ANTIBODIES HAVING SPECIFICITY FOR BTN2 AND USES THEREOF
20220162305 · 2022-05-26
Inventors
- Etienne FOUCHER (MARSEILLE, FR)
- Carla CANO (MARSEILLE, FR)
- Kieu Suong LE (MARSEILLE, FR)
- Christine PASERO (MARSEILLE, FR)
- Daniel OLIVE (Marseille, FR)
Cpc classification
C07K2317/73
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to antibodies having specificity for BTN2A and uses thereof, in particular for the treatment of cancer.
Claims
1-5. (canceled)
6. An anti-butyrophilin-2A (BTN2A) antibody comprising: a heavy chain variable region CDR1 comprising SEQ ID NO:3, a heavy chain variable region CDR2 comprising SEQ ID NO:4, a heavy chain variable region CDR3 comprising SEQ ID NO:5, a light chain variable region CDR1 comprising SEQ ID NO:6, a light chain variable region CDR2 comprising SEQ ID NO:7, and a light chain variable region CDR3 comprising SEQ ID NO:8, or a heavy chain variable region CDR1 comprising SEQ ID NO:21, a heavy chain variable region CDR2 comprising SEQ ID NO:22, a heavy chain variable region CDR3 comprising SEQ ID NO:23, a light chain variable region CDR1 comprising SEQ ID NO:24, a light chain variable region CDR2 comprising SEQ ID NO:25 and a light chain variable region CDR3 comprising SEQ ID NO:26.
7. The anti-BTN2A1 antibody of claim 6 which comprises: a heavy chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:1 and a light chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2, or a heavy chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:20.
8. The anti-BTN2A antibody of claim 6, wherein said anti-BTN2A antibody has at least one of the following functions: it activates secretion of cytolytic molecules of γ9Vδ2 T cells, it activates the cytolytic function of Vγ9Vδ2 T cells, and/or it activates the proliferation of Vγ9Vδ2 T cells.
9. The anti-BTN2A antibody of claim 8, which competes for binding to BTN2A1 with the reference murine antibody mAb 107G3 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.
10. (canceled)
11. The anti-BTN2A antibody of claim 6, having specificity for the human BTN2A1 isoform.
12. The anti-BTN2A antibody of claim 6, which is a human, chimeric or humanized antibody.
13. A nucleic acid molecule, which encodes a heavy chain and/or a light chain of the anti-BTN2A antibody of claim 6.
14. A host cell comprising the nucleic acid of claim 13.
15. (canceled)
16. A method of treating cancer or an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically efficient amount of the antibody of claim 6.
17. A pharmaceutical composition comprising the anti-BTN2A antibody of claim 6, and at least a pharmaceutically acceptable carrier.
18. The anti-BTN2A antibody of claim 6, wherein the anti-BTN2A antibody has at least one of the following functions: it inhibits the polarization of monocytes towards M2 macrophages, it induces reversion of M2 macrophages towards anti-tumoral M1 macrophages, it triggers NK cells activation directly, and/or it enhances NK cell-mediated cytotoxicity.
19. The anti-BTN2 antibody of claim 6 which competes for binding to BTN2A1 with the reference antibody mAb 107G3 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or the reference antibody mAb 101G5 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:20.
20. The anti-BTN2 antibody of claim 6, which binds an epitope comprising residues located in positions: 65, 68, 69, 72, 78; 84, 85, 95, 97, and 100 of SEQ ID NO. 17.
21. The anti-BTN2 antibody of claim 6, which binds an epitope comprising residues located in positions 212, 213, 218, 220, 224, and 229 of SEQ ID NO. 17.
22. The anti-BTN2 antibody of claim 6 which does not cross-react with the human BTN3 isoforms, and/or which cross-react with the cynomolgus BTN2A1 ortholog.
23. A method of treating cancer or an infectious diseases disease in a subject in need thereof, comprising: administering to the subject a therapeutically efficient amount an anti-butyrophilin-2A (BTN2A) antibody characterized in that it has at least one of the following functions: it inhibits the polarization of monocytes towards M2 macrophages, it induces reversion of M2 macrophages towards anti-tumoral M1 macrophages, it triggers NK cells activation directly, it enhances NK cell-mediated cytotoxicity.
24. The method of claim 23, wherein the antibody comprises: a heavy chain variable region CDR1 comprising SEQ ID NO:3, a heavy chain variable region CDR2 comprising SEQ ID NO:4, a heavy chain variable region CDR3 comprising SEQ ID NO:5, a light chain variable region CDR1 comprising SEQ ID NO:6, a light chain variable region CDR2 comprising SEQ ID NO:7, and a light chain variable region CDR3 comprising SEQ ID NO:8, or a heavy chain variable region CDR1 comprising SEQ ID NO:21, a heavy chain variable region CDR2 comprising SEQ ID NO:22, a heavy chain variable region CDR3 comprising SEQ ID NO:23, a light chain variable region CDR1 comprising SEQ ID NO:24, a light chain variable region CDR2 comprising SEQ ID NO:25 and a light chain variable region CDR3 comprising SEQ ID NO:26.
26. The method of claim 23, wherein the antibody binds an epitope comprising residues located in positions 212, 213, 218, 220, 224, and 229 of SEQ ID NO. 17.
Description
LEGENDS OF THE FIGURES
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EXAMPLES
[0326] Material and Methods
[0327] Cell Culture, Monocytes and NK Cell Sorting:
[0328] Peripheral blood mononuclear cells (PBMCs) were obtained from EDTA (Ethylene Diamine-tetraacetic acid)-buffy coats from healthy donors (HD) provided by the local Blood Bank (Etablissement Français du Sang (EFS)-Marseille-France) and isolated by centrifugation on density gradient (Eurobio). Fresh PBMCs were cultured at 37° C., 5% CO.sub.2 in Roswell Park Memorial Institute medium 1640 (RPMI; Lonza) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S).
[0329] Natural Killer (NK) cells were sorted from fresh PBMCs by negative selection using EasySep™ Human NK Cell Enrichment Kit (StemCell Technologies) following manufacturer's instructions. Human CD14+ monocytes were sorted by using CD14+ Microbead kit (Miltenyi) following manufacturer's instructions. Monocytes were cultured at a density of 10.sup.6 cells/mL in RPMI supplemented with 1% L-glutamine, 100 U/mL Penicillin/streptomycin, 1 mM Sodium Pyruvate, 10 mM HEPES, 0.1 mM non-essential amino acids and 10% FBS (all from Thermofisher), during 5 days at 37° C. Pancreatic adenocarcinoma cell line PANC-1 was cultured in RPMI supplemented with 10% FBS. Once grown to 90% confluence, culture medium was discarded, and cells were rinsed twice in PBS 1×. PANC-1 cells were then cultured in RPMI supplemented with 5% FBS for further 24 h (30 mL per 175 cm.sup.2 flasks to get a concentrated supernatant). PANC-1 conditioned-medium was then collected, filtered (0.2 μM) and stored at −20° C. until use. Other human cell lines and their corresponding culture media are summarized in the table 1 below:
TABLE-US-00003 TABLE 1 Cell Line Tissue Disease Cell Type Medium HEK-293T embryonic NA Epithelial DMEM 10% FBS kidney Glutamax 1 mM DU-145 prostate carcinoma Epithelial RPMI NaPyr Glutamax MDA-MB-231 breast Adenocarcinoma Epithelial DMEM Glutamax A-549 lung carcinoma Epithelial F-12K Medium HT-29 colon colorectal Epithelial DMEM adenocarcinoma Glutamax HCT-116 colon colorectal Epithelial McCoy's 5a 10% FBS carcinoma RAJI B lymphoblast Burkitt Lymphoma B lymphocyte RPMI 10% FBS Glutamax 1 mM HL60 Peripheral Acute Promyeloblast NaPyr blood promyelocytic leukemia
The following human cell lines were obtained from the American Type Culture Collection: Daudi (Burkitt's lymphoma), Jurkat (acute T cell leukemia), MDA-MB-134 (breast ductal carcinoma) and HEK-293T (embryonic kidney). The human pancreatic adenocarcinoma cell line L-IPC (PDAC087T) was kindly given by Dr. Juan IOVANNA. Daudi and Jurkat cells, as well as PBMCs, were cultured in RPMI 1640 medium supplemented with 10% foetal calf serum (FCS), 1% Na-Pyruvate, 1% L-glutamine (all from Life technologies). HEK-293T BTN2 KO cells were generated by CRISPR-Cas9-mediated inactivation of all isoforms of BTN2 (data not shown). MDA-MB-134, L-IPC, HEK-293 cells and HEK-293T BTN2 KO cells were cultured in DMEM medium (Life Technologies) with 10% FCS. Hybridomas were cultured in DMEM/Ham's F12 (1:1) (ThermoFisher Scientific), 4% FetalClone I (Hyclone), Chemically Defined Lipid Concentrate (1:250), 1% Glutamine, 1% sodium pyruvate and 100 μg/mL PenStrep (all from ThermoFisher Scientific). For collection of hybridoma supernatants, hybridomas were cultured for 4-5 days without Fetalclone.
[0330] For assessment of anti-BTN2A1 mAbs specificity, HEK-293T BTN2 KO cells were transfected independently with pcDNA3-Zeo-BTN2A1-CFP plasmids, which encode BTN2A1 and BTN2A2 CFP(Nter)-fusion proteins, using Lipofectamine 3000 reagent (Thermofisher Scientific) according to manufacturer's instructions.
Identification of the Reference Anti-BTN2A1 mAb 107G3
[0331] Mouse anti-human BTN2A1 antibodies were generated by immunizing 48 mice, bearing 6 different MHC combinations, with recombinant human BTN2A1-Fc fusion protein. Mice were bled after 21 days and serum titer of BTN2A1-specific polyclonal antibodies was determined via Luminex assay. Mice displaying the highest BTN2A1-specific antibodies titer were euthanized. Splenic B cells were isolated via positive selection and underwent PEG-induced fusion to myeloma cells for hybridoma generation.
[0332] Hybridomas were cloned by limiting dilution and hybridoma supernatants underwent two rounds of screening for target specificity and their capacity to induce Vγ9Vδ2-T cell degranulation (
Expansion of Vγ9Vδ2-T Cells
[0333] Effector Vγ9/Vδ2-T cells were established by culturing PBMCs from HV in presence of Zoledronate (Sigma, 1 μM) and recombinant human (rh)IL-2 (Proleukin, 200 IU/mL) starting at Day 0. From Day 5, rhlL-2 was renewed every other day and cell density was kept at 1.15×10.sup.6/mL for a total of 15 days. The last day, the purity of Vγ9/Vδ2-T cells was evaluated by flow cytometry. Only cell cultures that reached purity of Vδ9/Vδ2-T cells higher than 80% were selected to be used in functional tests. Purified Vδ9/Vδ2-T cells were frozen until use.
Luminex Assay
[0334] Magnetic COOH beads (Biorad) were conjugated to rhBTN2A1 protein (R&D) according to manufacturer's instructions and beads were stored in storage buffer (Biorad) at −20° C. until use. For titration of mouse sera, serial serum dilutions were made in Luminex assay buffer (Nanotools) starting at 1:50, by dilution steps 1:4; 100 μL bead suspension were mixed with 100 μL serum dilution and incubated for 1 hr at RT, after which beads were washed 3-times in washing buffer, incubated with 1 μg/mL biotinylated goat anti mouse IgG-Fc in Luminex assay buffer, and had 3 further washes in Luminex assay buffer. Finally, beads were incubated for 1 hr with 1 μg/mL streptavidin PE in Luminex assay buffer, before 3 final washes in Luminex read buffer (Nanotools). Beads were resuspended in Luminex read buffer and data were acquired on a Luminex 100/200 system. For hit identification, 30 μL supernatant were transferred into 96 well plates, and 90 μL Luminex assay buffer were added. One hundred microliters of bead suspension were mixed with 100 μL supernatant dilution and incubated for 16 hrs at RT, before proceeding to the protocol described above. For hit identification, those with the highest affinity for the target and the lowest affinity for an irrelevant control protein (Rank-Fc) were selected. For affinity/Kd calculation, hybridoma supernatants underwent serial dilution in Luminex assay buffer starting at 40.000 μM, by dilution steps 1:4, and were analyzed as described above. Kd corresponds to midpoint of the corresponding binding curve.
Flow Cytometry
[0335] PBMCs, purified Vγ9Vδ2-T cells-T cells or cell lines were incubated with specified mAbs before analysis on a BD LSRFortessa (BD Biosciences), CytoFlex LX or CytoFlex S (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo). Antibodies used for Vγ9Vδ2-T cell degranulation assay were: anti-CD107a-FITC (BD Biosciences), anti-CD107b-FITC (BD Biosciences), anti-CD3-PeVio700 (Miltenyi), anti-PanTγδ-PE (Miltenyi), live/dead near IR (Thermofisher). All immune stainings performed using 10 μg/mL of purified mAbs, in presence of FcR Block reagent (Miltenyi), goat anti-mouse-PE 1:100 (Jackson Immunoresearch), and live/dead near IR (Thermofisher). Mouse anti-human CD277 (also known as BTN3A; clone 103.2 with IgG2a isotype) was previously disclosed (WO2012/080351). For assessment of anti-BTN2A1 mAbs specificity, 24 hours after transfection, HEK-293T BTN2 KO cells (5×10.sup.4/sample) were collected and stained with the indicated concentrations (5 ng/mL to 75 μg/mL) of anti-human BTN2A1 107G3 mAb as described above. Mouse IgG1 antibody (Miltenyi) was used as isotype control for staining.
Functional Assay on Vγ9/Vδ2-T Cells
[0336] Purified Vγ9/Vδ2-T cells from HV were cultured overnight in rhlL-2 (200 UI/mL). Then, Vγ9/Vδ2-T cells were co-cultured at 37° C. with the indicated target cell lines (at effector: target (E:T) ratio of 1:1) with or without the following mAbs (50 μL of hybridoma supernatant or 10 μg/ml purified mAb, as indicated): anti-BTN2A1 mAbs, mIgG1 (isotype control antibody) or hybridoma culture medium. Phorbol 12-myristate 13-acetate (PMA, 20 ng/mL) with ionomycine (1 μg/mL) were used as positive control for Vγ9/Vδ2-T cell activation. For first round of hybridoma supernatant screening, culture supernatants were collected after 4 hours and tested for their content on IFNγ, as an indicator of Vγ9/Vδ2-T cell activation, using the Human IFNγELISA set (BD Biosciences). For second round of hybridoma supernatant characterization, Vγ9/Vδ2-T cell degranulation was assessed by a 4 hours incubation in presence of GolgiStop (BD Biosciences) and soluble CD107(a&b)-FITC. After 4 hours, cells were collected, fixed in PBS 2% paraformaldehyde and analyzed on a CytoFlex LX (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo).
Proliferation of Vγ9/Vδ2-T Cells
[0337] Vδ9/Vδ2-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, 5×10.sup.5 CellTrace-labeled cells were cultured in 96-well round-bottom plates in the presence of IL-2 (200 UI/ml), with or without pAg, and with or without purified anti-BTN2A1 107G3 antibody (10 μg/ml). After 5 days of culture, CellTrace dilution was evaluated by flow cytometry on a CytoFlex LX (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo).
Statistics:
[0338] For Vγ9/Vδ2-T cell degranulation, results are expressed as mean±SEM. EC.sub.50 of purified anti BTN2A1 mAb on BTN2A1-transfected HEK-293T BTN2 KO cells was determined based on log(dose) response curves after non-linear regression following a variable-slope model. All analyses were performed using GraphPad Prism 7.04 software (GraphPad).
Identification of the Reference Anti-BTN2A mAb 101G5
[0339] After VH and VL sequencing, 23 anti-BTN2A mAbs obtained from mouse hybridoma generation as described above, were produced under a chimeric IgG1 format. Briefly, murine VH and VK anti-BTN2A mAb sequences were synthesized in vitro and amplified by PCR using PrimeSTAR Max DNA Polymerase (Takara). PCR products were cloned in heavy chain and light chain expression vectors (MI-mAbs) using In Fusion system (Clontech), and plasmids were transformed into Stellar competent cells (Clontech). Vector sequencing (MWG Eurofins) was performed in order to validate anti-BTN2A mAbs, before large scale (maxi) preparation of plasmid for further transfection. Vectors encoding matched light and heavy chains for each anti-BTN2A clone were transiently transfected in HEK-293 cells (2.9×10.sup.6 of cells/mL) with a ratio heavy chain/light chain 1:1.2, and medium was renewed after 18h. Seven days after transfection, culture supernatants were harvested for mAb purification. Affinity purification of antibodies was performed with Protein A Sepharose Fast Flow (GE Healthcare), overnight at 44° C. Binding buffer was 0.5 M Glycine, 3M NaCl, pH8.9. Elution was performed with the following buffer: 0.1 M Citrate pH3. Samples were neutralized right after elution with 1M Tris-HCl, pH9 (10% v/v). Finally, chimeric anti-BTN2A mAbs were dialyzed into PBS 1× and filtered through 0.22 μM filters (Millex GV hydrophilic PVDF, Millipore). Chimeric anti-BTN2A mAb concentration was determined in a Nanodrop 2000 Spectrophotometer (ThermoScientific) taking into account the extinction coefficient of the antibodies. Purity, as defined by the fraction of mAb monomers, was determined by UPLC-SEC using an Acquity UPLC-HClass Bio (Waters), with an Acquity UPLC Protein-BEH-200A, 1.7 μm 4.6×50 mm column (Waters). Antibody mass was determined in a Xevo G2-S Q-T of mass spectrophotometer (Waters) using a reversed phase column (PLRP-S 4000 A, 5 μm, 50×2.1 mm (Agilent technologies). All samples were analyzed after de-glycosylation with PNGase F glycosidase (New England Biolabs) at 37° C. When an unexpected mass was found, the primary amino acid sequence was analyzed using bioinformatic tools to identify putative glycosylation sites within the Fab region. SDS-PAGE of purified antibodies allowed detection of fragmentation and/or aggregation of the final material stain free Mini protean TGX gel 4-15 (Biorad). Endotoxin level was determined using a Chromogenic LAL Limulus Amebocyte Lysate kinetic assay (Charles River Endosafe) using a ClarioStar spectrophotometer (BMG Labtech).
In Vitro Macrophage Polarization Assays:
[0340] M1 or M2 macrophages were polarized from sorted monocytes from healthy donors. To this end, sorted monocytes were cultured in presence of GM-CSF or M-CSF (40 ng/mL; Miltenyi) to generate M1 or M2 macrophages, respectively. After 5 days, the resulting macrophages were either collected for phenotype analysis, or stimulated with LPS (200 ng/mL) for further 2 days. In some experiments, IL-4 (20 ng/mL) was added to M2 macrophages at day 4, which resulted in generation of “M2+IL-4” or macrophages. In some experiments, M2 macrophages were generated by culturing monocytes in presence of PANC-1 cancer cell-conditioned medium (diluted 30% v/v in culture medium, at day 0 and day 3) without M-CSF supplementation. The resulting M2 macrophages are called “Tum-ind-M2” in this application. In order to screen anti-BTN2A mAbs for their ability to modulate M2 differentiation, M2 macrophages were generated from monocytes, as described above, with or without chimeric anti-BTN2A mAbs or their isotype control (human IgG1; Sigma) at the indicated concentrations. All mAbs are wet-coated (overnight at RT in PBS 1×). As control for M2 differentiation inhibition, GM-CSF (40 ng/mL) and IFNγ (100 ng/mL, BioTechne) were added to monocytes during M-CSF-induced M2 polarization. M1 macrophages polarized in presence of GM-CSF were used as phenotype control. After polarization, the resulting macrophages and their culture supernatants were collected, and the expression of M1- and M2-related markers at the plasma membrane was assessed by flow cytometry. In addition, cytokine content in culture supernatants was quantitated using IL-10 and TNFα ELISA kit (ThermoFisher Scientific) following manufacturer's instructions.
In Vitro M2 Macrophage Reversion Assays:
[0341] M2 macrophages were generated from monocytes in presence of M-CSF as described above in absence of the reference mAbs. M2 macrophages were collected and cultured for 2 days, with or without LPS, on culture wells that were previously wet-coated overnight with 10 μg/mL of the reference antibodies or their control isotype mAb (human IgG1 from Sigma). As controls of M2 reversion, GM-CSF (40 ng/mL) and IFNγ (100 ng/mL) were added to M2 macrophages culture for 2 days. M1 macrophages polarized in presence of GM-CSF were used as phenotype control. After reversion experiments, macrophages reverted without addition of LPS were collected for phenotype analysis by flow cytometry. Culture supernatants from LPS-stimulated reverted macrophages were harvested cytokine quantitation using ELISA.
In Vitro Assays on M2 Macrophage-Mediated Inhibition of T Cell Proliferation and IFNγ Production
[0342] M1, and M2 macrophages were generated with or without addition of the reference antibodies or their isotype control mAb as described above. CD3+ T cells were sorted from healthy donor PBMCs by using the CD3+ Microbead kit (Miltenyi) according to manufacturer's instructions and frozen until the co-culture. Activated CD3+ T cells were generated as follow: CD3+ T cells were stained with 5 μM CellTrace Violet dye (ThermoFisher Scientific), then 10.sup.5 such cells were cultured in X-Vivo 10 medium supplemented with 20 U/mL IL-2 (Miltenyi), LPS (200 ng/mL) and CountBright absolute counting beads (5×10.sup.3 per well, ThermoFisher Scientific) in 96-well flat-bottom plates, previously coated with 1 μg/mL anti-CD3 mAb (clone OKT3, BD biosciences). For co-culture with macrophages, 2×10.sup.4 allogeneic M1, M2, or macrophages polarized in presence of M-CSF and the reference mAbs or their control isotype, were added to activated allogeneic CD3+ cells. After 5 days of co-culture, 20 ng/mL PMA and 0.5 μg/mL ionomycin were added to the co-culture in order to enhance cytokine production, in presence of GolgiStop Protein transport inhibitor for 5 hours. Then, cells were recovered for phenotype analysis by flow cytometry. CellTrace dilution was used as an indicator of CD3+ T cell proliferation. Results of phenotype and proliferation were expressed in percentage or absolute cell number per mL (after calibration with absolute counting beads).
Natural Killer (NK) Challenge with the Reference Anti-BTN2A1 mAbs:
[0343] Sorted Natural Killer (NK) cells from healthy donors were labelled with and were then cultured at 37° C., 5% CO.sub.2 in RPMI supplemented with 10% FBS and 1% P/S, IL-2 (50 UI/mL) with or without IL-15 (10 ng/mL) stimulation. The reference anti-BTN2A mAbs or control isotype (10 μg/mL) were added to the culture on day 0. After 5 days, NK cells were extracellularly phenotyped for the expression of activation markers. NK activation was assessed by induction of CD69 and CD25 expression (percentage and Median Fluorescence Intensity MFI). Gating strategy for NK cells is shown in
Flow Cytometry:
[0344] Prior to staining, PBMCs/NK cells and monocytes/macrophages were incubated 10 min with human Fc block (Miltenyi) or human IgG1 (Sigma) for Fc receptor saturation. Labelled mAbs used were the following: CD14-FITC and -APC-Vio770 (Miltenyi), CD163-VioBlue (Miltenyi), DC-SIGN-PE-Vio770 (Miltenyi), CD80-PE (BD Biosciences), PDL1-APC (BD Biosciences), CD3-PE-CF594 (BD Biosciences) and CD3-BV605 (Biolegend), CD56-PE-Vio770 (Miltenyi) and -BV605 (BD Biosciences), CD69-BV421 (BD Biosciences), CD25-APC (BD Biosciences). Cells were incubated with the antibody cocktail 30 min at 4° C. Dead cells were excluded using a live/dead near IR dye (ThermoFisher Scientific) to define a “live” gate. For intracellular IFNγstaining, extracellularly stained cells were fixed and permeabilized using Intracellular Fixation & Permeabilization Buffer Set (eBioscience) and incubated with APC-labelled anti-IFNγ (BD Biosciences). Acquisition was performed on Fortessa flow cytometer (BD Biosciences) using FlowJo 10 software. For BTN2A1 and BTN2A2 phenotyping, purified anti-BTN2A1-specific (mAb5) and anti-BTN2A2-specific (mAb17) were used at 10 μg/mL and revealed with PE-labelled anti IgG (H+L) (Jackson lmmunoresearch). NK cells were CD45+CD14-CD3-CD56+ cells within the “live” gate, after selection of single cells. Monocytes were CD45+CD19-CD3-CD56-CD14+ cells within the “live” gate, after selection of single cells. Acquisition was performed on Cytoflex LS (Beckman Coulter), iQue Screener (Intellicyt) or Fortessa (BD Biosciences) flow cytometers, and data were analyzed using the FlowJo 10 software. Results are expressed as median fluorescence intensities (MFI) after subtraction of the value obtained with the corresponding staining control.
Octet-Based BTN2A1 Epitope Affinity Measurement and Binning Assay:
[0345] After generation of the reference anti-BTN2A antibodies in chimeric IgG1 format, affinities for the 2 different isoforms of this target (BTN2A1 and BTN2A2) were evaluated and competition assays were performed to determine whether these mAbs recognized the same epitope region of BTN2A1. Affinity and binning experiments were performed on an Octet Red96 platform (Fortebio/PALL), system based on Bio-layer interferometry (BLI) technology. For affinity experiments, recombinant human (rh)BTN2A1-Fc (GTP) was biotinylated using EZ-Link™ NHS-PEG4 Biotinylation Kit according to manufacturer's instructions, and biotinylated rhBTN2A2-Fc was purchased from R&D Systems. In the case of BTN2A1 affinity assays, biotinylated rhBTN2A1-Fc was loaded into streptavidin (SA) biosensors (ForteBio) diluted in Kinetic Buffer 1× (ForteBio) with a loading target level of around 1 nm, and chimeric anti-BTN2A antibodies were used as analytes. For BTN2A2 affinity assays, chimeric anti-BTN2A antibodies were loaded into FAB2G sensors (anti human CH1; Fortebio) as described above, and biotinylated rhBTN2A2-Fc was used as analyte. In both cases, analytes remained in solution and their working concentrations were diluted in Kinetic Buffer 10× (ForteBio). For the first run, the standard working concentration ranged from 200 to 3.125 nM. When necessary for the second run, working concentrations were adjusted from 80 to 1.25 nM. All runs (including loading, equilibration, association/dipping of sensors into analyte, dissociation and regeneration) were performed at 30° C. with shaking 1000 rpm. Analysis was performed using a 1:1 or 2:1 Langmuir model (for BTN2A1 or BTN2A2, respectively) calculated by Octet software, which allowed a better fitting calculation. For binning experiments, His-tagged BTN2A1 (rhBTN2A-His) was purchased from R&D Systems. The reference anti-BTN2A antibodies were tested in a pairwise manner against BTN2A1. Binning experiments were performed by following the«in-tandem»format, meaning that rhBTN2A-His was immobilized on the biosensor (anti-Penta-His«HIS1K biosensors; ForteBio/PALL) and presented to the 2 competing antibodies in consecutive steps. For this kinetic screening, the loading of rhBTN2A-His on HIS1K (signal intensity: 1 nm) was followed by an association step with 10 μg/mL of antibody for 3 min, then by a dissociation step of 3 min. rhBTN2A1-His activity was confirmed via a kinetic screening assay performed in the same format as the binning assay (BTN2A1 as ligand/capture on the sensor and antibody as analyte). All antibodies (saturating or competing one) were used at 10 μg/mL, diluted in Kinetic Buffer 1×. For this kinetic screening, the loading of rhBTN2A1-His on HIS1K (signal intensity: 1 nm) was followed by an association step with antibody for 3 min, then by a dissociation step of 3 min. Assay steps were as follow: Baseline->Antigen capture->Baseline->Saturating antibody->Baseline->Competing antibody->Regeneration following an “in-tandem” scheme. Binning data were analyzed using Octet Data Analysis HT 11.1 using epitope bin operation.
Epitope Mapping of the Reference mAbs 107G3 and 101G5
[0346] The interactions between BTN2A1 and the reference mAbs 107G3 and 101G5 were assessed by differential assessment of peptide mass fingerprint of BTN2A1 alone of with 107G3 or 101G5. Before starting the epitope mapping, a high-mass MALDI analysis has been performed on rhBTN2A1-Fc protein (GTP Technologies) in order to verify their integrity and aggregation level using an Autoflex II MALDI ToF mass spectrometer (Bruker) equipped with CovalX's HM4 interaction module (CovalX), which confirmed that no non-covalent aggregates or multimers of BTN2A1 were present in the sample. In order to characterize BTN2A1, and to determine the epitope of BTN2A1/107G3 and BTN2A1/101G5, we submitted the sample to trypsin, chymotrypsin, Asp-N, elastase and thermolysin proteolysis followed by nLC-LTQ-Orbitrap MS/MS analysis, using a nLC Ultimate 3000-RSLC system in line with a LTQ-Orbitrap mass spectrometer (Thermo Scientific). For BTN2A1/107G3 and BTN2A1/101G5 complexes, the protein complexes were incubated with deuterated cross-linkers prior to multi-enzymatic cleavage. After enrichment of the cross-linked peptides, the samples were and the data generated were analyzed using XQuest 2.0 and Stavrox 3.6 software. For sample preparation, reduction alkylation was performed as follows: BTN2A1 (4.04 μM) were mixed with DSS d0/d12 (2 mg/mL; DMF) before 180 minutes incubation time at room temperature. After incubation, reaction was stopped by adding 1 μL of Ammonium Bicarbonate (20 mM final concentration) before 1h incubation time at room temperature. Then, the solution was dried using a speedvac before H.sub.2O 8M urea suspension (10 μL). After mixing, 1 μL of DTT (500 mM) was added to the solution. The mixture was then incubated 1 hour at 37° C. After incubation, 1 μl of iodoacetamide (1M) was added before 1 hour incubation time at room temperature, in a dark room. After incubation, 100 μl of the proteolytic buffer were added. The trypsin buffer contains 50 mM Ambic pH 8.5, 5% acetonitrile, the chymotrypsin buffer contains Tris HCl 100 mM, CaCl.sub.2 10 mM pH 7.8; the ASP-N buffer contains Phopshate buffer 50 mM pH 7.8; the elastase buffer contains Tris HCl 50 mM pH 8.0 and the thermolysin buffer contains Tris HCl 50 mM, CaCl.sub.2 0.5 mM pH 9.0. For trypsin proteolysis, 100 μL of the reduced/alkyled BTN2A1 were mixed with 1 μL of trypsin (Roche Diagnostic) with the ratio 1/100. The proteolytic mixture was incubated overnight at 37° C. For chymotrypsin proteolysis, 100 μL of the reduced/alkyled BTN2A1 were mixed with 0.5 μL of chymotrypsin (Roche Diagnostic) with the ratio 1/200. The proteolytic mixture was incubated overnight at 25° C. For ASP-N proteolysis, 100 μL of the reduced/alkyled BTN2A1 were mixed with 0.5 μL of ASP-N(Roche Diagnostic) with the ratio 1/200. The proteolytic mixture was incubated overnight at 37° C. For elastase proteolysis 100 μL of the reduced/alkyled BTN2A1 were mixed with 1 μL of elastase (Roche Diagnostic) with the ratio 1/100. The proteolytic mixture was incubated overnight at 37° C. For thermolysin proteolysis, 100 μL of the reduced/alkyled BTN2A1 were mixed with 2 μL of thermolysin (Roche Diagnostic) with a ratio 1/50. The proteolytic mixture was incubated overnight at 70° C. After digestion formic acid 1% final was added to the solution. After proteolysis, 10 μL of the peptide solution generated by proteolysis were loaded onto a nano-liquid chromatography system (Ultimate 3000-RSLC) with the following settings: A: 95/05/0.1 H.sub.2O/ACN/HCOOH v/v/v; B: 20/80/0.1 H.sub.2O/ACN/HCOOH v/v/v, gradient 5-40% B in 35 minutes, injected volume 10 μL, precolumn 300-μm ID×5-mm C18 PepMap™, precolumn flow rate 20 μL/min, column 75-μm ID×15-cm C18 PepMapRSLC, column flow rate, 200 nL/min.
ELISA Assay for Human and Cynomolgus BTN2A1 Cross-Reactivity
[0347] Cynomolgus BTN2A1 ortholog sequence (XM_015448906.1) was identified after BLAST search using human BTN2A1 aminoacid sequence, and its extracellular domain was cloned into pFUSE-hIgG1FC2 vector (InvivoGen) using EcoRI/EcoRV restriction sites. Recombinant cynoBTN2A1-Fc fusion protein was produced by transfection of the resulting pFUSE-hIgG1FC2-cynoBTN2A1 plasmid into Expi293F™ cells with ExpiFectamine™ 293 (ThermoFisher) according to manufacturer's instructions. The cell culture supernatant collected on day 6 was used for purification through an affinity purification column. The purified cynoBTN2A1-Fc protein was analyzed by SDS-PAGE and Western blotting for molecular weight and purity measurements. cynoBTN2A1-Fc protein concentration was determined by Bradford assay with BSA as a standard. For ELISA, cynoBTN2A1-Fc protein or recombinant human BTN2A1-Fc (huBTN2A1-Fc, GTP Technologies) were coated (5 μg/mL in 1×PBS) overnight at 4° C. After 3 washes in PBS, plates were saturated with BSA 2% v/v in PBS for 1 h at room temperature, then saturating buffer was discarded. The reference mAbs 101G5 and 107G3 or a control human IgG1 were diluted in PBS BSA 2% in 10-fold cascade dilutions starting from 1 μM to 1 μM, and 100 μL of each dilution were added per well and incubated for 90 minutes at room temperature on a plate shaker. All wells were washed 3 times in PBS before addition of Goat anti-mouse IgG HRP (Jackson ImmunoResearch, 1:10000 dilution in PBS BSA 2%) and incubation for 1 h at room temperature. Then, all wells were washed 3 times in PBS and 1-step ABTS solution (ThermoFisher) was added for binding revelation, as assessed by absorbance at 405 nm in a Spark spectrometer (Tecan). All samples were assessed in duplicates.
Results:
Identification of the Reference Antibody Anti-BTN2A1 107G3
[0348] The reference anti-BTN2A1 107G3 antibody was identified as follows: mice were immunized with BTN2A1-Fc antigen and splenocytes from mice presenting with the highest titer of BTN2A1-specific sera were collected and fused with a myeloma cell line to obtain hybridomas. Hybridoma culture supernatants displaying the highest affinity for BTN2A1 were selected for a first round of screening based on their ability to modulate secretion of IFN-γ by Vγ9/Vδ2-T cells. Selected clones from this first round of screening were subcloned and tested for their ability to induce IFN-γ secretion and Vγ9/Vδ2-T cell degranulation and IFN-γ secretion, notably their ability to induce Vγ9Vδ2-T cell degranulation (
Anti-BTN2A1 107G3 Antibody Induces Vγ9Vδ2-T Cell Degranulation Against Different Cancer Cell Targets
[0349] Purified Vγ9/Vδ2 T cells were expanded from PBMCs of healthy donors and co-cultured with different cancer cell lines, including Daudi (Burkitt's lymphoma), Jurkat (acute T cell leukemia), L-IPC (pancreatic adenocarcinoma) and MDA-MB-134 (breast carcinoma) as target cells, with or without anti-BTN2A1 107G3 hybridoma culture supernatant. As shown in
[0350] The percentage of CD107+ cells induced by anti-BTN2A1 107G3 hybridoma supernatant ranged from 71.1±7.4% in Daudi cells to 17.1±2.9% in MDA-MB-134 cells vs. 24.9±4.7% and 4.9±0.4%, respectively, in co-cultures with control hybridoma culture medium. In co-cultures of Vγ9/Vδ2 T cells, with all of the cancer cell lines tested as targets, anti-BTN2A1 107G3 induced between 2-fold and 8-fold more Vγ9/Vδ2 T cell degranulation compared to the same co-cultures in the presence of control hybridoma culture medium.
[0351] Purified anti-BTN2A1 mAb 107G3 displayed an EC50 of 0.77 μg/mL (95% IC 0.32-13.22 μg/mL) for the induction of Vγ9/Vδ2 T cell degranulation, as depicted by the percentage of CD107+ cells, co-cultured with Daudi cells as targets in the presence of increasing concentrations (0 to 18 μg/ml) of the anti-BTN2A1 107G3 mAb (
TABLE-US-00004 TABLE 3 Control Anti-BTN2A1 hybridoma 107G3 supernatant EC.sub.50 on HEK-293T BTN2 0.32 (95% IC 0.21-0.46) not applicable KO + BTN2A1 μg/mL EC.sub.50 on Functional assay 0.77 (95% IC 0.32-13.22) not applicable on Daudi μg/ml Functional assay on Daudi 71.1 ± 7.4% 24.9 ± 4.7% (% CD107.sup.+ cells) Functional assay on MDA- 17.1 ± 2.9% 4.9 ± 0.4% MB-134 (% CD107.sup.+ cells) Functional assay on L-IPC 31.1 ± 3.6% 14.4 ± 0.5% (% CD107.sup.+ cells) Functional assay on Jurkat 25.7 +5.1% 3.7 ± 0.4% (% CD107.sup.+ cells)
Anti-BTN2A1 107G3 Antibody Recognizes the BTN2A1 but not BTN3
[0352] In order to establish the specificity of anti-BTN2A1 mAb 107G3 for the BTN2A1 isoform only, HEK-293T BTN2 KO cells, which bear a CRISPR-Cas9-mediated inactivation of both BTN2 isoforms, were transiently transfected with a plasmid encoding BTN2A1 as a CFP-fusion protein. As shown in
[0353] The anti-BTN3 mAb 103.2, which recognizes all BTN3 isoforms, readily detected BTN3 expression in HEK-293T BTN2 KO cells. Hence, anti-BTN2A1 107G3 is specific for the BTN2A1 isoform and does not cross-react with BTN3.
Affinity of Anti-BTN2A1 107G3 mAb for BTN2A1 in Cellulo
[0354] In order to measure the affinity of anti-BTN2A1 107G3 mAb for its target, HEK-293T BTN2 KO cells transfected with the BTN2A1-encoding plasmid were stained with increasing concentrations (5 ng/mL to 75 μg/mL) of purified anti-BTN2A1 107G3 mAb or control mIgG1 (
Production of Chimeric Anti-BTN2A mAbs, Affinity Measurements and BTN2A Isoform Specificity:
[0355] Twenty-three monoclonal antibodies were transiently produced in HEK-293T cells, achieving different ranges of productivity. Most anti-BTN2A antibodies were produced at high levels (>100 mg/L and up to 430 mg/L). One antibody, anti-BTN2A mAb3 (Table 3), presented with very poor production in HEK-293T cells, ranging from 6 to 8 mg/L. Amino acid sequence analysis revealed an N-glycosylation site within its Fab portion, in the CDR1 of its VH. Two other antibodies, anti-BTN2A mAb9 and mAb11, also exhibited an N-glycosylation site within their Fab region (in CDR1_VH for mAb9 and in CDR1_VL for mAb11). Six antibodies exhibited a lower purity level (<95% in monomer) but only anti-BTN2A mAb1 and mAb3 had a purity level <90% (86% and 75%, respectively). All the final purified anti-BTN2A mAb exhibited a very low level of endotoxin (in the range of 0.1 EU/mg). Only mAb3 had a higher endotoxin level (0.73 EU/mg) than the others, but still within the criteria of acceptance (<1EU/mg). The affinity constants (K.sub.D, k.sub.on and k.sub.off) of the 23 anti-BTN2A chimeric mAbs were determined for BTN2A1 and BTN2A2 isoforms using Octet technology, by using biotinylated recombinant Fc-fusion soluble proteins. Table 4 recapitulates K.sub.D constants for each anti-BTN2A mAb. For mAb6 and mAb9, K.sub.D calculation was not possible due to the absence of dissociation observed during the time of measurement (koff <10.sup.−7 s.sup.−1), which could be explained by an avidity effect of these antibodies slowing down their dissociation from the target. Eight anti-BTN2A mAbs were found to bind only the BTN2A1 isoform (mAb2, mAb3, mAb4, mAb5, mAb6, mAb8, mAb9 and mAb10), 8 anti-BTN2A mAbs were found to bind only the BTN2A2 isoform (mAb16, mAb17, mAb18, mAb19, mAb20, mAb21, mAb22 and mAb23), and 7 mAbs were found to bind both isoforms (mAb1, mAb7, mAb11, mAb12, mAb13, mAb14 and mAb15).
TABLE-US-00005 TABLE 4 Chimeric anti-BTN2A mAb production and affinity summary. Monomer Purity Theoretical Actual (%) Mass (Da) Mass (Da) (SEC- Endotox (reduced/ (reduced/ Productivity KD (nM) KD (nM) Antibody UPLC) (EU/mg) intact-Kterm) intact-Kterm) (mg/L) BTN2A1 BTN2A2 mAb1 85.66 0.09 MW = 144302 MW = 144331 158.11 0.041 4.4 (101G5) LC = 23236 LC = 23235 HC = 48941 HC = 48938 mAb2 98.41 0.04 MW = 144688 MW = 144661 0.41 No (107G3) LC = 23749 LC = 23747 246 binding HC = 48611 HC = 48591 mAb3* 74.9 0.73 MW = 146116 MW = 146093 6.75 0.14 No LC = 23451 LC = 23448 binding HC = 49623 HC = 49606 mAb4 100 0.1 MW = 146300 MW = 146275 58.22 0.1 No LC = 23556 LC = 23554 binding HC = 49610 HC = 49590 mAb5 98.08 0.06 MW = 146092 MW = 146067 82.22 0.16 No LC = 23498 LC = 23496 binding HC = 49564 HC = 49544 mAb6 100 0.07 MW = 146286 MW = 146262 28.6 <10.sup.−3 No LC = 23369 LC = 23366 binding HC = 49790 HC = 49770 mAb7 94.72 0.07 MW = 145098 MW = 145105 279.78 0.1 7 LC = 23460 LC = 23458 HC = 49105 HC = 49101 mAb8 98.93 0.15 MW = 146274 MW = 146248 44.22 0.11 No LC = 23556 LC = 23554 binding HC = 49597 HC = 49576 mAb9* 98.78 0.03 MW = 146790 MW = 146765 328.89 <10.sup.−3 No LC = 23476 LC = 23473 binding HC = 49935 HC = 49921 mAb10 98.24 0.12 MW = 147132 MW = 147141 105.4 0.79 No LC = 23383 LC = 23381 binding HC = 50199 HC = 50194 mAb11* 98.88 0.05 MW = 147422 MW = 147428 243.3 0.008 3.2 LC = 23995 LC = 23991 HC = 49732 HC = 49733 mAb12 93.36 0.09 MW = 143976 MW = 143950 127 0.03 3.3 LC = 23391 LC = 23388 HC = 48613 HC = 48593 mAb13 97.64 0.03 MW = 145140 MW = 145148 430 0.07 0.8 LC = 23679 LC = 23677 HC = 48907 HC = 48903 mAb14 93.57 0.08 MW = 144180 MW = 144154 75.78 0.004 2.8 LC = 23440 LC = 23338 HC = 48666 HC = 48647 mAb15 97.07 0.07 MW = 143972 MW = 143945 129.78 0.012 1.2 LC = 23558 LC = 23556 HC = 48444 HC = 48424 mAb16 98.15 0.06 MW = 146944 MW = 146953 75.5 No 0.2 LC = 23983 LC = 23981 binding HC = 49505 HC = 49503 mAb17 100 0.09 MW = 146976 MW = 146985 84.44 No 0.3 LC = 23983 LC = 23981 binding HC = 49521 HC = 49518 mAb18 97.87 0.12 MW = 146408 MW = 146415 152.56 No 0.8 LC = 23977 LC = 23975 binding HC = 49243 HC = 49239 mAb19 91.73 0.04 MW = 147130 MW = 147140 252.2 No 0.3 LC = 24000 LC = 23995 binding HC = 49581 HC = 49577 mAb20 98.69 0.06 MW = 146584 MW = 146594 238 No 0.8 LC = 23970 LC = 23968 binding HC = 49338 HC = 49334 mAb21 98.05 0.09 MW = 147088 MW = 147097 66.78 No 0.22 LC = 23992 LC = 23990 binding HC = 49568 HC = 49565 mAb22 96.98 0.05 MW = 146952 MW = 147087 312.22 No 4.7 LC = 23508 LC = 23506 binding HC = 49984 HC = 49981 mAb23 100 0.03 MW = 147080 MW = 147089 78.56 No 0.3 LC = 23969 LC = 23967 binding HC = 49587 HC = 49585 *presence of a site of N-glycosylation in VH or VL.
BTN2A1 and BTN2A2 Plasma Membrane Expression on Monocytes and NK Cells:
[0356] We sought to determine whether anti-BTN2A mAbs could target non-Vγ9Vδ2 T cell compartments of the peripheral blood, namely monocytes and NK cells, for therapeutic purposes. Hence, we used mAb5 and mAb17 that were found in our octet assays to bind only BTN2A1 or BTN2A2, respectively, for phenotyping monocytes and NK cells from the peripheral blood. As shown in
Screening of Anti-BTN2A mAbs for their Ability to Modulate Monocyte to Macrophage Polarization
[0357] In response to signals from their microenvironment, monocytes can be polarized into M1 or M2 macrophages. M1 macrophages present with pro-inflammatory and anti-tumoral properties, whereas M2 macrophages have anti-inflammatory properties and are associated with tumor development. Given that only BTN2A1 isoform was found at the plasma membrane of monocytes, anti-BTN2A mAbs that recognized BTN2A1-only or both BTN2A1/BTN2A2 isoforms were evaluated for their ability to interfere with monocyte polarization into M2 macrophages in vitro in the presence of M-CSF. M1 macrophages generated in the presence of GM-CSF (CD14+/−CD163-), and M2 (CD14+CD163+) macrophages generated in the presence of M-CSF, both without mAbs were used as controls for macrophage polarization. After 5 days of in vitro polarization, the expression of CD14 and CD163 at the plasma membrane of M1, M2, and M-CSF-induced macrophages polarized in the presence of anti-BTN2A mAbs or their control IgG1 were assessed by flow cytometry (Table 4). As expected, M1 cells presented with low CD14 expression and undetectable CD163 expression (Table 5 and
TABLE-US-00006 TABLE 5 Effect of anti-BTN2A mAbs on the expression of CD14 and CD163 after monocyte to M2-macrophage polarization. M2-related markers (MFI; n = 3) Polarizing CD14 CD163 Cytokine Median SEM Median SEM M1 GM-CSF 1047 463.6 64 17.27 M2 M-CSF 38878 3866 6405 1051 hlgG1 28070 2937 4524 839.4 mAb1 347 1892 38 60.87 (101G5) mAb2 4408 8448 289 1407 (107G3) mAb3 42997 3756 6757 1428 mAb4 43853 4650 6493 1129 mAb5 45119 4004 6450 949.7 mAb6 38277 4793 5299 932.3 mAb7 42071 3227 6337 1176 mAb8 39483 3564 6722 1199 mAb9 43203 4869 6432 1135 mAb10 45362 3078 6737 1097 mAb11 41519 3675 6605 1090 mAb12 44750 2845 6909 1113 mAb13 5684 9480 642 1173 mAb14 33889 10427 4614 1498 mAb15 17411 10372 2553 1429
[0358]
TABLE-US-00007 TABLE 6 IC.sub.50 and EC.sub.50 of the reference anti-BTN2A mAbs on M2 vs. M1-related phenotype and cytokine secretion Effect of the reference IC.sub.50/EC.sub.50 (μg/mL) Markers anti-BTN2A mAbs 101G5 107G3 CD14 inhibition 0.51 7.7 CD163 inhibition 0.43 6.2 CD86 induction 2.55 25 PDL1 induction 37.09 7 IL-10 inhibition 0.105 14.8 TNFα induction 1.12 19.8
[0359] Other stimuli from the tumor microenvironment have been described to induce M2 macrophage polarization (Mosser and Edwards, Nat Rev Immunol 2008; Mantovani and Allavena, J Exp Med 2015). In addition to M-CSF, one of the most commonly used stimuli to induce M2 polarization is IL-4. We determined the impact of 101G5 and 107G3 on the differentiation of the so called pro-tumoral “M2+IL-4” macrophages, generated from monocytes after stimulation with M-CSF and IL-4. After 5 days of culture in such conditions, 101G5 and 107G3 inhibited the expression of “M2+IL-4”-related markers (CD14, CD163 and DC-SIGN,
[0360] In addition, the effect of 101G5 and 107G3 on cancer cell-induced M2 polarization from monocytes was assessed by culturing sorted monocytes in the presence of PANC-1 (pancreatic adenocarcinoma cell line)-conditioned culture supernatants. When 101G5 or 107G3 was added in this setting, M2 polarization was inhibited, as shown by decreased expression of M2-related markers (CD14, CD163) and IL-10 secretion (
Effect of the Reference Anti-BTN2A mAbs 101G5 and 107G3 on M2-Macrophage Reprograming Towards M1
[0361] The potential of the reference 101G5 and 107G3 mAbs to revert M2-polarized macrophages towards an M1 phenotype was assessed. For this purpose, M2 macrophages previously polarized in the presence of M-CSF for 5 days were seeded into wells previously coated with 101G5 and 107G3 mAbs and cultured for further 2 or 4 days. Treatment of M2 macrophages with IFNγserved as positive control of M2->M1 reversion. As shown in
[0362]
TABLE-US-00008 TABLE 7 IC.sub.50 and EC.sub.50 of the reference anti-BTN2A mAbs on M2 reversion Effect of the reference IC.sub.50/EC.sub.50 (μg/mL) Markers anti-BTN2A mAbs 101G5 107G3 CD14 inhibition 3.77 nd CD163 inhibition 6.79 15.46 CD86 induction 1.14 nd IL-10 inhibition 0.87 3.03 TNFα induction 6.44 80.33
The Reference Anti-BTN2A mAbs 101G5 and 107G3 Release M2-Mediated Inhibition of T Cell Proliferation
[0363] The ability of the reference 101G5 and 107G3 mAbs to affect the function of M2 macrophages namely, inhibition M2-mediated T cell proliferation, was investigated. To this end, allogeneic pre-activated CD3+ T cells were co-cultured with M1, M2, or M2 generated in the presence of the mAbs. As expected, co-culture with conventional M2 macrophages resulted in a decreased number of the CD3+ T cells, as well as decreased T cell proliferation (as assessed by CTV dilution) and production of IFNγcompared to M1 macrophages (
[0364] Hence, in contrast to M2 macrophages induced by M-CSF alone, macrophages generated in presence of 101G5 or 107G3 in addition to M-CSF allow proliferation and Th1 function (IFNγproduction) of allogeneic CD3+ T cells, similar to M1 macrophages.
The Reference Anti-BTN2A mAbs 101G5 and 107G3 Trigger NK Cell Activation and Cytotoxicity
[0365] Since BTN2A1 was found at the plasma membrane of NK cells, the potential ability of 101G5 and 107G3 to modulate NK cell activation was investigated. Purified NK cells from healthy donors were cultured for 5 days in presence of 101G5 or 107G3, with or without further activation (IL-2 and IL-15 or IL-2 only, respectively). As shown in
TABLE-US-00009 TABLE 8 NK cell degranulation (% CD107+ cells) against different cancer cell lines without IL-2 and IL-15 stimulation IgG1 101G5 107G3 Stimulation Target cell Mean SEM Mean SEM Mean SEM None None 1.74 1.26 2.06 0.95 1.81 1.03 HL-60 24.57 0.71 22.43 1.31 27.23 2.29 Raji 8.70 1.43 9.19 0.84 11.68 0.56 HT-29 4.69 0.35 5.76 1.00 9.19 0.15 HCT116 2.79 0.46 3.98 1.81 5.62 0.47 MDA-MB-231 2.28 0.73 7.24 1.16 9.66 1.09 A549 1.68 0.07 6.78 2.44 12.57 1.15 DU-145 3.29 0.19 14.6 4.91 25.1 3.13 IL-2 + IL-15 None 3.32 0.84 6.14 2.01 5.22 2.01 HL-60 65.83 1.09 57.37 3.18 58.57 3.18 Raji 21.33 2.31 28.83 0.00 24.50 0.00 HT-29 22.43 2.11 20.73 2.18 19.83 2.18 HCT116 25.53 2.62 27.70 1.93 26.20 1.93 MDA-MB-231 11.85 3.26 29.00 4.00 30.70 4.00 A549 14.53 2.64 24.57 1.38 32.03 1.38 DU-145 23.73 4.71 41.07 0.67 56.80 0.67
[0366] Finally, we tested the capacity of 101G5 and 107G3 to enhance NK cell-mediated killing of cancer cells by assessing the percentage of caspase 3/7 cells after co-culture of purified NK cells with the leukemia cell line HL-60 or the lung adenocarcinoma cell line A459. As shown in
The Reference Anti-BTN2A 101G5 and 107G3 mAbs Recognize Different Epitopes of BTN2A1
[0367] Both 101G5 and 107G3 bind to BTN2A1 and share the ability to inhibit M2 macrophage polarization and to enhance NK cell activation and cytotoxicity. Hence, we investigated whether these mAbs recognized the same epitope region on the BTN2A1 protein. Therefore, octet-based binning experiments were performed where 101G5 and 107G3 competed for BTN2A1 binding using an “in-tandem” setting. As shown in
Epitope Mapping of the Reference mAbs 101G5 and 107G3
[0368] In order to characterize BTN2A1 we submitted the sample to trypsin, chymotrypsin, Asp-N, elastase and thermolysin proteolysis followed by nLC-LTQ-Orbitrap MS/MS analysis. After trypsin proteolysis, 32 peptides were identified in the sequence of BTN2A1, covering 79.84% of the sequence; 27 peptides were identified after chymotrypsin proteolysis, covering 94.76% of the BTN2A1 sequence; 2 peptides were identified after ASP-N proteolysis, covering 12.50% of the BTN2A1 sequence; 33 peptides were identified after elastase proteolysis, covering 89.11% of the BTN2A1 sequence; 29 peptides were identified after thermolysin proteolysis, covering 78.23% of the BTN2A1 sequence. Based on the results obtained, an overlap mapping of the trypsin, chymotrypsin, ASP-N, elastase and thermolysin peptides was designed (
TABLE-US-00010 TABLE 9 Sequences and positions of cross-links between BTN2A1/107G3 Sequence (sequence protein1- Position Position sequence Sequence Sequence protein 2) Enzyme Protein1 Protein2 Proteine 1 Proteine 2 LTNYV- Elastase 107G3_VH BTN2A1 29-33 60-74 EDMEVRWFRS QFSPA-a4-b6 LTNYV- Elastase 107G3_VH BTN2A1 29-33 60-74 EDMEVRWFRS QFSPA-a2-b9 LTNYV- Elastase 107G3_VH BTN2A1 29-33 60-74 EDMEVRWFRS QFSPA-a4-b9 WTGGDTNYNS- Elastase 107G3_VH BTN2A1 52-61 65-75 RWFRSQFSPAV- a8-b4 YCQHSRDLPYAF- Chymotrypsin 107G3_VL BTN2A1 91-102 67-76 FRSQFSPAVF- a10-b3 GLEWLGVIWTG Trypsin 107G3_VH BTN2A1 44-66 69-82 GDTNYNSALKS R- SQFSPAVFVYKG GR-a18-b4 LTNYV- Elastase 107G3_VH BTN2A1 29-33 60-74 EDMEVRWFRS QFSPA-a2-b13 TNYVI- Elastase 107G3_VH BTN2A1 30-34 60-74 EDMEVRWFRS QFSPA-a3-b13 YSYMHWYQQK Elastase 107G3 _VL BTN2A1 34-50 76-81 PGQPPKL- FVYKGG-a2-b3 YCARGGQLGL- Chymotrypsin 107G3_VH BTN2A1 94-103 77-98 VYKGGRERTEE QMEEYRGRTTF- a4-b8 KSRLS- Elastase 107G3_VH BTN2A1 64-68 82-99 RERTEEQMEEY RGRTTFV-a2-b4 ALKSR- Thermolysin 107G3_VH BTN2A1 62-66 77-98 VYKGGRERTEE QMEEYRGRTTF- a3-b9 SLTNYVISW- Chymotrypsin 107G3_VH BTN2A1 28-36 79-110 KGGRERTEEQM EEYRGRTTFVSK DISRGSVAL-a3- b17 YCARGGQLGL- Chymotrypsin 107G3_VH BTN2A1 94-103 77-98 VYKGGRERTEE QMEEYRGRTTF- a4-b21 GQRATISCRASK Chymotrypsin 107G3_VL BTN2A1 16-36 93-110 TVSSSGYSY- RGRTTFVSKDIS RGSVAL-a13-b5 TVSSSGYSYMH Trypsin 107G3_VL BTN2A1 28-49 96-101 WYQQKPGQPP K-TTEVSK-a11- b5 TVSSSGYSYMH Trypsin 107G3_VL BTN2A1 28-49 96-101 WYQQKPGQPP K-TTFVSK-a8-b5
[0369] Hence, our analysis indicates that the interaction between BTN2A1 and 107G3 mAb includes the following amino acids on BTN2A1: 65, 68, 69, 72, 78; 84, 85, 95, 97, 100. These results are illustrated in
[0370] After trypsin, chymotrypsin, ASP-N, elastase and thermolysin proteolysis of the protein complex BTN2A1/101G5, the nLC-orbitrap MS/MS analysis detected 14 crosslinked peptides between BTN2A1 and the antibody 101G5.
TABLE-US-00011 TABLE 10 Sequences and positions of cross-links between BTN2A1/101G5. Sequence (sequence Position Position protein1-sequence sequence sequence protein 2) Enzyme Protein1 Protein2 Protein 1 Protein 2 QSPEKSLEWIGEINP Trypsin 101G5_VH BTN2A1 39-65 211-215 STGGTTYNQKFK- DKSVR-a16-b2 QSPEKSLEWIGEINP Trypsin 101G5_VH BTN2A1 39-65 211-215 STGGTTYNQKFK- DKSVR-a17-b2 QSPEKSLEWIGEINP Trypsin 101G5_VH BTN2A1 39-65 211-215 STGGTTYNQKFK- DKSVR-a25-b2 FTVYYM- Thermolysin 101G5_VH BTN2A1 29-34 209-220 IRDKSVRNMSCS-a4- b5 FKAKATLTVDK- Trypsin 101G5_VH BTN2A1 64-74 216-230 NMSCSINNTLLGQK K-a2-b3 FKAKATLTVDK- Trypsin 101G5_VH BTN2A1 64-74 216-230 NMSCSINNTLLGQK K-a4-b3 TTYNQKFKAKA- Elastase 101G5_VH BTN2A1 58-68 214-220 VRNMSCS-a6-b5 TTYNQKFKAKA- Elastase 101G5_VH BTN2A1 58-68 214-220 VRNMSCS-a8-b5 INPSTGGTTYNQK- Thermolysin 101G5_VH BTN2A1 51-63 217-224 MSCSINNT-a10-b2 INPSTGGTTYNQK- Thermolysin 101G5_VH BTN2A1 51-63 217-224 MSCSINNT-a8-b2 FKAKATLTVDK- Trypsin 101G5_VH BTN2A1 64-74 216-230 NMSCSINNTLLGQK K-a4-b5 LLIYRTSNLASGVPGR- Trypsin 101G5_VL BTN2A1 47-62 213-229 SVRNMSCSINNTLLG QK-a7-b12 ISSNYLHWYRHKPGF Thermolysin 101G5_VL BTN2A1 29-46 217-225 SPK-MSCSINNTL- a2-b8 FKAKATLTVDK- Trypsin 101G5_VH BTN2A1 64-74 216-230 NMSCSINNTLLGQK K-a2-b14
[0371] Hence, our analysis indicates that the interaction between BTN2A1 and 101G5 includes the following amino acids on BTN2A1: 212, 213, 218, 220, 224, 229. These results are illustrated in
The Reference Anti-BTN2A 101G5 and 107G3 mAbs Present Cross-Reactivity with Cynomolgus BTN2A1 Ortholog
[0372] BTN2A1 orthologs are present in most non-human primates, including cynomolgus (Macaca fascicularis). In order to determine the cross-reactivity of the reference mAbs 101G5 and 107G3 with cynomolgus BTN2A1 ortholog (cynoBTN2A1; NCBI ref. XM_015448906.1, 93.31% identity to human BTN2A1), we generated a recombinant Fc-fusion protein containing the ectodomain of cynoBTN2A1 (cynoBTN2A1-Fc) and we performed ELISA assay for assessing the binding of the reference mAbs to this protein. We also performed ELISA using recombinant human BTN2A1-Fc protein in order to compare the affinity of the reference mAbs between human and cynomolgus BTN2A1 orthologs. As shown in
TABLE-US-00012 TABLE 11 Brief description of useful amino acid and nucleotide sequences for practicing the invention: Description of the SEQ ID NO: Type sequence Sequence 1 aa anti-BTN2A1 107G3 MAVLALLLCLMTFPSCALSQVQLKESGPGLVAPSQSLSITCTVSGFSLT mAb heavy chain NYVISWVRQPPGKGLEWLGVIWTGGDTNYNSALKSRLSISKDNSKSQ variable region VFLKMNSLQTGDTARYYCARGGQLGLRGYWGQGTLVTVSA 2 aa anti-BTN2A1 107G3 METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKTV mAb light chain SSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFT variable region LNIHPVEEEDAATYYCQHSRDLPYAFGGGTKLEIK 3 aa HCDR1 of 107G3 mAb NYVIS 4 aa HCDR2 of 107G3 mAb VIVVTGGDTNYNSALKS 5 aa HCDR3 of 107G3 mAb GGQLGLRGY 6 aa LCDR1 of 107G3 mAb RASKTVSSSGYSYMH 7 aa LCDR2 of 107G3 mAb LASNLES 8 aa LCDR3 of 107G3 mAb QHSRDLPYA 9 nt HCDR1 of 107G3 mAb AACTATGTTATAAGC 10 nt HCDR2 of 107G3 mAb GTAATTTGGACTGGTGGAGACACAAATTATAATTCAGCTCTCAAATC C 11 nt HCDR3 of 107G3 mAb GGGGGACAGCTCGGGCTACGTGGTTAT 12 nt LCDR1 of 107G3 mAb AGGGCCAGCAAAACTGTCAGTTCATCTGGCTATAGTTATATGCAC 13 nt LCDR2 of 107G3 mAb CTTGCATCCAACCTAGAATCT 14 nt LCDR3 of 107G3 mAb CAGCACAGTAGGGATCTTCCGTACGCG 15 nt 107G3 mAb heavy ATGGCTGTCCTGGCGCTACTCCTCTGCCTGATGACTTTCCCAAGC chain variable region TGTGCCCTGTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCT GGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCTGG GTTCTCATTAACCAACTATGTTATAAGCTGGGTTCGCCAGCCACCA GGAAAGGGTCTGGAGTGGCTTGGAGTAATTTGGACTGGTGGAGAC ACAAATTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCWGA CAACTCCAAGAGTCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTG GTGACACAGCCAGGTACTACTGTGCCAGAGGGGGACAGCTCGGG CTACGTGGTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 16 nt 107G3 mAb light chain ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTT variable region CCAGGTTCCACTGGTGACATTGTGCTAACACAGTCTCCTGCTTCC TTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCC AGCWACTGTCAGTTCATCTGGCTATAGTTATATGCACTGGTACC AACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATC CAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTC TGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGA TGCTGCAACCTATTACTGTCAGCACAGTAGGGATCTTCCGTACGC GTTCGGAGGGGGGACCAAGTTGGAAATAAAA 17 aa Human BTN2A1 MESAAALHFS RPASLLLLLL SLCALVSAQF IVVGPTDPIL 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 18 aa Human BTN2A2 MEPAAALHFS LPASLLLLLL LLLLSLCALV SAQFTVVGPA 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 19 aa Anti-BTN2A1 101G5 MGWNWIFILILSVTTGVHSEVQLQQSGPELVKP mAb heavy chain GASVKISCKASGYSFTVYYMLVVVKQSPEKSLE variable region WIGEINPSTGGTTYNQKFKAKATLTVDKSSSTAY (including leader MQLKSLTSEDSAVYYCARGPSFYALDYWGQGT sequence) SVTVSS- 20 aa Anti-BTN2A1 101G5 MDFQMQIISLLLISVTVIVSHGEIVLTQSPTTMAA mAb light chain SPGEKITITCSATSSISSNYLHVVYRHKPGFSPKL variable region LIYRTSNLASGVPGRFSGSGSGTSYSLTIGTMEA (including leader EDVATYYCQQGSSIPRTFGGGTKLEIK sequence) 21 aa HCDR1 of 101G5 VYYML mAb 22 aa HCDR2 of 101G5 EINPSTGGTTYNQKFKA mAb 23 aa HCDR3 of 101G5 GPSFYALDY mAb 24 aa LCDR1 of 101G5 SATSSISSNYLH mAb 25 aa LCDR2 of 101G5 RTSNLAS mAb 26 aa LCDR3 of 101G5 QQGSSIPRT mAb 27 nt HCDR1 of 101G5 GTCTACTACATGCTC mAb 28 nt HCDR2 of 101G5 GAGATTAATCCTAGCACTGGTGGTACTACCTA mAb CAACCAGAAGTTCAAGGCC 29 nt HCDR3 of 101G5 GGCCCGAGCTTTTATGCTCTGGACTAC mAb 30 nt LCDR1 of 101G5 AGTGCCACCTCTAGTATAAGTTCCAATTACTT mAb GCAT 31 nt LCDR2 of 101G5 AGGACATCCAATCTGGCTTCT mAb 32 nt LCDR3 of 101G5 CAGCAGGGTAGTAGTATACCACGCACG mAb 33 nt 101G5 mAb heavy ATGGGATGGAACTGGATCTTTATTTTAATCCT chain variable region GTCAGTAACTACAGGTGTCCACTCTGAGGTC (including leader CAGCTGCAGCAGTCTGGACCTGAGCTGGTGA sequence) AGCCTGGGGCTTCAGTGAAGATATCCTGCAA GGCTTCTGGTTACTCATTCACTGTCTACTACAT GCTCTGGGTGAAACAAAGTCCTGAAAAGAGC CTTGAGTGGATTGGAGAGATTAATCCTAGCAC TGGTGGTACTACCTACAACCAGAAGTTCAAGG CCAAGGCCACATTGACTGTAGACAAATCCTCC AGCACAGCCTACATGCAGCTCAAGAGCCTGA CATCTGAGGACTCTGCAGTCTATTACTGTGCA AGGGGCCCGAGCTTTTATGCTCTGGACTACT GGGGTCAAGGAACCTCAGTCACCGTCTCCTC A 34 nt 101G5 mAb light ATGGATTTTCAGATGCAGATTATCAGCTTGCT chain variable region GCTAATCAGTGTCACAGTCATAGTGTCTCAT (including leader GGAGAAATTGTGCTCACCCAGTCTCCAACCAC sequence) CATGGCTGCATCTCCCGGGGAGAAGATCACT ATCACCTGCAGTGCCACCTCTAGTATAAGTTC CAATTACTTGCATTGGTATCGACATAAGCCAG GATTCTCCCCTAAACTCTTGATTTATAGGACAT CCAATCTGGCTTCTGGAGTCCCAGGTCGCTTC AGTGGCAGTGGGTCTGGGACCTCTTACTCTCT CACAATTGGCACCATGGAGGCTGAAGATGTT GCCACTTACTACTGCCAGCAGGGTAGTAGTAT ACCACGCACGTTCGGAGGGGGGACCAAGCTG GAAATAAAA 35 aa Macaca fascicularis MQRQFSKASRPCLPWVLMEPAAALHFSLPASLI (Cynomolgus LLLLLLRLCALVSAQFTVVGPTDPILAMVGENTTL monkey) BTN2A1 RCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGG RERTEEQMEEYRGRTTFVSKDISRGSVALIIHNV TAQENGTYRCYFQEGRSYDEAILHLMVAGLGSK PLVEMRGHEDGGIRLECISRGWYPKPLTVWRD PYGRVVPALKEVFPPDTDGLFMVTTAVIIRDKSM RNMSCSISDTLLGQKKESVIFIPESFMPSVSPCV VALPIIVVFLMIIIAVCIYWINRLQKETKILSGEKES ERKTREIAVKELKKERVQKEKELQVKEQLQEEL RWRRTVLHAVDVVLDPDTAHPDLLLSEDRRSVR RCPLGHLGESVPDNPERFNSEPCVLGRESFAS GKHYWEVEVENVIEVVTVGVCRDSVERKEEVLL RPRNGFVVTLEMCKGQYRALSSPKRILPLKESLC RVGVFLDYEAGDVSFYNMRDRSHIYTCPRLAFS VPVRPFFRIGSDDSPIFICPALTGASGITVPEEGLI LHRVGTNQSLMPVGTRCYGHGMRPTGFIRMRE ERGIHRTTREEREPDMQNFDLGAHWSNNLPSA RSREFLNSDLVPDHSLESPVTPGLANKTGEPQA EVTCLCFSLPSSELRAFPSTATNHNHKATALGS DLHIEVKGYEDGGIHLECRSTGWYPQPQIQWSN TKGQHIPAVKAPVVADGVGLYAVAASVIMRGSS GEGVSCIIRNSLLGLEKTASISITDPFFRNAQPWI AALAGTLPISLLLLAGASYFLWRQQKEKIALSRET EREREMKEMGYAATKQEISLRGGEKSLAYHGT HISYLAAPERWEMAVFPNSGLPRCLLTLILLQLP KLDSAPFDVIGPPEPILAVVGEDAELPCRLSPNA SAEHLELRWFRKKVSPAVLVHRDGREQEAEQM PEYRGRATLVQDGIAEGRVALRIRGVRVSDDGE YTCFFREDGSYEEALVHLKVAALGSDPHISMQV QENGEIWLECTSVGWYPEPQVQWRTSKGEKFP STSESRNPDEEGLFTVAASVIIRDTSVKNVSCYI QNLLLGQEKEVEIFIPG
REFERENCES
[0373] 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.