-BTN2A2-CFP, which encode BTN2A1 and BTN2A2 CFP(Nter)-fusion proteins, using Lipofectamine™ 2000 reagent (Thermofisher scientific) according to manufacturer's instructions.

(20) Flow Cytometry

(21) Twenty-four hours after transfection, cells (5×10.sup.4/sample) were collected and stained in duplicate with the indicated concentrations (2-fold dilutions starting from 20 μg/mL to 64 pg/mL) of all 7 purified anti-human BTN2 mAbs in 50 μL of staining buffer (DPBS1X (Thermofisher Scientific) 1% FBS, 1 mM EDTA (Thermofisher Scientific)) during 30 minutes at 4° C. Equal concentrations of mouse IgG1 antibody (Miltenyi) were used as isotype control for staining. Then, cells were washed twice with 200 μL of staining buffer, and incubated with a 1:200 dilution of goat anti-mouse Ig-PE conjugated (Jackson Immunoresearch) in staining buffer for 30 min at 4° C. in the dark. Finally, cells were washed twice in staining buffer prior to fixation using BD Cytofix™ reagent (BD Bioscience) according to manufacturer's instructions. Mean fluorescence intensity (MFI) on PE channel within the CFP-positive population was assessed for each sample in a CytoFLEX LX (Beckman Coulter), and analyzed with a FlowJo™ V10.4.2 software (FlowJo™, LLC 2006-2018).

(22) Statistics

(23) EC.sub.50 of purified anti-human BTN2 mAbs on BTN2A1- and BTN2A2-transfected 293F BTN3/BTN2 KO cells were determined based on log(dose) response curves after non-linear regression following a variable-slope model. These analyses were performed using GraphPad Prism 7.04 software (GraphPad).

(24) Proliferation of γδ-T Cells

(25) γδ-T cells were isolated from PBMCs of healthy donors using anti-TCR γδ microbead kit (Miltenyi Biotec). The purity of γδ-T cells assessed by flow cytometry was greater than 80%. γδ-T cells were labeled with CellTrace™ Violet for 20 minutes at 37° C. Then, 5.Math.10.sup.5 CellTrace™-labeled cells were cultured in 96-well round-bottom plates in the presence of 200 UI/ml IL-2, with or without Pag, and with or without anti-BTN2 antibodies (10 μg/ml). After 5 days of culture, CellTrace™ dilution was evaluated by flow cytometry on a BD LSRFortessa™ (BD Biosciences, San Jose, Calif.).

(26) Statistics

(27) Results are expressed as median±SEM. Statistical analysis was performed using Spearman correlation, Wilcoxon test and Mann-Whitney t test. p values <0.05 were considered significant. Analyses were performed using GraphPad Prism program.

(28) Results

(29) Identification of the Reference Antibodies mAb1-mAb7

(30) The reference antibodies mAb 1-mAb7 were obtained as follows:

(31) Mice were immunized with BTN2A1-Fc antigen. The splenocytes of the mice were collected and fused with myeloma to obtain hybridomas. Hybridomas producing the antibodies with the highest affinity to BTN2 were screened and isolated, yielding the hybridomas as deposited under CNCM 1-5231, CNCM 1-5232, CNCM 1-5233, CNCM I-5234, CNCM 1-5235, CNCM 1-5236 and CNCM 1-5237 capable of producing mAb1-mAb7 respectively.

(32) The hybridoma producing mAb7 (mAb 8.33) which serves as a comparative control for the antibodies according to the present disclosure has been deposited at the Collection Nationale de Cultures des Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) in accordance with the terms of Budapest Treaty on Sep. 14, 2017.

(33) The deposited hybridoma for mAb 8.33 has CNCM deposit number CNCM 1-5237.

(34) Binding of mAbs 1-6 on HEK293F Cells.

(35) The graphs in FIG. 1 show a titration curve of the affinity of mouse anti-human BTN2 mAbs on HEK293F cells transfected with human BTN2A2 (FIG. 1A) or non-transfected HEK293F cells (FIG. 1B).

(36) The EC.sub.50 of each antibody is indicated in the table below. All the antibodies tested were able to recognize and bind to BTN2A2 onto the HEK cells, except mAb 8.33 (mAb7) that does not bind to BTN2A2 by flow cytometry.

(37) TABLE-US-00005 Antibody Clone Name EC.sub.50 (μg/ml) mAb1 mAb 4.15 0.07 mAb2 mAb 5.28 0.07 mAb3 mAb 7.28 31.3 mAb4 mAb 7.48 0.08 mAb5 mAb 8.15 0.1 mAb6 mAb 8.16 0.11 mAb7 mAb 8.33 —
BTN2 Polypeptide is Expressed on Cancer Cell Lines.

(38) Tumor cell lines were incubated with mAb6 (mouse anti-human BTN2 mAb 8.16) and then with secondary goat anti-mouse-PE. As shown in Figure A, a wide expression of BTN2 protein on panel of tumor cell lines, derivated from solid tumors or hematopoietic malignancies, was observed, including Daudi cell line (Burkitt lymphoma), a standard cell line used in Vγ9Vδ2 T cell degranulation assay.

(39) mAbs 1-6 Inhibit the Cytolytic Function of Vγ9Vδ2 T Cells.

(40) Purified Vγ9Vδ2 T cells were expanded from PBMCs of healthy donors. Vγ9Vδ2 T cells were co-cultured with Daudi target cells. As shown in FIG. 3, the addition of anti-BTN2 mAbs 1-6 lead to an inhibition of the cytolytic function of Vγ9Vδ2 T cells, as measured as CD107 degranulation, and the production of inflammatory cytokines (TNF-α, IFN-γ) against Daudi target cell line.

(41) Control antibody mAb 7 (mAb 8.33), which does not bind to BTN2A2 by flow cytometry, has no effect on Vγ9Vδ2 T cell degranulation (cytolytic function) or production of inflammatory cytokines.

(42) Anti-CD277 20.1 agonist antibody serves as a control example of an activating antibody of Vγ9Vδ2 T cell degranulation, and anti-CD277 103.2 antagonist antibody serves as a control example of an inhibiting antibody of Vγ9Vδ2 T cell degranulation.

(43) mAbs 1-6 Inhibit the Agonist Effect of Anti-CD277 (20.1) mAb on the Cytolytic Function of Vγ9Vδ2 T Cells.

(44) We also tested the effect of the combination of anti-BTN2 mAbs 1-6 in the presence of the agonist anti-CD277 20.1 mAb, (as disclosed in WO2012/080351) and known to increase the degranulation of Vγ9Vδ2 T cells.

(45) As shown in FIG. 4, mAbs 1, 2 and 4 (respectively anti-BTN2 mAbs 4.15, 5.28, 7.48) surprisingly inhibit the agonist effect of anti-CD277 20.1 antibody on the cytolytic function of Vγ9Vδ2 T cells against Daudi target cell line.

(46) mAbs 5 and 6 (respectively Anti-BTN2 8.15, 8.16) partially inhibit the agonist effect of anti-CD277 20.1 antibody on the cytolytic function of Vγ9Vδ2 T cells against Daudi target cell line.

(47) mAb 3 (anti-BTN2 7.28) and mAb 7 (anti-BTN2 8.33) does not show significant inhibition of the agonist effect of anti-CD277 20.1 antibody on the cytolytic function of Vγ9Vδ2 T cells against Daudi target cell line.

(48) The combination of antagonist 103.2 and agonist 20.1 anti-CD277 serves as a control: antagonist 103.2 antibody inhibits the agonist effect of 20.1 antibody on the cytolytic function of Vγ9Vδ2 T cells against Daudi target cell line, as previously described.

(49) The results of the characterization of mAbs 1-6 (and control mAb 7) are summarized hereafter:

(50) TABLE-US-00006 Effect on EC.sub.50 (μg/ml) Effect on γδ T cells/CD107 Clone on HEK-BTN2 cells γδ T cells/CD107 in presence of 20.1 4.15 0.07 Inhibition Inhibition 5.28 0.07 Inhibition Inhibition 7.28 31.30  Inhibition No effect 7.48 0.08 Inhibition Inhibition 8.15 0.10 Inhibition Partial inhibition 8.16 0.11 Inhibition Partial inhibition 8.33 — No No effect
mAbs 1-6 does not Cross-React with HEK293F Cells Transfected with CD277 Isoforms (BTN3A1, BTN3A2 or BTN3A3).

(51) Binding of BTN2 mAbs on HEK293F cells transfected with any one of the isoforms of CD277 (BTN3A1, BTN3A2 or BTN3A3) is similar as the one observed on non-transfected HEK293F cells (see FIG. 5). We can conclude that anti-BTN2 mAbs does not cross-react with one of the isoform of CD277.

(52) mAbs 1-6 has Binding Specificity with Human BTN2A1 and BTN2A2 Isoforms

(53) Binding of mAbs 1-6 on BTN3/BTN2 KO HEK293F Cells.

(54) EC50 of each antibody on BTN2A1 and BTN2A2 are indicated in the table below. All anti-BTN2 antibodies tested were able to bind both isoforms, excepting mAb 8.33 (mAb7), which does not display staining on flow cytometry.

(55) TABLE-US-00007 BTN2A1 BTN2A2 Antibody Clone Name EC.sub.50 (μg/ml) EC.sub.50 (μg/ml) mAb1 mAb 4.15 0.06 0.01 mAb2 mAb 5.28 0.04 0.01 mAb3 mAb 7.28 0.02 11.8 mAb4 mAb 7.48 0.02 0.01 mAb5 mAb 8.15 0.08 0.5 mAb6 mAb 8.16 0.08 2.4 mAb7 mAb 8.33 — —

(56) Summary Table:

(57) TABLE-US-00008 Tγδ degranulation (% of inhibition) Against target cells BTN2A1 BTN2A2 Against (Daudi) + Clone EC.sub.50 EC.sub.50 target cells anti-BTN3 Antibody Name (μg/ml) (μg/ml) (Daudi) (20.1) mAb mAb1 mAb 4.15 0.06 0.01 >90%  >90% mAb2 mAb 5.28 0.04 0.01 mAb3 mAb 7.28 0.02 11.8 ≤10% mAb4 mAb 7.48 0.02 0.01  >90% mAb5 mAb 8.15 0.08 0.5 ≤50% mAb6 mAb 8.16 0.08 2.4 mAb7 mAb 8.33 — — No effect No effect
Nucleotides and Amino Acid Sequences for Practicing the Claimed Invention

(58) TABLE-US-00009 SEQ ID Brief NO: Description 1 BTN2A1 aa MESAAALHFS RPASLLLLLL SLCALVSAQF IVVGPTDPIL sequence ATVGENTTLR CHLSPEKNAE DMEVRWFRSQ FSPAVFVYKG GRERTEEQME EYRGRTTFVS KDISRGSVAL VIHNITAQEN GTYRCYFQEG RSYDEAILHL VVAGLGSKPL ISMRGHEDGG IRLECISRGW YPKPLTVWRD PYGGVAPALK EVSMPDADGL FMVTTAVIIR DKSVRNMSCS INNTLLGQKK ESVIFIPESF MPSVSPCAVA LPIIVVILMI PIAVCIYWIN KLQKEKKILS GEKEFERETR EIALKELEKE RVQKEEELQV KEKLQEELRW RRTFLHAVDV VLDPDTAHPD LFLSEDRRSV RRCPFRHLGE SVPDNPERFD SQPCVLGRES FASGKHYWEV EVENVIEWTV GVCRDSVERK GEVLLIPQNG FWTLEMHKGQ YRAVSSPDRI LPLKESLCRV GVFLDYEAGD VSFYNMRDRS HIYTCPRSAF SVPVRPFFRL GCEDSPIFIC PALTGANGVT VPEEGLTLHR VGTHQSL 2 BTN2A2 aa MEPAAALHFS LPASLLLLLL LLLLSLCALV SAQFTVVGPA sequence NPILAMVGEN TTLRCHLSPE KNAEDMEVRW FRSQFSPAVF VYKGGRERTE EQMEEYRGRI TFVSKDINRG SVALVIHNVT AQENGIYRCY FQEGRSYDEA ILRLVVAGLG SKPLIEIKAQ EDGSIWLECI SGGWYPEPLT VWRDPYGEVV PALKEVSIAD ADGLFMVTTA VIIRDKYVRN VSCSVNNTLL GQEKETVIFI PESFMPSASP WMVALAVILT ASPWMVSMTV ILAVFIIFMA VSICCIKKLQ REKKILSGEK KVEQEEKEIA QQLQEELRWR RTFLHAADVV LDPDTAHPEL FLSEDRRSVR RGPYRQRVPD NPERFDSQPC VLGWESFASG KHYWEVEVEN VMVWTVGVCR HSVERKGEVL LIPQNGFWTL EMFGNQYRAL SSPERILPLK ESLCRVGVFL DYEAGDVSFY NMRDRSHIYT CPRSAFTVPV RPFFRLGSDD SPIFICPALT GASGVMVPEE GLKLHRVGTH QSL 3 mAb 4.15 SYDIN HCDR1 aa 4 mAb 4.15 WIFPGDDSIIQNEKFKG HCDR2 aa 5 mAb 4.15 LGPLRGFTY HCDR3 aa 6 mAb 4.15 RASESVDRYGSSFMH LCDR1 aa 7 mAb 4.15 RASNLES LCDR2 aa 8 mAb 4.15 QQSNEDPWT LCDR3 aa 9 mAb 4.15 VH MGWSWVFLFLLSVTAGVHSQVQLQQSGAELVKPGASVKLSCKAS aa GYIFTSYDINWVRQRPEQGLEWIGWIFPGDDSIIQNEKFKGKAT LITDKSSSTVYMQLSRLTSEDSAVYFCARLGPLRGFTYWGQGTL VTVSA 10 mAb 4.15 VL METDILLLWVLLLWVPGSTGDIVLIQSPASLAVSLGQRATISCR aa ASESVDRYGSSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSG SGSRTDFTLTINPVEADDVATYYCQQSNEDPWTFGGGTKLEIK 11 mAb 5.28 DYSMN HCDR1 aa 12 mAb 5.28 RINTETGEPTYADDFKG HCDR2 aa 13 mAb 5.28 DYAKR HCDR3 aa 14 mAb 5.28 KASQDVITAVA LCDR1 aa 15 mAb 5.28 STSYRYT LCDR2 aa 16 mAb 5.28 LQHYTTPWT LCDR3 aa 17 mAb 5.28 VH MAWVWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVKISCKAS aa GYTFTDYSMNWVKQAPGKGLKWVGRINTETGEPTYADDFKGRFA FSLETSASTAYLQIKNLKNEDTATYFCVRDYAKRWGQGTSVTVS S 18 mAb 5.28 VL MGIKMESQIQVFVFVSLWLSGVDGDIVMTQSHKFMSTSVGDRVS aa ITCKASQDVITAVAWYQQKPGQSPKLLIYSTSYRYTGVPDRFTG SGSGTDFTFTISSVQAEDLAVYYCLQHYTTPWTFGGGTKLEIK 19 mAb 7.28 SYWIE HCDR1 aa 20 mAb 7.28 EILPGSGSTKYNEKFRG HCDR2 aa 21 mAb 7.28 LKGYYGGGAMDY HCDR3 aa 22 mAb 7.28 RASKSISKYLA LCDR1 aa 23 mAb 7.28 SGSTLQS LCDR2 aa 24 mAb 7.28 QQHNEYPWT LCDR3 aa 25 mAb 7.28 VH MEWTWVFLFLLSVTAGVHSQVHLQQSGAELMKPGASVKISCKAT aa GYTFSSYWIEWVKQRPGHGREWIGEILPGSGSTKYNEKFRGKAT FAADTSSNTAYVQLSSLTSEDSAVYYCARLKGYYGGGAMDYWGQ GTSVTVSS 26 mAb 7.28 VL MRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCR aa ASKSISKYLAWYQEKPGKTNELLIYSGSTLQSGIPSRFSGSGSG TDFTLTISSLEPEDFAMYYCQQHNEYPWTEGGGTKLEIK 27 mAb 7.48 DEYMY HCDR1 aa 28 mAb 7.48 TISDGGSHTYYPDSVKG HCDR2 aa 29 mAb 7.48 DTTIITPY HCDR3 aa 30 mAb 7.48 RSSTGAVTTSNYAN LCDR1 aa 31 mAb 7.48 GTNNRAP LCDR2 aa 32 mAb 7.48 GLWYSNHWV LCDR3 aa 33 mAb 7.48 VH MNFGLSLIFLVLVLKGVQCEVQLVESGGDLVKPGGSLKLSCAAS aa GFTFSDFYMYWVRRTPEKRLEWVATISDGGSHTYYPDSVKGRFT ISRDNAKNNLYLQMRSLKSEDTAMYYCGRDTTIITPYWGQGTLV TVSA 34 mAb 7.48 VL MAWISLILSLLALSSGAISQSVVTQESALTTSPGETVTLTCRSS aa TGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLI GDKAALTITGAQTEDEAIYFCGLWYSNHWVFGGGTKLTVL 35 mAb 8.15 GYWMT HCDR1 aa 36 mAb 8.15 EINPDSSTINYTPSLRD HCDR2 aa 37 mAb 8.15 GSYYPSY HCDR3 aa 38 mAb 8.15 RASKSVSSSGYSYMN LCDR1 aa 39 mAb 8.15 LASNLES LCDR2 aa 40 mAb 8.15 QHSRELPHT LCDR3 aa 41 mAb 8.15 VH MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCAASG aa FDFSGYWMTWVRQAPGKGLEWIGEINPDSSTINYTPSLRDKFII SRDNAKNTLYLQMSKVRSEDTALYFCARGSYYPSYWGQGTLVTV SA 42 mAb 8.15 VL METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCR aa ASKSVSSSGYSYMNWYQQKPGQPPKLLIYLASNLESGVPARFSG SGSGTDFTLNIHPVEDEDAATYYCQHSRELPHTFGGGTKLEIK 43 mAb 8.16 GYWMT HCDR1 aa 44 mAb 8.16 EINPDSSTINYTPSLRD HCDR2 aa 45 mAb 8.16 GSYYPSY HCDR3 aa 46 mAb 8.16 RASKSVSSSGYSYMN LCDR1 aa 47 mAb 8.16 LASNLES LCDR2 aa 48 mAb 8.16 QHSRELPHT LCDR3 aa 49 mAb 8.33 VH MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCAASG aa FDFSGYWMTWVRQAPGKGLEWIGEINPDSSTINYTPSLRDKFII SRDNAKNTLYLQMSKVRSEDTALYFCARGSYYPSYWGQGTLVTV SA 50 mAb 8.33 VL METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCR aa ASKSVSSSGYSYMNWYQQKPGQPPKLLIYLASNLESGVPARFSG SGSGTDFTLNIHPVEDEDAATYYCQHSRELPHTFGGGTKLEIK 51 mAb 8.33 SGYYWN HCDR1 aa 52 mAb 8.33 YISYDGNNNYNPSLKN HCDR2 aa 53 mAb 8.33 PLYDGYYWYFDV HCDR3 aa 54 mAb 8.33 ITSTDIDDDMN LCDR1 aa 55 mAb 8.33 EANTLRP LCDR2 aa 56 mAb 8.33 LQSDNLPYT LCDR3 aa 57 mAb 8.33 VH MKVLSLLYLLTAIPGILSDVQLQESGPGLVKPSQSLSLTCSVTG aa YSITSGYYWNWIRQFPGNKLEWMGYISYDGNNNYNPSLKNRISI TRDTSKNQFFLKLNSVTTEDTATYYCASPLYDGYYWYFDVWGAG TTVTVSS 58 mAb 8.33 VL MTMFSLALLLSLLLLCVSDSRAETTVTQSPASLSLAIGEKVTIR aa CITSTDIDDDMNWYQQKPGEPPKLLISEANTLRPGVPSRFSSSG RGTDFVFTIENMLSEDVADYYCLQSDNLPYTFGGGTKLEIK 59 mAb 4.15 AGCTATGATATAAAC HCDR1 nt 60 mAb 4.15 TGGATTTTTCCTGGAGATGATAGTATTATTCAGAATGAGAAGTT HCDR2 nt CAAGGGC 61 mAb 4.15 TTGGGCCCATTACGAGGGTTTACTTAC HCDR3 nt 62 mAb 4.15 AGAGCCAGTGAAAGTGTTGATCGTTATGGCAGTAGTTTTATGCA LCDR1 nt C 63 mAb 4.15 CGTGCATCCAACCTAGAATCT LCDR2 nt 64 mAb 4.15 CAGCAAAGTAATGAGGATCCGTGGACG LCDR3 nt 65 mAb 4.15 VH ATGGGATGGAGCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGC nt AGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAAC TGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCT GGCTACATCTTCACAAGCTATGATATAAACTGGGTGAGGCAGAG GCCTGAACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAG ATGATAGTATTATTCAGAATGAGAAGTTCAAGGGCAAGGCCACA CTGACTACAGACAAATCCTCCAGCACAGTCTACATGCAGCTCAG CAGGCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGAT TGGGCCCATTACGAGGGTTTACTTACTGGGGCCAAGGGACTCTG GTCACTGTCTCTGCAGGGTTTACTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCTGCA 66 mAb 4.15 VL ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGT nt TCCAGGTTCCACAGGTGACATTGTGCTGACCCAATCTCCAGCTT CTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGA GCCAGTGAAAGTGTTGATCGTTATGGCAGTAGTTTTATGCACTG GTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATC GTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGC AGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGA GGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGG ATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 67 mAb 5.28 GACTATTCAATGAAC HCDR1 nt 68 mAb 5.28 AGGATCAACACTGAGACTGGTGAGCCAACATATGCAGATGACTT HCDR2 nt CAAGGGA 69 mAb 5.28 GACTACGCTAAGCGG HCDR3 nt 70 mAb 5.28 AAGGCCAGTCAGGATGTGATTACTGCTGTAGCC LCDR1 nt 71 mAb 5.28 TCGACATCCTACCGGTACACT LCDR2 nt 72 mAb 5.28 CTGCAACATTATACTACTCCGTGGACG LCDR3 nt 73 mAb 5.28 VH ATGGCTTGGGTGTGGACCTTGCTATTCCTGATGGCAGCTGCCCA nt AAGTATCCAAGCACAGATCCAGTTGGTACAGTCTGGACCTGAGC TGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCT GGTTATACCTTCACAGACTATTCAATGAACTGGGTGAAACAGGC TCCAGGAAAGGGTTTAAAGTGGGTGGGCAGGATCAACACTGAGA CTGGTGAGCCAACATATGCAGATGACTTCAAGGGACGGTTTGCC TTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAA AAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGTTAGAG ACTACGCTAAGCGGTGGGGTCAAGGAACCTCAGTCACCGTCTCC TCA 74 mAb 5.28 VL ATGGGCATCAAAATGGAGTCACAGATTCAGGTCTTTGTATTCGT nt GTCTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGACCC AGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGC ATCACCTGCAAGGCCAGTCAGGATGTGATTACTGCTGTAGCCTG GTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACT CGACATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGC AGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCA GGCTGAAGACCTGGCAGTTTATTACTGTCTGCAACATTATACTA CTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 75 mAb 7.28 AGCTACTGGATAGAG HCDR1 nt 76 mAb 7.28 GAGATTTTACCTGGAAGTGGAAGTACTAAGTACAATGAGAAGTT HCDR2 nt TAGGGGC 77 mAb 7.28 TTGAAGGGTTACTACGGAGGAGGTGCTATGGACTAC HCDR3 nt 78 mAb 7.28 AGGGCAAGTAAGAGCATTAGCAAATATTTAGCC LCDR1 nt 79 mAb 7.28 TCTGGATCCACTTTGCAATCT LCDR2 nt 80 mAb 7.28 CAACAGCATAATGAATACCCGTGGACG LCDR3 nt 81 mAb 7.28 VH ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGC nt AGGTGTCCACTCCCAGGTTCACCTGCAGCAGTCTGGAGCTGAGC TGATGAAGCCTGGGGCCTCAGTGAAAATATCCTGCAAGGCTACT GGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAG GCCTGGACATGGCCGTGAGTGGATTGGAGAGATTTTACCTGGAA GTGGAAGTACTAAGTACAATGAGAAGTTTAGGGGCAAGGCCACA TTCGCTGCAGATACATCCTCCAACACAGCCTACGTGCAACTCAG CAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGAT TGAAGGGTTACTACGGAGGAGGTGCTATGGACTACTGGGGTCAA GGAACCTCAGTTACCGTCTCTTCA 82 mAb 7.28 VL ATGAGGTTCCAGGTTCAGGTTCTGGGGCTCCTTCTGCTCTGGAT nt ATCAGGTGCCCAGTGTGATGTCCAGATAACCCAGTCTCCATCTT ATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGG GCAAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAA ACCTGGGAAAACTAATGAGCTTCTTATCTACTCTGGATCCACTT TGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGT ACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTT TGCAATGTATTACTGTCAACAGCATAATGAATACCCGTGGACGT TCGGTGGAGGCACCAAGCTGGAAATCAAA 83 mAb 7.48 GACTTTTACATGTAT HCDR1 nt 84 mAb 7.48 ACCATTAGTGATGGTGGTAGTCACACCTACTATCCAGACAGTGT HCDR2 nt GAAGGGG 85 mAb 7.48 GATACTACGATAATTACTCCTTAC HCDR3 nt 86 mAb 7.48 CGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAAC LCDR1 nt 87 mAb 7.48 GGTACCAACAACCGAGCTCCA LCDR2 nt 88 mAb 7.48 GGTCTTTGGTACAGCAACCATTGGGTG LCDR3 nt 89 mAb 7.48 VH ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAA nt AGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGACT TAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCT GGATTCACTTTCAGTGACTTTTACATGTATTGGGTTCGCCGGAC TCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTGATGGTG GTAGTCACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACC ATCTCCAGAGACAATGCCAAGAACAACCTCTACCTACAAATGAG AAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGGAAGAG ATACTACGATAATTACTCCTTACTGGGGCCAAGGGACTCTGGTC ACTGTCTCTGCA 90 mAb 7.48 VL ATGGCCTGGATTTCACTTATACTCTCTCTCCTGGCTCTCAGCTC nt AGGGGCCATTTCCCAGTCTGTTGTGACTCAGGAATCTGCACTCA CCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGT ACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGA AAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACA ACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATT GGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGA TGAGGCAATATATTTCTGTGGTCTTTGGTACAGCAACCATTGGG TGTTCGGTGGAGGAACCAAACTGACTGTCCTA 91 mAb 8.15 GGATACTGGATGACT HCDR1 nt 92 mAb 8.15 GAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCT HCDR2 nt AAGGGAT 93 mAb 8.15 GGGAGCTACTATCCCTCTTAC HCDR3 nt 94 mAb 8.15 AGGGCCAGCAAAAGTGTCAGTTCATCTGGCTATAGTTATATGAA LCDR1 nt C 95 mAb 8.15 CTTGCATCCAACCTAGAATCT LCDR2 nt 96 mAb 8.15 CAGCACAGTAGGGAGCTTCCGCACACG LCDR3 nt 97 mAb 8.15 VH ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGG nt GGTCCAGTGTGAAGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGG TGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGA TTCGATTTTAGTGGATACTGGATGACTTGGGTCCGGCAGGCTCC AGGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCA GTACGATAAACTATACGCCATCTCTAAGGGATAAATTCATCATC TCCAGAGACAACGCCAAGAATACGCTGTACCTGCAAATGAGCAA AGTGAGATCTGAGGACACAGCCCTTTATTTCTGTGCAAGAGGGA GCTACTATCCCTCTTACTGGGGCCAAGGGACTCTGGTCACTGTC TCTGCA 98 mAb 8.15 VL ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGT nt TCCAGGTTCCACTGGGGACATTGTGCTGACACAGTCTCCTGCTT CCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGG GCCAGCAAAAGTGTCAGTTCATCTGGCTATAGTTATATGAACTG GTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATC TTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGC AGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGA GGATGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGC TTCCGCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 99 mAb 8.16 GGATACTGGATGACT HCDR1 nt 100 mAb 8.16 GAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCT HCDR2 nt AAGGGAT 101 mAb 8.16 GGGAGCTACTATCCCTCTTAC HCDR3 nt 102 mAb 8.16 AGGGCCAGCAAAAGTGTCAGTTCATCTGGCTATAGTTATATGAA LCDR1 nt C 103 mAb 8.16 CTTGCATCCAACCTAGAATCT LCDR2 nt 104 mAb 8.16 CAGCACAGTAGGGAGCTTCCGCACACG LCDR3 nt 105 mAb 8.33 VH ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGG nt GGTCCAGTGTGAAGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGG TGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGA TTCGATTTTAGTGGATACTGGATGACTTGGGTCCGGCAGGCTCC AGGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCA GTACGATAAACTATACGCCATCTCTAAGGGATAAATTCATCATC TCCAGAGACAACGCCAAGAATACGCTGTACCTGCAAATGAGCAA AGTGAGATCTGAGGACACAGCCCTTTATTTCTGTGCAAGAGGGA GCTACTATCCCTCTTACTGGGGCCAAGGGACTCTGGTCACTGTC TCTGCA 106 mAb 8.33 VL ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGT nt TCCAGGTTCCACTGGGGACATTGTGCTGACACAGTCTCCTGCTT CCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGG GCCAGCAAAAGTGTCAGTTCATCTGGCTATAGTTATATGAACTG GTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATC TTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGC AGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGA GGATGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGC TTCCGCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 107 mAb 8.33 AGTGGTTATTACTGGAAC HCDR1 nt 108 mAb 8.33 TACATAAGCTACGACGGTAACAATAACTACAACCCATCTCTCAA HCDR2 nt AAAT 109 mAb 8.33 CCTCTCTATGATGGTTATTACTGGTACTTCGATGTC HCDR3 nt 110 mAb 8.33 ATAACCAGCACTGATATTGATGATGATATGAAC LCDR1 nt 111 mAb 833 GAAGCCAATACTCTTCGTCCT LCDR2 nt 112 mAb 8.33 TTGCAAAGTGATAACTTGCCGTACACG LCDR3 nt 113 mAb 8.33 VH ATGAAAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGG nt TATCCTGTCTGATGTACAGCTTCAGGAGTCAGGACCTGGCCTCG TGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGC TACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTT TCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGACG GTAACAATAACTACAACCCATCTCTCAAAAATCGAATCTCCATC ACTCGTGACACGTCTAAGAACCAGTTTTTCCTGAAGTTGAATTC TGTGACTACTGAGGACACAGCTACATATTACTGTGCAAGTCCTC TCTATGATGGTTATTACTGGTACTTCGATGTCTGGGGCGCAGGG ACCACGGTCACCGTCTCCTCA 114 mAb 8.33 VL ATGACCATGTTCTCACTAGCTCTTCTCCTCAGTCTTCTTCTCCT nt CTGTGTCTCTGATTCTAGGGCAGAAACAACTGTGACCCAGTCTC CAGCATCCCTGTCCCTGGCTATAGGAGAAAAAGTCACCATCAGA TGCATAACCAGCACTGATATTGATGATGATATGAACTGGTACCA GCAGAAGCCAGGGGAACCTCCTAAGCTCCTTATTTCAGAAGCCA ATACTCTTCGTCCTGGAGTCCCATCCCGATTCTCCAGCAGTGGC CGTGGTACAGATTTTGTTTTTACAATTGAAAACATGCTCTCAGA AGATGTTGCAGATTACTACTGTTTGCAAAGTGATAACTTGCCGT ACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

REFERENCES

(59) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.