LILRB3 ANTIBODY MOLECULES AND USES THEREOF

Abstract

Described are anti-LILRB3 antibody molecules, such as agonistic anti-LILRB3 antibody molecules for use in treatment of graft rejection or autoimmunity via reprograming of human myeloid cells. Described are also specific anti-LILRB3 antibody molecules and use of such antibody molecules in medicine, for example in treatment of graft rejection, autoimmune disorders or inflammatory disorders.

Claims

1. A method for the treatment of graft rejection, an autoimmune disorder and/or an inflammatory disorder in a patient comprising administering to the patient an antibody molecule that binds specifically to LILRB3 (ILT5).

2. A method according to claim 1, wherein said antibody molecule is an agonistic antibody molecule.

3. An antibody molecule that binds specifically to LILRB3 (ILT5), wherein the antibody molecule is selected from the group consisting of antibody molecules comprising 1-6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, wherein VH-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 1, 9, 17 and 25; wherein VH-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 2, 10, 18 and 26; wherein VH-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 3, and 19 and 27; wherein VL-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 4, 12, 20 and 28; wherein VL-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 5, 13, 21 and 29; and wherein VL-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 6, 14, 22 and 30.

4. An antibody molecule according to claim 3, wherein the antibody molecule comprises a variable heavy chain (VH) comprising the following CDRs: (i) SEQ. ID. NO: 1, SEQ. ID. NO: 2 and SEQ. ID. NO: 3; or (ii) SEQ. ID. NO: 9, SEQ. ID. NO: 10 and SEQ. ID. NO: 11; or (iii) SEQ. ID. NO: 17, SEQ. ID. NO: 18 and SEQ. ID. NO: 19; or (iv) SEQ. ID. NO: 25, SEQ. ID. NO: 26 and SEQ. ID. NO: 27 and/or wherein the antibody molecule comprises a variable light chain (VL) comprising the following CDRs: (v) SEQ. ID. NO: 4, SEQ. ID. NO: 5 and SEQ. ID. NO: 6; or (vi) SEQ. ID. NO: 12, SEQ. ID. NO: 13 and SEQ. ID. NO: 14; or (vii) SEQ. ID. NO: 20, SEQ. ID. NO: 21 and SEQ. ID. NO: 22; or (viii) SEQ. ID. NO: 28, SEQ. ID. NO: 29 and SEQ. ID. NO: 30.

5. An antibody molecule according to claim, wherein the antibody molecule comprises a variable heavy chain (VH) amino acid sequence selected from the group consisting of SEQ. ID. NOs 7, 15, 23 and 31; and/or wherein the antibody molecule comprises a variable light chain (VL) amino acid sequence selected from the group consisting of SEQ. ID. NOs: 8, 16, 24 and 32.

6. An antibody molecule according to claim 3, wherein the antibody molecule is an agonistic antibody molecule.

7. A method according to claim 1, or an antibody molecule according to any one of the claims 3-6, wherein the antibody molecule is selected from the group consisting of a wild-type or Fc engineered human IgG antibody molecule, a humanized IgG antibody molecule, and an IgG antibody molecule of human origin.

8. A method according to claim 7 or an antibody molecule according to claim 7, wherein the antibody molecule is a human IgG1, IgG2 or IgG4 antibody.

9. An antibody molecule for use according to claim 1 or 2, or an antibody molecule according to any one of the claims 3-8, wherein the antibody molecule is a monoclonal antibody.

10. A method according to claim 1, wherein the antibody is selected from the group consisting of antibody molecules comprising 1-6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, wherein VH-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 1, 9, 17 and 25; wherein VH-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 2, 10, 18 and 26; wherein VH-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 3, and 19 and 27; wherein VL-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 4, 12, 20 and 28; wherein VL-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 5, 13, 21 and 29; and wherein VL-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 6, 14, 22 and 30.

11. The method according to claim 1, wherein the antibody molecule is an antibody molecule that is capable of competing for binding to LILRB3 (ILT5) with an antibody molecule selected from the group consisting of antibody molecules comprising 1-6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, wherein VH-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 1, 9, 17 and 25; wherein VH-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 2, 10, 18 and 26; wherein VH-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 3, and 19 and 27; wherein VL-CDR1, if present, is selected from the group consisting of SEQ. ID. NOs: 4, 12, 20 and 28; wherein VL-CDR2, if present, is selected from the group consisting of SEQ. ID. NOs: 5, 13, 21 and 29; and wherein VL-CDR3, if present, is selected from the group consisting of SEQ. ID. NOs: 6, 14, 22 and 30.

12. An isolated nucleotide sequence encoding an antibody molecule as defined in claim 3.

13. A plasmid comprising a nucleotide sequence as defined in claim 12.

14. A cell comprising a nucleotide sequence as defined in claim 12.

15. A method for the treatment of a graft rejection, an autoimmune disorder and/or an inflammatory disorder in a patient comprising administering to the patient nucleotide sequence according to claim 12.

16-17. (canceled)

18. A pharmaceutical composition comprising or consisting of an antibody molecule as defined in claim 3, optionally a pharmaceutically acceptable diluent, carrier, vehicle and/or excipient.

19. A pharmaceutical composition according to claim 18, for use in the treatment of graft rejection, an autoimmune disorder and/or an inflammatory disorder.

20. (canceled)

21. The method according to claim 1, wherein the antibody molecule is an agonistic antibody molecule binding specifically to LILRB3 (ILT5).

22. A method for treatment of graft rejection, an autoimmune disorder and/or an inflammatory disorder in a patient comprising administering to the patient a therapeutically effective amount of a nucleotide sequence according to claim 12.

23. A pharmaceutical composition comprising or consisting of a nucleotide sequence according to claim 12, and optionally a pharmaceutically acceptable diluent, carrier, vehicle and/or excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0133] In the examples below, reference is made to the following figures:

[0134] FIG. 1. Generation of fully human mAb against human LILRB3. FIG. 1A: Screening of generated LILRB3 clones. FMAT was performed and scFv clones screened against LILRB3 target and LILRB1 non-target-transfected cells. MFI was calculated, with target-specific scFvs depicted in lighter color and non-target scFvs in darker color. FIG. 1 B: Screening of LILRB3 mAb by flow cytometry. Peripheral blood mononuclear cells (PBMCs) or LILR-transfected CHO-S cells were incubated with His-tagged scFv supernatants, followed by anti-His-AF647 staining. Where transfected CHO-S cells were used, LILRB1- and LILRB2-transfected CHO-S cells were used as non-targets for LILRB3. Antibody clones were compared against both gated monocytes and target transfected CHO-S cells using the TIBCO Spotfire software, with LILRB3 specific clones highlighted in light grey, non-specific clones in dark grey, and the irrelevant isotype control in grey. FIG. 1 C: Specificity of LILRB3 clones against human LILR-transfected 2B4 cells. LILRB3 mAb were tested against cells transfected with the indicated LILR family members by flow cytometry; a representative clone (A16) is presented. FIGS. 1 D and E: Testing the specificity of LILRB clones against primary cells by flow cytometry. PBMCs (FIG. 1 D) or whole blood (FIG. 1 E) stained with either APC-labelled LILRB3 (clone A16) or hIgG1 isotype as well as various leukocyte surface markers, as indicated. Histograms are representative plots of multiple donors: monocytes and B cells (n=12), T cells and NK cells (n=12) and neutrophils (n=6).

[0135] FIG. 2. Characterization of LILRB3 antibodies. FIG. 2A: LILRB3 mAb affinity assessed by SPR. LILRB3-hFc recombinant protein was immobilized and various LILRB3 mAb flowed across the chip. KD values were calculated using the Biacore™ T100 Evaluation Software. Representative LILRB3 clones are shown. FIG. 2 B: Ability of generated mAb to cross-block binding of LILRB3 mAb (a commercial LILRB3 mAb; (clone 222821, R&D Systems, UK)). PBMCs were stained with unconjugated LILRB3 antibody clones and subsequently stained with a directly-conjugated commercial LILRB3 mAb and analyzed by flow cytometry; representative clones displayed, as indicated. The isotype control (iso ctrl) is shaded in grey, clone 222821 alone in black and in combination with indicated LILRB3 clones in grey line. FIG. 2 C: LILRB3 domain epitope mapping. HEK293F cell transfected with either WT LILRB3 (full-length extracellular portion), LILRB3-D1-3, LILRB3-D1-2 or LILRB3-D1 were stained with LILRB3 clones, followed by an anti-hIgG-PE secondary antibody staining. Schematic of domain constructs generated and restriction digest of each construct shown. Histograms showing staining of two representative clones differentially binding to WT (D4), D3, D2 and D1-expressing cells, as indicated (n=3 independent experiments). FIG. 2 D: LILRB3 2B4 reporter cells were treated with 10 μg/ml LILRB3 antibodies overnight to assess agonism or antagonism. GFP expression was then measured by flow cytometry; representative clones shown.

[0136] FIG. 3. LILRB3 ligation regulates T cell activation and proliferation. CFSE-labelled PBMCs were stimulated with antibodies against human CD3 and CD28 in the presence or absence of isotype control (iso ctrl) or LILRB3 mAb (10 μg/ml) and proliferation measured through CFSE dilution after 3-5 days. FIG. 3A: Assessing T cell activation and proliferation following treatment. Light microscopy images following PBMC stimulation in culture. CD8+ T cell proliferation was assessed through CFSE dilution; representative histograms shown. FIG. 3 B: LILRB3 mAb were deglycosylated (Degly) through PNGase-treatment, as confirmed by SDS-PAGE; representative clones shown. FIG. 3 C: Assessing the effects of deglycosylated LILRB3 mAb on T cell proliferation. CFSE dilution of CD8+ T cells, treated with various LILRB3 mAb was assessed by flow cytometry. Data normalized to anti-CD3/CD28-treated samples and mean represented by solid line; representative clones shown. Two-tailed paired T-test performed and stars represent level of significant difference compared to iso ctrl (*** p<0.005); n=13-20 independent donors (each dot represents an individual donor).

[0137] FIG. 4. LILRB3 ligation modulates macrophage phagocytosis. FIG. 4A: Human MDMs were stained with anti-CD14 and anti-LILRB3 (A16) and analyzed by flow cytometry. FIG. 4 B: MDMs were treated with deglycosylated isotype control (iso ctrl) or LILRB3 mAb (10 μg/ml) prior to co-culture with CFSE.sup.+ rituximab-opsonized target cells; and phagocytosis was defined as the number of gated live cells that were double positive (CD16.sup.+ CFSE.sup.+ cells), as a percentage of total MDMs (CD16.sup.+ cells), using the following equation:

(Double positive MDM/Total MDM)×100=% positive MDMs

FIG. 4 C: The effect of deglycosylated LILRB3 mAb on phagocytosis. Each donor was performed in triplicate and the mean is represented by a solid line (n=4-6 healthy donors); representative clones shown. Two-tailed paired T-test was performed and stars represent level of significant difference compared to isotype control (* p<0.05, *** p<0.0005). FIG. 4 D: The effect of deglycosylated LILRB3 mAb on phagocytosis assessed by confocal microscopy. LILRB3-treated MDMs (grey) were co-cultured with CFSE-labelled B cells (light grey), fixed in 4% PFA and membrane glycoproteins stained with biotinylated WGA. Cells were then incubated with a secondary streptavidin-conjugated AF635 and analyzed by confocal microscopy.

[0138] FIG. 5. LILRB3 ligation induce induces tolerance in vivo. Fully reconstituted humanized mice (≥50% circulating hCD45+ leukocytes) were generated and the expression of human LILRB3 was confirmed on CD14+ myeloid cells. FIG. 5A: Representative flow cytometry histogram showing LILRB3 expression on hCD45+ bone marrow hCD14.sup.+ myeloid cells; isotype control in solid dark grey, LILRB3 in solid light grey. FIG. 5 B: Humanized mice were injected with 200 μg LILRB3 mAb (clone A1) or an isotype-matched (hIgG1) control mAb on day 0 and 4, i.v. and intraperitoneal (i.p.), respectively. On day 7, mice were injected i.p. with 1×10.sup.7 non-autologous luciferase.sup.+ human lymphoma cells. Lymphoma cell growth was monitored over time using an IVIS imager, representative images from 3 independent experiments shown (n=3 mice/group).

[0139] FIG. 6. Human LILRB3 ligation reprograms human primary myeloid cells. Freshly isolated human peripheral CD14.sup.+ monocytes were treated with an isotype control (iso ctrl) or a human LILRB3 mAb (clone A1). FIG. 6A: Monocyte morphology following treatment. Light microscopy images following overnight treatment of freshly-isolated CD14.sup.+ monocytes with indicated mAb in culture. FIG. 6 B: Transcriptomic analysis of LILRB3-treated monocytes. RNA was extracted from cells following mAb treatment (˜18 hours) and subjected to RNA sequencing. The left panel depicts a list of genes that were significantly upregulated and the right panel depicts genes that were significantly downregulated compared to iso ctrl treated-cells (n=4; each row represents an individual donor). FIG. 6 C: Ligation of LILRB3 on primary human CD14.sup.+ monocytes induces M2-polarized genes. GSEA graphs showing a significant enrichment for M2-polarizing genes in LILRB3-treated monocytes versus isotype control, respectively. UP; upregulated, NES; normalized enrichment score=−1.68; FWER; familywise-error rate p<0.001. FIG. 6 D: qPCR analysis of selected genes following LILRB3 ligation on monocytes. Data were normalized to GAPDH mRNA levels and standardized to the levels of isotype control-treated monocytes. Fold difference data were log 10 transformed. One-way ANOVA with Bonferroni's multiple comparisons test was performed, n=3 independent donors (** p<0.005, *** p<0.0005). FIG. 6 E: GSEA analysis showing negative correlation with ‘IFN-γ’ (NES=−2.17; FWER p<0.001), ‘IFN-α’ (NES=−2.3; FWER p<0.001) and ‘allograft rejection’ (NES=−1.58; FWER p=0.14) signaling elements and positive correlation with ‘oxidative phosphorylation’ (NES=2; FWER p<0.001). FIG. 6 F: Schematic diagram demonstrating the immunosuppressive function of LILRB3 following ligation on APCs.

EXAMPLES

[0140] Specific, non-limiting examples which embodies certain aspects of the invention will now be described.

Materials and Methods

Ethics Statement

[0141] All research with human samples and mice was performed in compliance with institutional guidelines, the Declaration of Helsinki and the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals. The Committee on Animal Care at Massachusetts Institute of Technology (MIT) reviewed and approved the studies described here. All human samples (adult peripheral blood and fetal liver) were collected anonymously with informed consent by a third party and purchased for research. For human peripheral blood, ethical approval for the use of clinical samples was obtained by the Southampton University Hospitals NHS Trust; from the Southampton and South West Hampshire Research Ethics Committee following provision of informed consent. Primary chronic lymphocytic leukemia (CLL) samples were released from the Human Tissue Authority licensed University of Southampton, Cancer Science Unit Tissue Bank as part of the LPD study LREC number 228/02/T.

Hematopoietic Stem/Progenitor Cells (HSPCs) Isolation and Generation of Humanized Mice

[0142] Human fetal livers were obtained from aborted fetuses at 15-23 weeks of gestation, in accordance with the institutional ethical guidelines (Advanced Bioscience Resources, Inc., Calif., USA). All women gave written informed consent for the donation of their fetal tissue for research. Fetuses were collected within 2 hours of the termination of pregnancy. Fetal liver tissue was initially cut into small pieces and digested with collagenase VI (2 mg/ml in Dulbecco's modified Eagle's medium [DMEM]) for 30 minutes at 37° C. with periodic mixing. Single-cell suspensions were prepared by passing the digested tissue through a 100 μm cell strainer (BD Biosciences, NJ, USA). CD34.sup.+ cells were purified with the use of a CD34.sup.+ selection kit (Stem Cell Technologies, Vancouver, BC, Canada); the purity of CD34.sup.+ cells was 90%-99%. Viable cells were counted by trypan blue exclusion of dead cells. All cells were isolated under sterile conditions.

[0143] NSG mice were purchased from the Jackson Laboratories (Bar Harbor, Me., USA) and maintained under specific pathogen-free conditions in the animal facilities at MIT. To reconstitute mice, newborn pups (less than 2 days old) were irradiated with 100 cGyusing a Gamma radiation source injected intracardially with CD34+CD133+ cells (approximately 2×10.sup.5 cells/recipient), as reported previously (25). Around 12 weeks of age, human leukocyte reconstitution was determined by flow cytometry of peripheral blood mononuclear cells (PBMCs). Chimerism, or the level of human leukocyte reconstitution, was calculated as follows: % CD45+ human cell/(% CD45+ human cell+% CD45+ mouse cell). Mice with 40% human CD45+ leukocytes were used in the study.

Cell Culture

[0144] Cell lines were grown at 37° C. in either RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich, UK), 100 U/ml Penicillin-Streptomycin, 2 mM glutamine and 1 mM pyruvate (Thermo Fisher Scientific, UK) in a humidified incubator with 5% CO.sub.2, Freestyle 293F media, in 8% CO.sub.2, shaking at 130 rpm, or Freestyle CHO media (Thermo Fisher Scientific, UK) with 8 mM glutamine, in 8% CO.sub.2, shaking at 140 rpm.

Antibody Generation and Production

Generation of LILRB3 Antibodies.

[0145] Selection of various LILRB3-specific mAb was performed using the n-CoDeR® phage display library (26). Three consecutive panning rounds were performed, as well as a pre-panning step. In the panning, Fc fusion proteins containing the extracellular domains of LILRB1, LILRB2 or LILRB3 (LILRB-Fc) were used as non-targets or targets, respectively. These proteins were produced in transiently transfected HEK293 cells followed by purification on protein A, as described previously (27). CHO-S cells transiently transfected to express the various LILRB proteins were also used as targets/non-targets in the panning.

[0146] In panning 1, BioInvent n-CoDeR® scFv were selected using biotinylated in-house produced recombinant LILRB-human (h) Fc recombinant fusion proteins (captured with streptavidin-coated Dynabeads®) with or without competition or LILRB-hFc coated to etched polystyrene balls (Polysciences, US)/plastic immunotubes. Binding phages were eluted by trypsin digestion and amplified on plates using standard procedures (28). The amplified phages from panning 1 were used for panning 2, the process repeated, and the amplified phages from panning 2 used in panning 3. In the third panning round however, amplified phages from all 3 strategies were combined and selected against LILRB transiently transfected CHO-S cells.

[0147] Next, the LILRB3-positive scFv cassettes from the enriched phage repertoires from panning 3 were re-cloned to allow soluble scFv expression in E. coli. The soluble scFv fragments expressed by individual clones were tested for binding against LILRB-transfected CHO-S cells using Flourometric Microvolume Assay Technology (FMAT), and recombinant LILRB protein by Enzyme-linked immunosorbent assay (ELISA). This allowed the identification of clones binding specifically to LILRB3. Clones were then further reduced in a tertiary screen against CHO-S cells expressing LILRB1-3 and primary cells (PBMCs). Clones showing specific patterns of binding to a single LILRB were sequenced, yielding LILRB1-3-specific mAb.

Production of Full-Length IgG's.

[0148] The unique scFv identified above were cloned into a eukaryotic expression system allowing transient expression of full-length IgG in HEK293-EBNA cells. The antibodies were then purified from the culture supernatants using Protein A-based affinity chromatography as previously described (29).

Production of Deglycosylated IgG.

[0149] To allow dissection of Fc- and Fab-dependent effector functions, IgG were deglycosylated using PNGase F (Promega) with 0.05 U of PNGase/μg of IgG, at 37° C. for at least 15 hours. Deglycosylation was confirmed by reduction in size of the heavy chain on SDS-PAGE.

Production of Domain Mutant Constructs

[0150] Using wild-type LILRB3 cDNA isolated from a healthy donor PBMCs, a series of domain mutant DNA constructs were generated by overlap PCR to express 1, 2 or 3 LILRB3 Ig-like domains (with domains identified based on annotations in Uniprot). The gene constructs were then cloned into pcDNA3.

Cell Transfections

[0151] 10×10.sup.6 HEK293F cells were transiently transfected with 10 μg of plasmid DNA by lipofection using 233 fectin with Optimem 1 Media (Thermo Fisher Scientific, UK).

Preparation of Human Leukocytes and Generation of Monocyte-Derived Macrophages (MDMs)

[0152] Whole blood was acquired with informed consent from healthy volunteers. PBMC were isolated from leukocyte blood cones (Blood Transfusion Services, Southampton General Hospital). Isolation was performed by gradient density centrifugation using lymphoprep (Axis Shield, UK). MDMs were generated from healthy peripheral blood human monocytes as before (30). Briefly, PBMCs were plated at 2×10.sup.7 cells/well in a 6-well plate (Corning, UK) with 1% human AB serum (Sigma-Aldrich, UK) and incubated at 37° C. for 2 hours. Non-adherent cells were washed away and the adherent monocytes (>90% CD14.sup.+) were incubated at 37° C. overnight with 5% CO.sub.2. The following day 100 ng/ml human recombinant M-CSF (in house) was added to each well. Media and cytokine were replenished twice during culture and cells were then harvested on day 7-8.

Macrophage Phagocytosis Assay

[0153] Human MDMs generated as described above, were plated at 1×10.sup.5 cells/well in a 96-well flat-bottom plate. MDMs were treated with 10 μg/ml LILRB3 antibodies for 2 hours and washed. Primary chronic lymphocytic leukemia (CLL) cells, labelled with 5 μM CFSE (Sigma-Aldrich, UK), were opsonized with rituximab for 25 minutes at 4° C. (or herceptin as an isotype control). MDMs and target CLL cells were then co-cultured for 1 hour at 37° C., at a 1:5 ratio, respectively, before staining with 10 μg/ml CD16-APC (BioLegend, UK) for 15 minutes at room temperature in the dark. Cells were washed, harvested, analyzed by flow cytometry and % phagocytosis calculated as follows: (% double-positive MDM)/(% total MDM)×100.

Flow Cytometry

[0154] For cell surface staining of PBMCs or whole blood, cells were blocked with 2% human AB serum (Sigma-Aldrich, UK) for 10 minutes on ice and then stained with the relevant APC-labelled mAb or hIgG1 isotype (BioInvent, Sweden), alongside the following cell surface markers: CD14-PE (eBioscience, UK), CD20-A488 (fluorescent labelled rituximab, in house), CD3-PE-Cy7, CD56-APC-Cy7 or CD15-Pacific Blue and CD66B-FITC mAb (all BioLegend, UK). Cells were stained for 30 minutes at 4° C. and then were washed twice, first in 10% red blood cell (RBC) lysis buffer (Serotec, UK) and then FACS wash (PBS, 1% BSA, 10 mM NaN3), before acquisition on a FACSCalibur or FACSCanto II (BD Biosciences, USA) and analyzed with FCS Express V3 (De Novo Software).

[0155] For assays to determine if mAb bound to similar cross-blocking epitopes 1×10.sup.6 PBMCs were blocked with 2% human AB serum for 10 minutes and stained with 10 μg/ml unconjugated LILRB3 mAb for 30 minutes at 4° C. The cells were then stained with directly-conjugated commercial LILRB3 mAb (clone 222821; R&D Systems, UK) for 20 minutes at 4° C., before washing and acquisition using a FACSCalibur.

[0156] For LILRB3 epitope mapping studies, LILRB3-domain mutant-transfected HEK293F cells were stained with the relevant LILRB3 mAb for 25 minutes at 4° C., washed twice, stained with an anti-human-PE secondary (Jackson ImmunoResearch, USA) for 20 minutes at 4° C., before washing and acquisition using a FACSCalibur.

[0157] For staining of 2B4 reporter cells expressing LILR-A1, -A2, -A5, -B1, -B2, -B3, -B4, or -B5 (or non-transfected controls) cells were stained with 10 μg/ml LILRB mAb and incubated at 37° C. with 5% CO.sub.2, overnight. The following day, the cells were washed and stained with a secondary anti-hIgG antibody (Jackson ImmunoResearch, USA) at 4° C., for 45 minutes. The cells were washed and acquisition performed using a FACScan (BD Biosciences, USA) and analysis using Cell Quest (BD Biosciences, USA).

[0158] Flow cytometry data were analyzed with FCS Express V3 (De Novo Software) and FlowJo.

Surface Plasmon Resonance (SPR)

[0159] SPR was performed with the Biacore T100 (GE Healthcare, UK) as per the manufacturer's instructions. LILRB3-hFc recombinant protein (the extracellular LILRB3 domain with a human Fc tag) was used as the ligand and immobilized by amine coupling onto a series S sensor chip (CM5). Various LILRB3 mAb were used as “analytes” and flowed across the chip, and SPR measured. KD values were calculated from the ‘Univalent’ model of 1:1 binding by Kd [1/s]/Ka [1/Ms], using the Biacore™ T100 Evaluation Software (GE Healthcare, UK).

T Cell Proliferation Assay

[0160] PBMCs (1-2×10.sup.7) were labelled with 2 μM CSFE at room temperature for 10 minutes. Cells were subsequently resuspended in serum-free CTL medium (Immunospot, Germany) and plated at 1×10.sup.5 cells/well in a 96-well round-bottom plate (Corning, UK). Cells were then stimulated with 0.02 μg/ml CD3 (clone OKT3, University of Southampton), 5 μg/ml CD28 (clone CD28.2; BioLegend, UK) and 10 μg/ml LILRB3 antibodies or a relevant isotype. Plates were then incubated at 37° C. for 4 days, after which time cells were stained with 5 μg/ml CD8-APC (clone SK1; BioLegend, UK), harvested and CSFE dilution measured by flow cytometry, as a readout for T cell proliferation.

In Vivo Allograft Assay

[0161] Fully reconstituted humanized mice (≥40% circulating hCD45+ leukocytes) were injected with 200 μg LILRB3 mAb (clone A1) or an isotype-matched (hIgG1) control on day 0 and day 4, i.v. and i.p, respectively. On day 7 cohorts of mice were injected i.p. with 1×10.sup.7 luciferase-positive human ‘double-hit’ B cell lymphomas (25, 31), derived from unrelated unmatched donors. Lymphoma cell growth was monitored over time using an IVIS Spectrum-bioluminescent imaging system, as before (25).

Transcriptome Analysis

[0162] To assess LILRB3-mediated transcriptional changes on monocytes, human peripheral blood monocytes were isolated from freshly prepared PBMCs taken from healthy donors using an EasySep™ Human Monocyte Enrichment Kit (negative selection cell; Stem-Cell Technologies, USA). Cells were incubated in CTL medium supplemented with 100 U/ml Penicillin-Streptomycin, 2 mM Glutamine and HEPES buffer and treated with 10 μg/ml of an isotype control or an agonistic LILRB3 mAb (clone A1; hIgG1). 18 hours later cells were lysed in RLT lysis buffer containing β-mercaptoethanol and total RNA extracted using the RNeasy micro kit (Qiagen, USA). Total RNA was assessed for quality and quantified using a total RNA 6000 Nano LabChip on a 2100 Bioanalyzer (Agilent Inc., USA) and cDNA libraries prepared and sequenced according to the Illumina TruSeq RNA Sample Preparation Guide for SMARTer Universal Low Input RNA Kit (Clontech, USA) and a HiSeq 2000 system (Illumina, USA). RNAseq outputs were aligned to hg19, using Bowtie2 v2.2.3 (32). The number of mapped reads were quantified by RSEM v1.2.15 (33). Differential expression analysis between paired samples before and after treatment was performed using edgeR (34) with p<0.05 and >2 fold-change cut-offs. Differentially expressed genes were annotated using online functional enrichment analysis tool DAVID (http://david.ncifcrf.gov/) (35). Gene set enrichment analysis (GSEA) was performed using Broad Institute Software (36), with the gene list pre-ranked according to log FC values from the edgeR output. For comparison of gene-set expression, M1 and M2 macrophage gene sets (37) were obtained from the Molecular Signature Database (http://software.broadinstitute.org/gsea/msigdb/). Heatmaps were visualized with MeV (38).

Quantitative PCR (qPCR)

[0163] Probe-based qPCR was used to amplify cDNA in 20 μl reactions performed in triplicate for each sample condition in a 96-well PCR plate (Bio-Rad, UK). Each reaction comprised of 48 ng cDNA, 10 μl platinum qPCR mix (Life Technologies, UK), 8 μl DEPC water and 1 μl of gene-specific 20× PrimeTime probe/primer mix, as per manufacturer's protocol. The 96-well plate containing the PCR reagents were run in a C1000 Thermal Cycler CFX96 Real-time System PCR machine (Bio-Rad, Kidlington, UK). The CFX manager software (Bio-Rad, Kidlington, UK), was used for data acquisition and analysis of gene expression initially recorded as cycle threshold values (Ct). The Ct values were normalized to housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and standardized to gene expression levels in isotype control-treated cells.

Statistics

[0164] Paired two-tailed T-tests were performed for both the phagocytosis and T cell proliferation data; straight bars indicate median values. On bar graphs, where at least 3 experiments were performed, error bars represent standard deviation. One-way ANOVA with Bonferroni's multiple comparisons test was performed for qPCR data analysis. Statistical analysis was performed using GraphPadPrism (v5 or 6).

Results

Generation of a Panel of Specific LILRB3 mAb

[0165] To study the protein expression and function of human LILRB3, antibodies against LILRB3 were identified using a human phage display library. Selection was performed using target LILRB3 protein (in solution, coated to a plastic surface or expressed on cells) and by selecting against homologous non-target LILRB1 and LILRB2 proteins. After three rounds of phage panning and enrichment, successful selection of clones specific for LILRB3 was confirmed by flow cytometry and ELISA (data not shown). Subsequently, target-specific phage-bound scFv clones were converted to soluble scFv and screened by FMAT and ELISA. Fluorescent intensity for each clone was plotted and target versus non-target specificity displayed (FIG. 1A). Successful clones were selected based on binding LILRB3 and lack of cross-reactivity to LILRB1 and LILRB2. Selected clones were then sequenced and tested for binding against primary cells and transfectants (FIG. 1B). Once the target-specific clones were chosen and converted to IgG, specificity was reconfirmed by screening against a panel of LILR-expressing 2B4 reporter cell lines (FIG. 10). In total 16 LILRB3-specific antibodies were identified for further study. Staining of PBMCs or whole blood with these LILRB3 mAb showed predominant staining of monocytes (FIG. 1D) and neutrophils (FIG. 1E), in agreement with previous reports (39). LILRB3-specific clones were further tested and confirmed to have no cross reactivity to the mouse orthologue, PIR-B (data not shown).

LILRB3 mAb Bind with High Affinity and Map to Different Epitopes

[0166] The LILRB3-specific mAb were then tested for their binding properties. SPR analysis showed that all LILRB3 clones bound to recombinant LILRB3-hFc protein in a dose-dependent manner (FIG. 2A) and displayed a range of affinities, represented by A16 (8.16×10.sup.−10). Interestingly, all of the mAb had similar association rates (˜105), but varied in their dissociation rates by three orders of magnitude (˜10.sup.−3-10.sup.−6).

[0167] Epitope mapping studies were then performed. Some mAb were able to block the binding of a commercial LILRB3 mAb (e.g., A35), suggesting a shared or proximally-related epitope; whilst others could not (e.g., A1), indicating binding elsewhere (FIG. 2B). Binding specificities were further confirmed with a series of LILRB3 domain (FIG. 2 D) mutants displaying either all four extracellular domains (WT), three, two or one domain, transiently transfected into HEK293F cells. Binding to these cells showed two groups of mAb: those that bound to the WT, D3 and D2 expressing cells; and those that bound only the WT-transfected cells (FIG. 2B), indicating binding within D4 (exemplified by A1), respectively (FIG. 2C). These data demonstrate that highly specific, fully human IgG1 mAb were raised against LILRB3. Epitope mapping revealed that, although conserved residues seem to be present in all 4 domains, LILRB3 mAb bind to either of the two distinct extracellular dominant epitopes, located within D2 and D4, respectively.

[0168] Furthermore, reporter cells transfected with the extracellular domain of LILRB3, fused with the human CD3 cytoplasmic domain were used to investigate whether the generated mAb were able to crosslink the receptor. Signaling through these hybrid cells results in the expression of GFP under the NFAT promoter (40). We were able to identify two distinct groups of LILRB3 mAb, those capable of inducing signaling (e.g. A1) and those being inert (e.g. A28) upon binding to the receptor (FIG. 2D).

LILRB3 Ligation Modulates T Cell Activation and Proliferation

[0169] Next, we sought to investigate the effect of these mAb on cellular effector functions. LILRB1 has previously been shown to inhibit T cell responses; either by causing dephosphorylation in the CD3 signaling cascade, or competing with CD8 for HLA-I binding (41, 42). LILRBs have also been shown to inhibit T cell responses indirectly by rendering antigen-presenting cells (APC), such as monocytes and DCs tolerogenic, through the induction of CD8+ T suppressor cells (10, 12, 43). In order to investigate the immunomodulatory potential of LILRB3 and its ability to regulate adaptive immune responses, we tested LILRB3 mAb in PBMC assays, measuring T cell proliferation in response to anti-CD3/CD28 stimulation. T cell activation and proliferation was successfully driven by CD3 and CD28 antibodies, demonstrated by cell clustering and CFSE dilution (FIG. 3A).

[0170] Fcγ receptors (FcγRs) are known to mediate the effects of human IgG (29, 44-46), therefore, to study the direct F(ab):receptor-mediated effects of the LILRB3 mAb on T cell proliferation, they were first deglycosylated to eliminate effects mediated by FcγR-IgG interactions (47). SDS-PAGE confirmed a decrease in molecular weight of deglycosylated mAb (Degly) compared to wild-type (WT) controls, indicative of successful deglycosylation (FIG. 3 B). The mAb were then introduced to the T cell proliferation assay detailed above. Successful T cell proliferation driven by CD3 and CD28 antibodies was assessed in 20 different donors, showing a significant increase in CD8+ T cell proliferation, compared to controls (p<0.0001) (FIG. 3 C). The majority of the LILRB3 mAb significantly inhibited CD8+ T cell proliferation, represented by clone A1, when compared to the human IgG1 isotype control (p=0.0001; FIG. 3 C). A28 also exhibited a trend for inhibited proliferation, but A16 appeared to have no inhibitory effect. These data demonstrate that targeting LILRB3 can modulate T cell responses in either direction in a clear mAb-specific manner, with some delivering LILR3B-agonistic properties (enhanced inhibition) such as A1. When the assay was repeated with isolated T cells in the same manner, no inhibition was seen confirming that the APCs within the PBMCs, most likely monocytes, were responsible for the effects observed (data not shown). This result was expected, given the lack of expression of LILR3B on T cells.

LILRB3 mAb Modulate Macrophage Effector Function

[0171] The above findings indicated that the LILRB3 mAb were able to agonize or antagonize LILRB3 to regulate T cell proliferation, likely through regulating APC function. Hence, as macrophages also express high levels of LILRB, and are known to be regulated by them (13), the effects of LILRB3 ligation on macrophage phagocytosis were studied. Staining with representative LILRB3 mAb confirmed high expression levels of LILRB3 on human MDMs (FIG. 4A). To assess any modulation of their effector function, CFSE-labelled primary CLL B cells were opsonized with anti-CD20 mAb (rituximab) and used as targets for macrophages in a phagocytosis assay (FIG. 4 B-C). The deglycosylated anti-LILRB3 clones significantly decreased the extent of phagocytosis (p<0.05 in all cases) (FIG. 4 C). These findings were further confirmed by confocal microscopy, showing lower number of CFSE+ target cells in LILRB3-treated macrophages, compared to isotype control (FIG. 4 D). These data demonstrate that the majority of LILRB3 mAb delivered inhibitory signals to reduce macrophage effector function. Importantly, the LILRB3 mAb were deglycosylated, capable of mediating only Fab-dependent effects without complications arising as a result of Fc:FcγR interactions (48).

LILRB3 Ligation Induces Immune Tolerance in Humanized Mice

[0172] Given these data showing both adaptive (T cells) and innate (myeloid) activities can be suppressed following LILRB3 ligation, we next tested the possible effects of LILRB3 modulation in an allogeneic engraftment model using humanized mice (reconstituted with primary human HSC). Characterization of the humanized mice demonstrated that LILRB3 was expressed on the myeloid cells in a similar manner to human peripheral blood (FIG. 5 A). Allogeneic human lymphoma cells are readily rejected in humanized mice due to the HLA mismatch (data not shown; 49). To test the potential of LILRB3 ligation to suppress the allogeneic immune response, we pre-treated reconstituted adult humanized mice with an agonistic LILRB3 mAb (A1) and assessed the engraftment of allogeneic human ‘double-hit’ B cell lymphoma cells (31, 50) derived from unrelated donors. LILRB3 mAb treatment was able to induce a state of tolerance in the mice and led to a successful engraftment of human lymphoma cells (FIG. 5 B). LILRB3-treated tumor-bearing mice had to be humanely culled due to high tumor burden, whereas, isotype control-treated mice readily rejected the lymphoma cells without any morbidity. These observations further corroborate our in vitro functional assays and identify LILRB3 a key regulator of myeloid cells during an immune response.

LILRB3 Ligation Leads to Transcriptional Modifications and M2-Skewing of Human APCs

[0173] To investigate the pathways and factors involved in LILRB3-mediated immunosuppression we investigate the transcriptomic changes in primary APCs following LILRB3 engagement. Short-term (˜18 hour) in vitro treatment of isolated human peripheral CD14+ monocytes with agonistic LILRB3 mAb (A1) caused a dramatic shift in their phenotype (FIG. 6A), with the cells displaying an elongated morphology resembling “M2”, immunosuppressed IL4/IL-13 treated macrophages (51). RNAseq analysis revealed that ligation of LILRB3 on monocytes induced a signature resembling “M2”-skewed immunosuppressive macrophages (FIG. 6 B). Likewise, the expression of genes associated with “M1”-skewed immunostimulatory macrophages was downregulated in LILRB3 mAb-treated compared to isotype control-treated monocytes (FIG. 6 B-C). We confirmed these data by performing qPCR on a further 3 donors for a select number of differentially regulated genes (FIG. 6 D). Treatment of monocytes with a less/non-agonistic LILRB3 mAb (A28) did not affect monocyte phenotype and gene expression (data not shown and FIG. 6 C). Gene-set enrichment analysis (GSEA) showed a positive correlation with gene signatures reported for suppressive macrophages, e.g., oxidative phosphorylation (52). Conversely, LILRB3-ligated monocyte gene signatures negatively correlated with the gene signatures reported for inflammatory macrophages, e.g., IFN-γ and IFN-α responsive elements, as well as allograft rejection (FIG. 6 E). Taken together, these data confirm that LILRB3 activation results in significant phenotypic and transcriptional alterations in APCs, such as monocytes, leading to potent inhibition of downstream immune responses (FIG. 6 F).

DISCUSSION

[0174] We previously demonstrated that ligation of LILRB1 on human monocytes induces a tolerogenic phenotype, subsequently hindering T cell responses (12, 53). In this study, we investigated another LILR family member, LILRB3, whose function is not yet determined, due to lack of suitable reagents and experimental systems. We, therefore, generated and characterized an extensive panel of fully human mAb with specificity for LILRB3. Staining of different leukocyte populations with the specific mAb confirmed that LILRB3 is mainly restricted to human myeloid cells (3). This was confirmed in several independent donors, suggesting that although these receptors are polymorphic (LILRB3 has at least ten variants (3, 54)), the antibodies recognize many if not all variants, which is important for the development of these reagents for therapeutic use. Subsequent analysis showed that the LILRB3 mAb displayed a range of affinities, all of which were in the nanomolar (nM) range with similar on-rates, but off-rates differing over three orders of magnitude. KD values that are of low nM range are generally considered to be viable drug candidates; rituximab, for example, has an 8 nM affinity for its target, CD20 (55). This suggests that the LILRB3 mAb generated here have potential as therapeutic agents. Some of the selected LILRB3 clones showed unexpected cross-reactivity to other human LILR-transfectants and were excluded from subsequent analysis. However, it should be noted that as LILR3B shares >95% sequence homology in its extracellular domain with LILRA6, LILRB3 mAb may well interact with LILRA6 if co-expressed (56). Furthermore, epitope mapping experiments revealed that the specific LILRB3 mAb were generated against two distinct epitopes, as they bound to either Ig-like extracellular domain two or four. None of the generated LILRB3 mAb bound to domains one or three, suggesting that these domains may not contain conserved unique epitopes.

[0175] The ability of the LILRB3 mAb to influence T cell responses was observed through either inhibition or enhancement of proliferation, indicating agonistic or antagonistic properties, respectively. Similar to LILRB1 (12, 13, 57), this is likely through an effect on APCs, as they are the only cells expressing LILRB3 in the culture. Unlike LILRB1 (42, 53, 583, 59), LILRB3 is not expressed on T cells, and can only affect T cell responses indirectly. Based on the LILRB3 expression pattern and frequency of the cells within the PBMC culture, monocytes represent the most likely cell type influenced. In support of this, agonistic LILRB3 mAb did not suppress T cell proliferation in the absence of monocytes. Binding epitopes influence the ability of mAb to modulate receptor function in many systems (29, 60) and so it was unsurprising to see LILRB3 mAb capable of opposing functions. The majority of LILRB3 mAb that bound to the second Ig-like domain of LILRB3 were able to inhibit T cell proliferation. Conversely, some clones that bound to domain four enhanced proliferation. However, a D4-binding mAb (A1) was one of the strongest inhibitors of proliferation and another D4-binding (A28) induced less inhibitory effect. Therefore, domain-specific epitopes do not seem to correlate directly with LILRB3 mAb-mediated effector cell functions.

[0176] Although the LILRB3 mAb showed variation in their ability to inhibit or enhance T cell proliferation, the majority of clones inhibited phagocytosis by macrophages or had no effect. This suggests that the majority of mAb are agonistic in this context, stimulating inhibitory signaling and suppressing effector function, akin to the inhibition of T cell responses.

[0177] Our observations demonstrating immunoinhibitory activities downstream of LILR3B were further confirmed in the reconstituted humanized mouse model. In this system, where LILRB3 is present only on the monocytic cells, ligation of LILRB3 with an agonistic LILRB3 mAb prior to engraftment of allogeneic lymphoma cells (31) induced tolerance in vivo and enabled subsequent tumor growth. This demonstrates the capacity of LILRB3 to exert profound immunosuppressive effects that may be exploited in therapeutic settings, such as autoimmunity and transplantation, where induction of immune tolerance will be beneficial. Although regarded as an orphan receptor, it has been suggested that LILRB3 associates with cytokeratin (CK)-associated proteins (exposed on necrotic cancer cells), angiopoietin-like protein 5 and bacteria, such as Staphylococcus aureus (S. aureus)(40, 61, 62). Therefore, our functional data suggest that certain pathogens (61) may be able to subvert immune responses by actively ligating LILRB3 during an active response.

[0178] To investigate the pathways and factors involved in LILRB3-mediated immunosuppression, we investigated the transcriptomic changes in isolated peripheral myeloid cells following LILRB3 activation. Over one hundred genes were differentially regulated in primary human monocytes following LILRB3 ligation, some of which are known to be modulated in M2 macrophages and TAMs. Amphiregulin was among the genes whose expression was significantly upregulated in LILRB3-ligated monocytes. Amphiregulin is an epidermal growth factor-like growth factor, responsible for inducing tolerance and immunosuppression, via various mechanisms including enhancement of Treg activity (63). Furthermore, amphiregulin is overexpressed in tumor-associated DCs (64) and suppressive/M2 macrophages (65) and has been suggested to play a crucial role in immunosuppression and cancer progression (66). Such LILRB3-inducible factors may be responsible for the suppression observed in our T cell assays. Our ongoing efforts aim to test this and fully understand the mechanisms responsible for LILRB3-mediated suppression of myeloid cells. A recent study investigating the mode of action of Glatiramer acetate (Copaxone), a peptide-based drug used to treat patients with the relapsing-remitting form of multiple sclerosis that ameliorates autoimmunity, identified LILRB2 and LILRB3 as potential ligands (67). Targeting human LILRB2 with antagonistic mAb on human myeloid cells is able to promote their pro-inflammatory activity and enhance antitumor responses in vivo (13). Furthermore, recent data by Zhang and colleagues suggests that LILRB4 signaling in leukemia cells mediates T cell suppression of supports tumor cell dissemination to distal organs (68). These data further support our findings, demonstrating that activation of human LILRBs induce immunosuppression via reprogramming myeloid cells (i.e., reducing M1-like maturation and promoting MDSC-suppressive function).

[0179] Together the findings presented here show that LILRB3 activation on primary human myeloid cells exerts potent immunoinhibitory functions and that LILRB3-specific mAb are potentially powerful immunomodulatory agents, with a broad application ranging from transplantation to autoimmunity to inflammatory disorders, and beyond.

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