Anti-HERV-K envelope protein antibodies and methods for detecting HERV-K envelope protein in patients with sporadic amyotrophic lateral sclerosis

Abstract

The present invention relates to a novel antibody against HERV-K envelope that targets a conserved region not affected by glycosylation or by native conformation, and its use in diagnostics and/or is therapy.

Claims

1. An antibody that recognizes human endogenous retrovirus K (HERV-K) envelope protein having an epitope comprising the amino acid sequence of SEQ ID NO: 9, wherein said antibody comprises a light chain comprising a light chain variable region (VL) which comprises three complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), and a heavy chain comprising a heavy chain variable region (VH) which comprises three complementary determining regions (CDR-H1, CDR-H2 and CDR-H3), wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 1, the CDR-L2 comprises the amino acid sequence of SEQ ID NO:2, the CDR-L3 comprises the amino acid sequence of SEQ ID NO:3, the CDR-H1 comprises the amino acid sequence of SEQ ID NO:4, the CDR-H2 comprises the amino acid sequence of SEQ ID NO:5, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO:6.

2. The antibody according to claim 1, wherein: the light chain variable region (VL) comprises the amino acid sequence of SEQ ID No: 7; and the heavy chain variable region (VH) comprises the amino acid sequence of SEQ ID No: 8.

3. The antibody according to claim 1, wherein said antibody is a murine monoclonal antibody, a chimeric monoclonal antibody or a humanized monoclonal antibody.

4. A pharmaceutical composition comprising the antibody according to claim 1 and a pharmaceutically acceptable excipient.

5. A method for detecting HERV-K envelope protein in a biological sample obtained from a subject comprising the steps of contacting said biological sample with an anti-HERV-K envelope protein antibody as defined in claim 1; and detecting binding of the anti-HERV-K envelope protein antibody to the HERV-K envelope protein; wherein the HERV-K envelope protein has an epitope comprising the amino acid sequence of SEQ ID NO: 9.

6. A method for detecting an increased level of HERV-K envelope protein in a sporadic amyotrophic lateral sclerosis (ALS) patient comprising the steps of contacting a biological sample obtained from said patient with an anti-HERV-K envelope protein antibody as defined in claim 1; detecting binding of the anti-HERV-K envelope protein antibody to the HERV-K envelope protein; and comparing a level of binding in the biological sample to a healthy control sample, wherein the level of binding in the biological sample is increased as compared to the healthy control sample, wherein the biological sample is serum or cerebrospinal fluid, and wherein the HERV-K envelope protein has an epitope comprising the amino acid sequence of SEQ ID NO: 9.

Description

FIGURE LEGENDS

(1) FIG. 1: GN_mAb_Env_K01 is an IgG2b murine antibody and has a kappa light chain.

(2) Isotyping was performed by ELISA on 1:10 diluted supernatant from GN_mAb_Env_K01 hybridoma (monoclonal stage) captured by anti-murine immunoglobulin. Detection with various anti-mouse light (A) or heavy (B) chains antibodies showed that GN_mAb_ENV_K01 is an IgG2b/kappa murine antibody. Results are plotted as OD.sub.450nm mean of duplicate values±SD.

(3) FIG. 2: GN_mAb_Env_K01 light and heavy chains sequences

(4) RNA from hybridoma cells was extracted and reverse transcribed into cDNA that was amplified by PCR before sequencing with primers targeting cDNA encoding murine antibody heavy (A) and light (B) chains.

(5) (A) CDR1 (Bold), CDR2 (underlined) and CDR3 (bold italics) sequences from kappa light chain (SEQ ID NO: 11);

(6) (B) CDR4 (Bold), CDR5 (underlined) and CDR6 (bold italics) sequences from heavy chain; constant murine IgG2 Sequences (grey italics) (SEQ ID NO: 12).

(7) FIG. 3: GN_mAb_Env_K01 binds to HERV-K-Env SLDKHKHKKLQSFYP (SEQ ID NO: 9) epitope.

(8) The intensity profile (left) of each peptide from HERV-K-Env (MyBiosource) (right; SEQ ID NO: 13) are: displayed. These overerlapping peptides of 15 amino acids with an offset of one residue showed that GN_mAb_Env_K01 binds to the linear SLDKHKHKKLQSFYP (SEQ ID NO:9) epitope. Results are presented as the intensity of signal (mAU) obtained on the CCD camera used, similar to a standard 96-well plate ELISA-reader.

(9) FIG. 4: Glycosylated HERV-K Env is detected by GN_mAb_ENV_K01 in ELISA.

(10) GN_mAb_Env_K01 (A) or Anti-HERV-K-Env from AMSBIO (B) (1 μg/ml) were used as primary antibodies in ELISA on HEK cell lysates at various dilutions (1:25, 1:50, 1:100, 1:200, 1:400). Contrary to Anti-HERV-K-Env (AMSBio) GN_mAb_Env_K01 recognized glycosylated HERV-K-Env. Results are plotted as OD.sub.450nm mean of duplicate values±SD.

(11) FIG. 5: Non-glycosylated HERV-K-Env is detected by GN_mAb_ENV_K01 in ELISA

(12) GN_mAb_Env_K01 or Anti-HERV-K-Env from AMSBIO (1 μg/ml) were used as primary antibodies in ELISA on 1 μg/ml of his-SUMO-HERV-K-Env recombinant protein from E. coli. Both anti-HERV-K-Env recognized the non-glycosylated HERV-K-Env protein. Results are plotted as OD.sub.450 nm mean of duplicate values±SD.

(13) FIG. 6: HERV-K Env detection by Western Blotting with GN_mAB_ENV_K01

(14) Anti-HERV-K-Env (1 μg/ml) from AMSBIO (wells #1 & 2) or diluted (1:3) supernatant from GN_mAb_Env_K01 hybridoma (wells #3 & 4) were used as primary antibodies in western blotting. 0.2 μg of his-SUMO tagged HERV-K-Env were deposited in wells #1 & 3 and 24.5 μg of protein extract from HERV-K-Env transfected HEK cells were deposited in wells #2 & 4. Non-glycosylated his-SUMO tagged HERV-K-Env protein is detected at 75 kDa by both antibodies, along with multimers of high MW and cleaved fragments of lower MW. Glycosylated HERV-K-Env (90 kDa) is only detected with GN_mAb_Env_K01.

(15) MW: molecular weight.

(16) FIG. 7: GN_mAb_Env_K01 (GN K01) antibody specifically protects human neuronal cells from cytotoxicity induced by extracellular HERV-K envelope protein: Cell survival assay.

(17) Neuronal cultures were treated with differentiation media (see example 2) and with IgG samples GN K01 or control non-Immune IgG (Thermo Product #MA 1-10418) at a final concentration of 3 ug/mL. After 60 minute pre-incubation, recombinant HERV-K Env protein (My BioSource, amino acid 90-632, Cat #MBS1391552) was added to a final concentration of 100 nM. One sample of GN K01 Ig was pre-incubated with HERV-K Env for 30 minutes, and then added together to the human neurons. The neuronal cultures were observed with a GE INCell Analyzer 2000 BioImager to acquire images of each well (4 images per well) at various time points, in this experiment 5 days post Env treatment. The neuronal cell count was determined with GE Investiaator high content imaging software. Arrow on top of histogram bar is showing the results with GN_mAb_Env_K01 (GN K01) antibody.

(18) FIG. 8: GN_mAb_Env_K01 (GN K01) antibody specifically protects human neuronal cells from cytotoxicity induced by extracellular HERV-K envelope protein: neurite length.

(19) Neuronal cultures were treated with differentiation media (described above) and with IgG samples GN K01 or control non-Immune IgG (Thermo Product #MA 1-10418) at a final concentration of 3 ug/mL. After 60 minute pre-incubation, recombinant HERV-K (Env protein (My BioSource, amino acid 90-632, Cat #MBS1391552) was added to a final concentration of 100 nM. The neuronal cultures were obsemed with a GE INCell Analyzer 2000 BioImager to acquire images of each well (4 images per well) at various time points, in this experiment at 5 days post Env post treatment. The mean neurite fiber length was determined with GE investigator high content imaging software.

(20) FIG. 9: GN_mAb_Env_K01 (GN K01) antibody specifically protects human neuronal cells from cytotoxicity induced by extracellular HERV-K envelope protein: neuron global electrophysiological activity.

(21) Electrophysiological activity, noted by increased spike rate in the wells, increased significantly by 21 days in vitro and was monitored by recording spontaneous electrical activity in all wells for 5 minutes per day. At this point, the human neuronal cultures were treated with differentiation media (described above) and with GN K01 or control non-Immune IgG (Thermo Product #MA 1-10418) at a final concentration of 3 ug/mL. After 60 minute pre-incubation, recombinant HERV-K Env protein (My BioSource) was added to a final concentration of 100 nM. Spontaneous electrical activity was recorded daily afterward, beginning at 24 h post treatment. The mean firing rate was determined for each treatment group at 24 h post HERV-K Env exposure.

(22) Arrow on top of histogram bar is showing the results with GN_mAb_Env_K01 (GN K01) antibody. Arrow on top of histogram bar is showing the results with GN_mAb_Env_K01 (GN K01) antibofy.

EXAMPLES

Example 1: Development and Characterization of the GN_mAb_Env_K01 Antibody

(23) 1. Materials and Methods

(24) 1.1, Monoclonal Antibody Development 1.1.1, Immunization and Immune Cells Recovery
Three female OF mice (Charles River) were immunized with his-SUMO tagged HERV-K-Env protein (75 kDa) from provided by Mybiosource (MBS1391552) following the confidential RAD (Rapid Antibody development) protocol from Biotem company.
Briefly, at day+10 (D+10) blood sample from immunized mice were analyzed by direct ELISA on the recombinant HERV-K Env protein (MyBiosource, MBS1391552) or Escherichia coli lysate as negative control. At D+13, immunized mice were sacrificed and immune cells from lymph nodes were collected and washed thrice with 45 of Dulbecco's Modified Eagle's medium (DMEM, SIGMA, D5671). Immune cells from immunized mice (420×10.sup.6 cells) were mixed with myeloma cells (107×10.sup.6 cells) in the exponential phase of growth according to a 1:3.9 ratio. Cells were centrifuged at 244 g for 7 minutes and the pellet was resuspended in 1 ml of the Polyethylene glycol (PEG) used as fusing agent (SIGMA, P7181). After the washing step including a centrifugation at 108 g for 12 minutes cells were resuspended in 10 ml of DMEM (SIGMA, D5671), 1× Hypoxanthine Aminopterine Thymidine (HAT, SIGMA, H0262-10VL), 20% FCS (PAA, A15-251), 4 mM L-Glutamine (SIGMA, G7513) and stored for 2 hours at room temperature. 1.1.2. Fusion
At D-1, immunodeficient Nude mice (BIOTEM) were injected with 5 ml of DMEM (SIGMA, D5671) containing 20% FCS (PAA, A15-251) and 2+/−1 minutes later, macrophages from peritoneal fluid have been collected and cultivated in DMEM medium (SIGMA, D5671).
BALB/c spleen cells from mouse immunized with sheep red blood cells fused with P3X63Ag8 myeloma have already been selected, characterized and stored by BIOTEM. At D-10, these myeloma have been thawed and cultivated in DMEM (SIGMA, D5671)—8-Azaguanine (AZA, SIGMA, A5284)—10% FCS (PAA, A15-251).
Macrophages from Nude mice were counted and resuspended at 10.sup.4 macrophages/ml in DMEM (SIGMA, D5671), 1× HAT (SIGMA, H0262-10VL), 20% FCS (_AA, A15-251), 4 mM L-Glutamine (SIGMA, G7513), 1% penicillin/streptomycine (SIGMA, P0781). Then, 50 μl of macrophage suspension (corresponding to 500 macrophages) used as a growth factor has been plated in 96-wells plates with 50 μl of hybridomas cells suspension. These cells were cultivated at 37° C., 5% CO.sub.2 for 21 days. 1.1.3. Cloning
Hybridoma cells were thawed and cultiated with DMEM (SIGMA, D5671). HT (hypoxantine 100 μM, thymidine 16 μM—SIGMA H0137), 20% FCS (PAA, A15-251), 2% Hybridoma Enhancing Supplement (HES, SIGMA, H6020), 4 mM L-Glutamine (SIGMA, G7513), 1% pencillin/streptomycine (SIGMA, P0781) in 24-wells plate during 1 week at 37° C., 5% C02. The day before cloning, hybridoma cells were split.
At D0, after serial dilutions at 10.sup.−1, 50, 25, 5 and 2.5 cells/ml in culture medium hybridoma suspension were plated at 5, 1 and 0.5 cells/200 μl into the wells of a 96-wells plate. At D+6, 100 μl of supernatant from cell containing wells (selected through a screening with an optical microscope) was replaced by fresh DMEM (SIGMA, D5671), HT (hypoxantine 100 μM, thymidine 16 μM—SIGMA H0137, 20% FCS (PAA, A15-251), 2% HES (SIGMA, H6020) 1% penicillin/streptomycine (SIGMA, P0781).
After the first ELISA screening corresponding to D+10, anti-HERV-K-Env positive hybridoma were cultivated in 24-wells plates (0.5 ml/well).
After the second ELISA screening corresponding to D+14, anti-HERV-K Env positive hybridoma were cultivated in plates or culture flask (Corning) and 5 vials containing 4 at 5×10.sup.6 cells were frozen at −196° C. (liquid azote) in DMEM (SIGMA, D5671), 15% FCS (PAA, A15-251), 4 mM L-Glutamine (SIGMA, G7513), 1% HES (SIGMA, H6020), 1% penicillin/streptomycine (SIGMA, P0781), 20% Dimethylsulfoxyde (DMSO, Sigma, D2650) media.

(25) 1.2. Anti-HERV-K-Env ELISA

(26) The maxisorp 96 conical bottom well plates (NUNC, 449824) were coated with 50 μl of 1 μg/ml HERV-K Env protein (Mybiosource, MBS1391552), E. coli lysate (XL1-Blue MRF, Stratagene.), HEK cell lysate in 1× Phosphate Buffered Saline (PBS, BIOTEM) overnight at room temperature. Plates were washed with [1× PBS+0.05% Tween20 (VWR, 28829.296)] washing buffer (300 μl/well). Non-specific binding sites were blocked with [1× PBS+0.05% Tween20+2.5% milk (Regilait)] blocking buffer (150 μl/well) for one hour at room temperature. Plates were washed with [1× PBS+0.05% Tween20] washing buffer (300 μl/well).
Antibody samples have been diluted in [1× PBS+0.05% Tween20+0.5% BSA (VWR, 1.12018.0100) dilution buffer. Antibody samples or purified anti-HERV-K-Env from AMSBIO (1 μg/ml) (50 μl/well) were incubated for two hours at room temperature. The plates have been washed thrice with [1× PBS+0.05% Tween20] washing buffer (300 μl/well) and were incubated with 50 μl/well of polyclonal Peroxidase-conjugated affiniPure F(ab′2 fragment Goat anti-mouse IgG+IgM (Jackson, 115-036-068) (1/10000 in 1× PBS+0.05% Tween20+0.5% BSA) for one hour at room temperature. Plates were washed thrice and the revelation was performed with Tetramethybenzidine (TMB, Eurobio, 52-00-01) substrate solution (50 μl/well) for 10 minutes at room temperature. The reaction was blocked with 0.1M H2SO4 (Merck, 1.12080.1000) (50 μl/well). Optical density (OD) was measured at 450 nm using the optical density (OD) reader (Dynex).

(27) 1.3. Production, Purification, Dialysis

(28) GN_mAb_Env_K01 hybridoma cells were thawed and cultivated first in T75 cm.sup.2 and then in T300 cm.sup.2 tissue culture flasks (Corning). Finally, 10 at 12×10.sup.6 cells were cultivated in 500 ml of DMEM (SIGMA, D5671), 15% FCS (PAA, A15-251), 4 mM L-Glutamine (SIGMA, G7513), 1% HES (SIGMA H6020), 1% penicillin/streptomycine (SIGMA, P0781) medium in Hyperflask (Corning, 10030) at 37° C., 5% CO2 within 10+/−1 days.

(29) The culture supernatant were centrifuged at 244 g for 7 minutes and filtered through a 11 μm nylon net filter (SIGMA, NY1104700) Protein A chromatography column (GE Healthcare, Mab Select Xtra) were washed twice with demineralized water and equilibrated with 5 volumes of 1× PBS (Biotem). Then 0.5 L of culture supernatant free of cells was loaded. The column was washed with 5 volumes of 1× PBS. Immunoglobulin elution was performed at acid pH with 3.5+/−0.5 volumes of acetic acid (SIGMA, A6283). Eluted fractions containing immunoglobulins were neutralized with 100 μl of 1M Tris pH 8.8 buffer (Biotem) and stored at 4° C.

(30) IgG purified fractions were dialyzed twice on 0.5 ml micro dialysis capsule Quixsep® (Roth, H448-1) with 10 kDa SnakeSkin Dialysis Tubing, 22 mm (Thermofischer, 68100) for 2 hours in 1× PBS at 4° C. and concentrated by centrifugation at 4° C. on Vivaspin 20 (30 Kda) (Sartorius, ref). Antibodies were filtered on 0.22 μm Minisart® filter (Sartorius, ref) and the protein concentration was measured by spectrophotometry at 280 nm.

(31) 1.4. Purity Analysis by SDS PAGE Gel Electrophoresis

(32) Antibody (5 μl at 0.2 μg/μl) previously diluted in Laemmli buffer (Biotem) were heated for 5 minutes at 95° C. and separated on a 13.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing a stacking gel with 5% SDS-PAGE. Gel was run for 30 minutes at 90V and then at 120V for 2 hours. Protein detection was done using colorizing solution (Biotem) for 1 hour with agitation. The gel was washed with decolorizing solution (Biotem) for 1 hour with agitation.

(33) 1.5. Isotyping 1.5.1. Isotyping by ELISA
The maxisorp 96 conical bottom well plates (NUNC, 449824) was coated with 50 μl of 1 μg/ml anti-murine Immunoglobulin (Clinisciences, 1010-01) overnight at room temperature. Plates were washed with [1× PBS+0.05% Tween20] washing buffer (300 μl/well). Non-specific specific binding sites were blocked with [1×PBS+0.05% Tween20+2.5% milk] blocking buffer (150 μl/well) for one hour at room temperature. Plates were washed with [1×PBS+0.05% Tween20] washing buffer (300 μl/well). Hybridoma supernatant was 1:10 diluted in [1×PBS+0.05% Tween20+0.5% BSA] dilution buffer. Sample (50 μl/well) was incubated for two hours at room temperature. The plates were washed once with [1×PBS+0.05% Tween20] washing buffer (300 μl/well) and were incubated with 50 μl/well of peroxidase conjugated goat anti-mouse heavy chain (IgA, IgG1, IgG2a, IgG2b, IgG3, IgM) (Clinisciences, 5300-05) (1/2000 in 1×PBS+0.05% Tween20+0.5% BSA) for one hour at room temperature. Plates were washed once and the revelation was performed with TMB (Eurobio, 52-00-01) substrate solution (50 μl/well) for 10 minutes at room temperature. The reaction was blocked with 0.1M H.sub.2SO.sub.4 (Merck, 1.12080.1000) stop solution (50 μl/well). Optical density (OD) was measured at 450 nm using the optical density (OD) reader (Dynex). 1.5.2. Isotyping by Lateral-Flow Immunoassay
Light chain (kappa or lambda) were characterized with a lateral-flow immunoassay (LFIA) (ThermoFisher, 26179).

(34) 1.6. HEK Transfection

(35) Human embryonic cells (1.10.sup.6 cells/mL) were transfected with 1 μg of HERV-K-Env (accession number AY037928.1) expressing plasmid Transfected cells were cultivated at 37° C., 8% CO.sub.2, 120 rpm agitation.

(36) 1.7. Western Blot Analysis

(37) The recombinant HERV-K-Env protein (Mybiosource) at 12.5 ng/μl and the protein lysate from HEK transfected cells at 1.5 μg/μl were diluted (1:1) in 2× Laemmli buffer (SIGMA, S3401) and heated for 5 minutes at 90° C. Then, 32 μl of samples were loaded on a 8-16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Novex, EC60452BOX), Gels were run for 1 hour at 160 mA in 1× Tris-Glycine SDS running buffer (Novex, LC2675). After the protein transfer onto a 0.2 μm nitrocellulose membrane (Biorad, 162-0146) in 1× Tris-Glycine transfer buffer (Novex, LC3675), the membrane was blocked for 1 hour with [1×PBS+5% milk (La Vie Claire)] blocking buffer on a rotating platform at room temperature. Cell supernatant from GN_mAb_Env_K01 hybridoma was 1.5 diluted in [1×PBS+1% milk] antibody diluent and used as primary antibody by incubation for 1 hour. The membrane was then washed thrice for 5 minutes in [1×PBS+0.05% Tween20 (SIGMA, P7949)] washing buffer and incubated for 30 minutes with 1:1000 diluted HRP-conjugated goat anti-mouse IgG antibody (Jackson, 115035-146). Membrane was washed thrice and protein of interest was detected with a colorometric reaction (Opti 4-CN, Biorad, 170-8235), according to the provided protocol.

(38) 1.8. Epitope Mapping

(39) Epitope mapping was conducted at Pepscan Presto BV, (Zuidersluisweg 2, 8243RC Lelystad, The Netherlands).

(40) 1.8.1. Peptides Library Synthesis
To reconstruct epitopes of the target molecule a library of peptides was synthesized. An amino functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with Nhydroxybenzotriazole (HOB.sub.1) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA). Standard Fmoc-peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS liquid handling stations (Perkin Elmer). Synthesis of structural mimics was done using Pepscan's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows to structure peptides into single loops, doubleloops, triple loops, sheet-like folds, helix-like folds and combinations thereof. CLIPS templates are coupled to cysteine residues. The side-chains of multiple cysteines in the peptides are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of the P2 CLIPS (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8)/acetonitrile (1:3(v/v)). This solution is added onto the peptide arrays. The CLIPS template will bind to side-chains of two cysteines as present in the solid-phase bound peptides of the peptide-arrays (455 wells plate with 3 μl wells). The peptide arrays are gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrays are washed extensively with excess of H2O and sonicated in disrupt-buffer containing 1% SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70° C. for 30 minutes, followed by sonication in H.sub.2O for another 45 minutes. The T3 CLIPS carrying peptides were made in a similar way but now with three cysteines. 1.8.2. ELISA Screening
The binding of antibody to each of the synthesized peptides was tested in a Pepscan-based ELISA. The peptide arrays were incubated with primary antibody solution (GN_mAb_Env_K01 at 1 μg/ml in Pepscan Buffer) overnight at 4° C. After washing, the peptide arrays were incubated with a 1/1000 dilution of a rabbit anti-mouse IgG(H+L) HRP conjugate antibody (Southern Biotech; Table 4) for one hour at 25° C. After washing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl/ml of 3 percent H2O2 were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD)—camera and an image processing system. The values obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader.

(41) 1.9. Sequencing,

(42) High quality RNA was extracted and purified from hybridoma cells using PureLink RNA Mini Kit (Life technologies, 12183018A) and controlled on agarose gel. Starting from purified total RNA, Superscript enzyme (Invitrogen, 18064022) was then used to synthesize first-strand complementary DNA (cDNA) followed by a Polymerase chain reaction (PCR) using HotStar HiFidelity Polymerase Kit (Qiagen, 202602) and degenerate primers (Biotem design) targeting specifically cDNA encoding for murine antibody heavy and light chains. PCR products were controlled on agarose gel and sequenced (double strand sequencing). The resulting sequences were analyzed for assembling and quality controls using dedicated bioinformatics tools (Blast-ClustalW) and translated to peptidic sequences.

(43) TABLE-US-00003 TABLE 1 BIOTEM Buffer composition Solution Composition 10 X 80 g NaCl PBS 2 g KCl 28.7 g Na2HPO4•12H2O 2.4 g KH2PO4 into 1 L of deionized water 1 X 100 ml 10X PBS PBS 900 ml deionized water Laemmli 0.5 ml of 2.5M Tris pH 6.8 (ICN, 103133) buffer 0.6 g of 12% Sodium Dodecyl Sulfate (ICN, 102918) (4X) 0.01 g of 0.2% Bleu de Bromophénol 2 ml of 40% Glycérol (SIGMA, G7757) qsp 5 ml H2O (reducing conditions: 1 ml of 20% 2-Mercaptoéthanol (SIGMA, M7154) 13.5% 9 ml of 30% acrylamide (SIGMA, A3699) Separating 5 ml of 1.5M Tris pH 8.8 (ICN, 103133) SDS-PAGE 200 μl of 10% Sodium Dodecyl Sulfate (ICN, 102918) 5.8 ml H.sub.2O 105 μl Ammonium persulfate (APS, ICN, 802811) 15 μl TEMED (ICN, 805615) 5% stacking 1.6 ml of 30% acrylamide (SIGMA, A3699) SDS-PAGE 2.5 ml of 0.5M tris pH 6.8 (ICN, 103133) 100 μl of 10% Sodium Dodecyl Sulfate (ICN, 102918) 5.8 ml H.sub.2O 150 μl Ammonium persulfate (APS, ICN 802811) 15 μl TEMED (ICN, 805615) 10X Running 288 g Glycine (SIGMA, G7126) buffer 60 g Tris base (ICN, 103133) 20 ml SDS (ICN, 102918) qsp 2 L H.sub.2O colorating 45 ml acetic acid (Sigma, A6283) solution 278 ml ethanol 90% 177 ml H.sub.2O 0.75 g Brilliant blue Reagent (SIGMA, B7920) decolorating 75 ml acetic acid (SIGMA, A6283) solution 56 ml éthanol 90% 869 ml H.sub.2O

(44) 2.Results

(45) Using specific anti-immunoglobulin antibodies that are capable of detecting the different heavy and light chains of monoclonal antibodies we have shown that GN_mAb_Env_K01 was detected by anti-Kappa light chain antibodies (FIG. 1A) and anti-IgG2b heavy chain (FIG. 1B).
GN_mAb_Env_K01 heavy and light chains were sequenced, showing their three CDR regions (FIG. 2 and confirming both IgG2b heavy and kappa light chains.

(46) 2.1. GN_mAb_Env_K01 Recognizes the Linear Epitope SLDKHKHKKLQSFYP (SEQ ID NO: 9) from HERV-K-Env Surface Unit.

(47) Using a panel of 529 peptides among the sequence of truncated HERV-K-Env protein from Mybiosource (no signal peptide and truncated (transmembrane domain) epitope mapping test under high stringency conditions reveals that GN-mAb-Env_K01 antibody bound to linear peptide with SLDKHKHKKLQSFYP (SEQ ID NO: 9) core sequence (FIG. 3, left panel). This epitope is contained within the extracellular domain of HERV-K-Env protein (FIG. 3 right panel), corresponding to the 298-312 region of the HERV-K-Env protein described by Dewannieux et al (Dewannieux, Blaise, and Heidmann 2005).
Blasting this epitope into Blastp from the National Center for Biotechnology Information (NCBI) reveals that it is a highly conserved epitope with 100% homology within the top 100 blast hits corresponding to HERV-K-Env sequences (data not shown).

(48) 2.2. GN_mAb_Env_K01 Recognizes Glycosylated and Non-Glycosylated HERV-K-Env Proteins in Native Conditions.

(49) We have further analyzed the ability of GN_mAb_Env_K01 to recognize glycosylated HERV-K-Env. For this purpose, human embryonic kidney (HEK) cells were transfected with a plasmid encoding HERV-K-Env protein. Despite several attempts with various buffers, we failed to extract soluble HERV-K-Env protein (data not shown). However this insoluble fraction of glycosylated HERV-K-Env protein from transfected HEK cell lysate could show that GN_mAb_Env_K01 specifically recognizes the glycosylated HERV-K-Env antigen by ELISA (FIG. 4, left panel). Conversely, the classical commercial anti-HERV-K-Env mAb (HERM-1821-5, IgG2b) from AMSBIO (FIG. 4, right panel) did not detect the glycosylated HERV-K-Env.
The present results indicate that GN_mAb_Env_K01 is biologically active in ELISA and confirm that SLDKHKHKKLQSFYP (SEQ ID NO: 9) epitope is accessible in native conditions.
Though both GN_mAb_Env_K01 and Anti-HERV-K-Env mAb (HERM-1821-5, IgG2b) from AMSBIO recognized native his-SUMO tagged recombinant HERV-K-Env from E. coli (FIG. 5), it appeared that GN_mAb_Env_K01 gave a much higher detection compared to Anti-HERV-K-Env mAb counterpart from AMSBIO (FIG. 5) when both are tested at the same concentration (1 μg/ml). This evidences a higher affinity of GN_mAb_Env_K01 for HERV-K Env.
As shown in FIG. 6, both GN_mAb_Env_K01 and Anti-HERV-K-Env mAb (HERM-1821-5, IgG2b) from AMSBIO recognized denatured his-SUMO tagged recombinant HERV-K-Env from E. coli as observed with the 75 kDa signal.
Importantly, GN_mAb_Env_K01 also detects denatured HERV-K-Env glycosylated proteins from HEK transfected cells as observed with the signal at 90 kDa, while no signal was detected with anti-HERV-K-Env antibody from AMSBIO. In addition to previous ELISA results with non-denatured proteins, GN_mAb_Env_K01 is biologically active in Western blot. The SLDKHKHKKLQSFYP (SEQ ID NO: 9) epitope is therefore also accessible in denaturing conditions.

(50) 4. Conclusions

(51) The present report reveals that, after mouse immunization, antibody screening and monoclonal hybridoma selection, a murine monoclonal antibody (named GN_mAb_EnvK-01) recognizing HERV-K-Env SLDKHKHKKLQSFYP (SEQ ID NO: 9) epitope has been developed and has been revealed to display unexpected properties.

(52) Biological comparison with another anti-HERV-K Env mAb (HERM-1821-5, IgG2b) from AMSBIO confirms that, despite their similar origin (murine), isotype (IgG2b, kappa) and protein target (HERV-W-Env protein), GN_mAb_EnvK-01 is advantageous since recognizing both glycosylated and non-glycosylated proteins, while displaying high affinity in both native and denaturing conditions. In addition, the GN_mAb_EnvK-01 antibody targets a stable and conserved epitope among HERV-K envelope sequences from numerous and various copies described in the databases.

(53) GN_mAb_EnvK-01 is therefore a useful tool, not only for immunoassays, but also for therapeutic purpose against HERV-K Env proteins as therapeutic targets in, e.g. ALS. Its stable epitope sequence among HERV-K copies. Its high affinity and its efficient binding to native glycosylated forms, fulfill important requirements for a valuable therapeutic in patients with, e.g. ALS in which different HERV-K copies seem to be significantly expressed.

Example 2: GN mAb Env_K01 (GN K01) Antibody Efficiently Neutralizes HERV-K Envelope Neurotoxicity to Human Neuronal Cells

(54) 1. Materials and Methods

(55) 1.1. Human Neuronal Cells

(56) Human neural stem cell (NSC)-derived neuronal cultures ere prepared as described (Efthymiou, Shaltouki et al. 2014). Briefly, NSCs were split into a 96-well plate coated with 0.002% poly-L-omithine (Sigma, St. Louis, Mo.) and 10 μg/mL laminin (Life Technologies) at 7500 to 10,000 cells/cm2, and neuronal differentiation medium was added 24 h after plating. The differentiation medium contained DMEM/F12 with GlutaMax, 1.8% bovine serum albumin (BSA), 1 StemPro hESC supplement (all from Life Technologies), 10 ng/mL brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF; R&D Systems, Minneapolis, Minn.), and cells received fresh medium and growth factors every other day. Neurons day 7-12 in vitro were utilized in neurotoxicity assays.

(57) 1.2 Neurotoxicity Assays: 1.2.1. Neuronal Morphology and Viability
Human neuronal cultures (15-20,000 cells per well), stably expressing Td-Tomato fluorescent protein to label the cells and processes, were plated onto 96 well plates as described above and were maintained at 37 0° C. in a humidified tissue culture incubator at 5% CO2. Neuronal cultures were treated with differentiation media (described above and with IgG samples GN K01 or control non-Immune IgG (Thermo Product #MA 1_10418) at a final concentration of 3 μg/mL. After 60 minute pre-incubation, recombinant HERV-K Env protein (My BioSource, amino acid 90632, Cat #MBS1391552) was added to a final concentration of 100 nM. One sample of GN K01 Ig was pre-incubated with HERV-K Env for 30 minutes, and then added together to the human neurons. The neuronal cultures were observed with a GE INCell Analyzer 2000 BioImager to acquire images of each well (4 images per well) at various time points, 24, 48, 72 h post treatment. High content imaging/analysis of these cultures was achieved with GE Investigator 1.93 analysis software. Neuronal viability, neurite length and other morphological parameters were quantitated for each sample. The data were depicted with Graph Pad Prism 7.02. 1.2.2 Electrophysiological Analysis
Electrophysiological analysis with Axion Maestro Microelectrode array (MEA) assays.
48-well t-MEA plates were utilized to plate the human neuronal cultures for analysis. These plates contain 16 active recording electrodes per well, 200,000 neurons were applied to each well of the t-MEA plate and cultures were maintained at 37° C. in a humidified tissue culture incubator at 5% CO.sub.2. Electrophysiological activity, noted by increased spike rate in the wells, increased significantly by 21 days in vitro and was monitored by recording spontaneous electrical activity in all wells for 5 minutes per day. At this point, the human neuronal cultures were treated with differentiation media (described above) and with IgG samples GN K01 or control non-Immune IgG (Thermo Product #MA 1-10418) at a final concentration of 3 ug/mL. After 60 minute pre-incubation, recombinant HERV-K Env protein (My BioSource) was added to a final concentration of 100 nM. In these conditions, the antibody is first added to the cells without Env, thus reproducing the conditions of a treated patients with presence of therapeutic antibody that diffused in his brain tissue. Thereafter the active Env protein (not-pre-incubated with the antibody, not “pre-neutralized and not added as an inactive protein) is added, thereby reproducing the expression of the pathogenic protein with an extracellular secretion in the extracellular space. One sample of GN K01 Ig was pre-incubated with HERV-K Env for 30 minutes, and then added together to the human neurons. Spontaneous electrical activity was recorded daily afterward beginning at 24 h post treatment. Quantitation of electrical activity was completed with Axion Axis software. Parameters such as number of

(58) 2. Results

(59) 2.1. Extracellular HERV-K Envelope Protein is Toxic to Human Neuronal Cells and its Toxicity is specifically Inhibited by GN_mAb_Env_K01 (GN K01) Antibody 2.1.1. Neuronal Viability
Human neuronal cultures treated with 100 nM recombinant HERV-K Env protein displayed significant neurotoxicity, resulting in important neuronal cell loss within next days. The effects of HERV-K Env were quantitated at 5 days after exposure to HERV-K Env protein. We observed that the Env+GN K01 Ig (3 μg/mL) treated neurons had increased survival compared to Env treated neurons. When analyzed five days after the HERV-K envelope protein was added to the culture medium, cells treated with Env plus 3 μg/mL of non-immune IgG control antibody exhibited similar toxicity. Neurons treated with GN K01 3 μg/mL, either prior to HERV-K Env or pre-incubated with Env and then applied to the neuronal cultures showed significantly more viable neurons, thereby confirming the efficacy of GN K01 antibody in neutralizing the toxicity of HERV-K Env protein (FIG. 7). 2.1.2. Neuronal Neurite Length
The effect of HERV-K Env on mean neurite length was analyzed in parallel, and addition of GN K01 (3 μg/mL) to Env exposed neurons significantly increased neurite length compared to Env or Env+ control non-Immune Ig treated neurons (FIG. 8). 2.1.3. Neuronal Functional Activity
Electrophysiological studies with the Axion Maestro MEA system were conducted to evaluate whether HERV-K Env treatment resulted in functional changes to spontaneous electrical activity, which is a major feature of normal neuronal activity. After plating the human neuronal cultures on the 48 well MEA plates for 21 days, the cultures were incubated in differentiation media (described above) and with GN K01 or control non-Immune IgG (Thermo Product #MA 1-10418) at a final concentration of 3 μg/mL. After 60 minute pre-incubation, incubation, recombinant HERV-K Env protein (My BioSource, Cat #MBS1391552) was added to a final concentration of 100 nM. One sample of GN K01 Ig was pre-incubated with HERV-K Env for 30 minutes, and then added together to the human neurons. Spontaneous electrical activity was recorded beginning at 24 h post treatment. 24 h post HERV-K Env treatment, the number of spikes and mean firing rate decreased by 40% with Env treatment. Neurons exposed to HERV-K Env plus control non-immune IgG treatment showed about 30% decrease in the number of spikes and mean firing rate. Most interestingly, neurons exposed to HERV-K Env plus GN K01 showed number of spikes and mean firing rates similar to the wells incubated with control media only, thereby showing complete inhibition of HERV-K Env pathogenic effects on global neuronal functional activity (FIG. 9).

(60) 3. Conclusions

(61) The efficacy of GN K01 antibody was therefore specific and its beneficial effects against the pathogenic consequences of neuronal exposure to HERV_K Env protein were evidenced by its significant (i) inhibition of cell death, (II) maintenance of neuron cell morphology and neurite length and (iii) full recovery of neuronal functional activity as measured by electrophysiological activity in the presence of pathogenic of HERV-K Env protein, versus same exposure to HERV-K Env with an irrelevant control antibody or with out antibody.
Therefore, (i) after the specific detection of HERV-K Env protein in the altered neurons within the brain parenchyma of patients with sporadic ALS was shown and (ii) after the proof of concept that this HERV-K Env protein alone drives the pathogenicity has been brought by clinical and histological signs of sporadic ALS reproduced in transgenic mice expressing HERV-K env gene encoding this unique protein (Li, Lee et al. 2015), the specific efficacy of GN K01 antibody, as demonstrated by the inventors on relevant human neuron cellular and functional aspects, is evidencing its therapeutic value in ALS, in particular in sporadic ALS.
The specific activity of such an antibody appears to neutralize (i.e: treat) the pathogenic effects of the HERV-K envelope (Env) protein, which itself was shown to be associated with ALS pathognomonic neuronal lesions in the brain of patients with sporadic ALS and to reproduce the same neuronal alterations when added to cultured neurons, or when expressed as a single transgene in mice along with the same clinical signs as in ALS. Moreover, the addition of the GN K01 antibody in the presence of the HERV-K Env pathogenic protein shows more significant efficacy that pre-incubation of GN K01 with HERV-K Env recombinant protein in vitro, which shows its optimal efficacy in physiological conditions, therefore in a therapeutic application.
HERV_K protein toxicity not only involves intracellular expression in transfected target cells or in neurons from transgenic animals from previous knowledge, but also involves secreted extracellular HERV-K protein pathogenic to naive (non-HERV-K transfected or overexpressing neurons. Therefore, an antibody of the invention also protects human neuronal cells from paracrine spreading of cytotoxicity induced by neuronal exposure to secreted and/or extracellular HERV_K Env protein. Implicitly, this also applies to autocrine cytotoxicity by HERV_K Env protein produced in neurons expressing or overexpressing HERV-K env coding gene(s)
As a conclusion, the antibody specifically targeting HERV-K envelope protein according to the invention can neutralize its pathophysiological properties as observed in ALS, notably in sporadic ALS, or in HERV-K env mouse transgenic models reproducing SLS features.

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