IMMUNOGENIC CONSTRUCT COMPRISING AN EBV-CELL ANTIGEN AND A TARGETING MOIETY AND APPLICATIONS THEREOF
20180360950 · 2018-12-20
Assignee
- Institut National De La Sante Et De La Recherche Medicale (Inserm) (Paris, FR)
- Centre National De La Recherche Scientifique (Cnrs) (Paris, FR)
- Universite Paris Diderot Paris 7 (Paris, FR)
- Institute National Transfusion Sanguine (Paris Cedex 15, FR)
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
C07K2319/74
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61K2039/60
HUMAN NECESSITIES
C12N2710/16234
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C12N2710/16211
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
C07K2319/01
CHEMISTRY; METALLURGY
C07K16/2896
CHEMISTRY; METALLURGY
International classification
C07K16/28
CHEMISTRY; METALLURGY
Abstract
The present invention generally relates to an immunogenic construct,useful for redirecting an EBV-existing immune response towards an undesired target cell and/or microorganism, to methods for preparing said conjugate, to a pharmaceutical applications comprising said conjugate, and to medical applications thereof.
Claims
1. An immunogenic conjugate comprising: i) a moiety binding to a target cell and/or microorganism; and ii) an Epstein-Barr Virus (EBV) B-cell antigen.
2. The immunogenic conjugate according to claim 1, wherein said target-binding moiety is covalently coupled to said Epstein-Barr Virus B-cell antigen.
3. The immunogenic conjugate according to claim 1 or 2, wherein said moiety is a ligand-binding protein selected from the group consisting of antibodies, binding fragments thereof, antibody mimetics, cell-surface receptors, cell-surface ligands and any combination thereof.
4. The immunogenic conjugate according to claim 3, wherein said antibody fragments are selected from the group consisting of Fab antibodies, Fab antibodies, F(ab)2 antibodies, Fv antibodies, single chain antibodies (scFv), and single-domain antibodies, and any combination thereof.
5. The immunogenic conjugate according to claim 1 or 2, wherein said moiety is a non-proteic moiety selected from the group consisting of vitamins, carbohydrates, glycosaminoglycans, small nucleic acids, small chemical compounds, and any combination thereof.
6. The immunogenic conjugate according to any one of claims 1 to 5, wherein said Epstein-Barr Virus B-cell antigen is selected from the group consisting of the P18 antigens of sequence SEQ ID NO: 15, the P23 antigens of sequence SEQ ID NO: 16, functional variants and functional fragments thereof, and any combination thereof.
7. The immunogenic conjugate according to claim 6, with the proviso that, when said moiety comprises a thiol group, the functional variants and functional fragments of the P18 and/or P23 antigens do not comprise any cysteine residue.
8. The immunogenic conjugate according to claim 7, wherein said functional variants are selected from the group consisting of the P18 antigen functional variants of sequence SEQ ID NO: 39 and the P23 antigen functional variants of sequence SEQ ID NO: 40.
9. The immunogenic conjugate according to claim 6 or 7, wherein said functional fragments comprise between about 10 amino acid residues and about 150 amino acid residues of said antigens.
10. The immunogenic conjugate according to claim 9, wherein said functional fragments are P18 antigen functional fragments comprising at least the sequence selected from the group consisting of SEQ ID NO:75 and SEQ ID NO: 43 to SEQ ID NO: 49, and/or P19 antigen functional fragments comprising at least the sequence selected from the group consisting of SEQ ID NO: 50 and SEQ ID NO: 51.
11. The immunogenic conjugate according to claim 10, wherein said P18 antigen functional fragments comprise or consist of the sequence selected from the group consisting of SEQ ID NO:76 and SEQ ID NO: 52 to SEQ ID NO: 58, and/or said P23 antigen functional fragments comprise or consist of the sequence selected from the group consisting of SEQ ID NO: 59 and SEQ ID NO: 60.
12. The immunogenic conjugate according to any one of claims 1 to 11, wherein the target cell is a diseased cell, preferably a cancer cell or a cell infected by a pathogen such as a virus, a bacterium, a fungus and/or a parasite, and/or the target microorganism is a pathogenic microorganism, preferably a virus, a bacterium, a fungus, and/or a parasite.
13. The immunogenic conjugate according to claim 12, wherein: said diseased cell is a malaria infected erythrocyte and/or said pathogenic microorganism is Plasmodium falciparum; or said diseased cell is a cancer cell.
14. A pharmaceutical composition comprising at least one immunogenic conjugate as defined in any one of claims 1 to 13 and at least one pharmaceutically acceptable excipient.
15. The immunogenic conjugate as defined in any one of claims 1 to 13, or the pharmaceutical composition as defined in claim 14, for use as a medicament.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0230]
[0231]
[0232]
[0233]
[0234]
[0235]
[0236]
[0237]
[0238]
[0239]
[0240]
[0241]
[0242]
[0243]
[0244]
[0245]
[0246]
[0247]
EXAMPLES
1. Material and Methods
[0248] 1.1. Construction of the Immunogenic Conjugates According to the Invention
[0249] The DNA sequences of BFRF3 and BLRF2 encoding for the EBV (B95-8 strain) P18 and P23 proteins were codon-optimized (Integrated DNA Technology) to maximize their expression in E. coli. Full length P18 and P23 recoded nucleotide sequences as well as truncated fragments thereof were cloned into the pET28a plasmid (Novagen) in order to express C-terminal His-tagged proteins, for purification purpose.
[0250] In some conjugates, a flexible linker of 20 amino-acids (GGGGS).sub.4 (SEQ ID NO: 7) has been added between the binding moiety (VHH, scFv, cell surface receptor, or cell-surface ligand) and the EBV-B cell P18 or P23 antigen in order to maintain, and if possible enhance, the proper functional attributes of the 2 different modules (i.e. of the moiety binding to the target cell and/or microorganism and of the EBV-B cell antigen) (Hu et al., 2004; Chen et al., 2013). The binding moiety sequences (except EPCR) were then inserted between the NcoI and NheI restriction sites of the modified pET28a plasmid. For the EPCR conjugate, the gene encoding soluble EPCR (residues S18-S210;[Uniprot:Q9UNN8; SEQ ID NO:14) was amplified by PCR from a human lung endothelium cDNA library and fused to a recoded gene fragment encoded for P18F3. The EPCR-P18F3 sequence was cloned into a pTT3 vector with a hexa-His C-terminal tag.
[0251] In the present study, the following conjugates were thus designed: [0252] EBV P18 full length antigen fused in frame to DARC.sub.VHH, with linker; [0253] EBV P23 full length antigen fused in frame to DARC.sub.VHH, with linker; [0254] EBV P18 mutated antigen fused in frame to DARC.sub.VHH, with and without linker; [0255] EBV P23 mutated antigen fused in frame to DARC.sub.VHH, with and without linker; [0256] various EBV P18 fragments fused in frame to DARC.sub.VHH, VAR2CSA.sub.VHH, VAR2CSA, hCD20scFv or EPCR, without linker; and [0257] various EBV P23 fragments fused in frame to DARC.sub.VHH, without linker.
[0258] These linkers can be summarized in the following Table 1.
TABLE-US-00015 TABLE1 Immunogenicconjugates targeting entire moeity EBVantigen corresponding (inN-terminal) linker (inC-terminal) sequence DARG.sub.VHH (GGGGS)4 P18(SEQIDNO:17) SEQIDNO:61 (SEQIDNO:9) (SEQIDNO:7) Smolareket (GGGGS)4 P18-C565(SEQIDNO:41) SEQIDNO:62 al.,2010 (SEQIDNO:7) none P18-C565(SEQIDNO:41) SEQIDNO:63 none P18F2(SEQIDNO:58) SEQIDNO:64 none P18F3(SEQIDNO:76) SEQIDNO:77 none P18F4(SEQIDNO:52) SEQIDNO:66 VAR2CSA.sub.VHH none P18F2(SEQIDNO:58) SEQIDNO:67 (SEQIDNO:11) none P18F3(SEQIDNO:76) SEQIDNO:78 Nunesetal., none P18F4(SEQIDNO:52) SEQIDNO:69 2014 DARC.sub.VHH (GGGGS)4 P23(SEQIDNO:23) SEQIDNO:70 (SEQIDNO:9) (SEQIDNO:7) Smolareket (GGGGS)4 P23-C465(SEQIDNO:42) SEQIDNO:71 al.,2010 (SEQIDNO:7) none P23-C465(SEQIDNO:42) SEQIDNO:72 none P23F2(SEQIDNO:60) SEQIDNO:73 none P23F3(SEQIDNO:59) SEQIDNO:74 CD20scFv none P18F3(SEQIDNO:76) SEQIDNO:81 Otzetal.,2009; Liuetal.,1987 (SEQIDNO:80) VAR2CSA none P18F3(SEQIDNO:76) SEQIDNO:83 (SEQIDNO:82) EPCR none P18F3(SEQIDNO:76) SEQIDNO:79 (SEQIDNO:14)
[0259] 1.2. Expression and Purification of the Conjugates
[0260] For protein expression, SHuffle E. coli (New England Biolabs) allowing cytoplasmic disulphide bonds formation were transformed with the different pET28a-based conjugates. Bacteria cultures were induced with 0.2 mM IPTG at OD.sub.600nm 0.5 and protein expression was carried out at 20 C. for 16 h. For protein purification, bacteria suspensions were thawed on ice and lysis was achieved by passing the cell suspensions through an EmulsiFlex-C5 high-pressure homogenizer. Soluble proteins were subjected to a 2 step purification process. His-tagged proteins were first purified on Ni-NTA Superflow (Qiagen) then passed through a Superdex 200 10/300 GL gel filtration column (GE Healthcare).
[0261] In particular, for the EPCR conjugate, FreeStyle 293-F cells (Invitrogen) were grown in Freestyle 293 serum free expression medium and transfected with the pTT3 vector containing the EPCR-P18F3 sequence following Invitrogen's recommendations. 72 hours post-transfection, cells were centrifuged and the culture medium was harvested. After filtration on a 0.22 m filter, supernatants were concentrated five times using a 10 kDa cut-off Vivaflow 200 System (Vivasciences). Soluble proteins were subjected to a 2 step purification process. His-tagged proteins were first purified on Ni-NTA Superflow (Qiagen) then passed through a Superdex 200 10/300 GL gel filtration column (GE Healthcare).
[0262] 1.3. Affinity Determination by Surface Plasmon Resonance
[0263] Interactions between the conjugates and their respective molecular targets were studied by surface plasmon resonance (SPR), using a Biacore X100 instrument (GE Healthcare). All experiments were performed in HBSEP buffer (GE Healthcare) at 25 uC. Recombinant full-length DARC.sub.325 or VAR2CSA protein was immobilized on the analysis Fc2 channel of a CM5 chip (GE Healthcare) by amine coupling to a total loading of 800 RU. Reference channel Fc1 was blocked with 1 M ethanolamine-HCl pH 8.5 using the same chemistry. Conjugates were injected at 30 L/min in dilution series over the coated chips. The highest concentration of conjugates was 1 mM and ten twofold serial dilutions were also injected. Between the injections, the chip surface was regenerated with 2 injections of 15 ml of 10 mM HCl pH2. The specific binding response was obtained by subtracting the response given by the analytes on Fc2 by the response on Fc1. The kinetic sensorgrams were fitted to a global 1:1 interaction Langmuir model using the manufacturer's software.
[0264] 1.4. Immune Recognition of the Conjugates
[0265] ELISA plates (Nunc) were coated with 100 ml per well of conjugates diluted in PBS at 1 g/ml and incubated at 4 C. overnight. BSA (2% in PBS) was coated for background measurement. After coating, the wells were blocked with PBS 2% BSA at room temperatureT for 1 h. After removing the blocking solution, human plasma dilutions in PBS 2% BSA were added and the plates were incubated at room temperature for 1 h. The wells were then washed 3 times with 150 ml of PBS 0.05% Tween20 (PBST). Human IgG binding was detected with a horseradish peroxidase-conjugated (HRP) anti-human IgG (Jackson Immunoresearch 709-036-098), diluted 1:4000 in PBS 2% BSA, incubated at room temperature for 1 h. After washing with PBST, the plates were developed with 100 ml per well of TMB (3,39,5,59-tetramethylbenzidine) substrate (Biorad) and absorbance was measured at 655 nm.
[0266] 1.5. Cell Surface Recognition by the Conjugates
[0267] The capacity of the DARC.sub.VHH-EBV conjugates to recognize the native DARC expressed at the surface of erythrocytes, the one of the hCD20.sub.ScFv-P18F3 conjugate to recognize the native CD20 protein expressed at the surface of B cells, the one of the VAR2CSA-P18F3 conjugate to recognize the native glycosaminoglycan chondroitin sulfate A (CSA) expressed at the surface of cancer cells, and the one of the EPCR-P18F3 conjugate to recognize the native VAR19 protein expressed at the surface of Plasmodium falciparum-infected cells, were tested by flow cytometry.
[0268] To do so, DARC+ erythrocytes (for DARC.sub.VHH-EBV conjugates), CD20.sup.+ RAJI cells (for the hCD20.sub.ScFv-P18F3 conjugate), CSA.sup.+ RAJI cells (for the VAR2CSA-P18F3 conjugate) or VAR19-expressing erythrocytes (for the EPCR-P18F3 conjugate) were incubated with serial dilution of conjugate in PBS 2% BSA, for 1 h at room temperature. The cells were then washed twice with PBS 2% BSA and incubated for 30 min at room temperature with a mouse anti-PentaHis IgG (Qiagen, Cat.no.34660) at 5 mg/ml diluted in PBS 2% BSA. After 30 min, cells were washed twice with PBS 2% BSA and incubated for 30 min at room temperature with a PE-conjugated goat F(ab)2 anti-mouse IgG (Beckman Coulter, IM0855, diluted 1:100 in PBS 2% BSA). Cells were washed with PBS and subjected to flow cytometry analysis. Data was acquired using a BD FACScanto II flow cytometer (Becton-Dickinson, San Jose, Calif.) and analysis was performed using the FLOWJO 8.1 software (Tree Star Inc.). Cellular debris were excluded from the analysis by appropriated gating using the forward and side scatters.
[0269] The reactivity of the tested conjugates to surface-expressed DARC (for DARC.sub.VHH-EBV conjugates), to surface of RAJI cells (for the hCD20.sub.ScFv-P18F3 and VAR2CSA-P18F3 conjugates), or to surface-expressed VAR19 (for the EPCR-P18 conjugate) was reflected by an increase geometric mean in fluorescence intensity in the PE channel (Geometric Mean PE).
[0270] 1.6. Opsonization Assays
[0271] For opsonization assays, DARC+ erythrocytes were incubated with a constant concentration of the DARC.sub.VHH-EBV conjugates in PBS 2% BSA, for 1 h at room temperature. The cells were then washed twice with PBS 2% BSA and incubated for 1 h at room temperature with serial dilutions of human plasma in PBS 2% BSA. Cells were then washed twice with PBS 2% BSA and incubated for 30 min at room temperature with a PE-conjugated donkey F(ab)2 anti-human IgG (Jackson Immunoresearch 709-116-098, diluted 1:100 in PBS 2% BSA). Cells were washed with PBS and subjected to flow cytometry analysis.
[0272] By contrast, RAJI cells were incubated with serial dilutions of the hCD20.sub.ScFv-P18F3 or VAR2CSA-P18F3 conjugate in PBS 2% BSA, for 1 h at room temperature. These cells were then washed twice with PBS 2% BSA and incubated for 1 h at room temperature with a constant human plasma dilution (1:100) in PBS 2% BSA. Said cells were then washed according to the same protocol as the one applied for DARC+ erythrocytes.
[0273] 1.7. Opsonic Phagocytosis Assays
[0274] The non-adherent human monocyte cell line THP-1 (Sigma) was maintained in 150 cm.sup.2 flasks with RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco, Grand Island, N.Y.), 2 mM L-Glutamine, 100 units/ml penicillin and 100 ug/ml streptomycin (THP-1 culture medium). The cells were subcultured every 3 days and density was maintained at less than 210.sup.5 cells per ml; cultures were kept in a humidified 37 C. incubator with 5% (v/v) CO.sub.2 and 95% (v/v) air.
[0275] THP-1 cells were seeded at 2.510.sup.5 cells per well in 6-well plates and the volume of each well was made to 3 ml with THP-1 culture media. To obtain macrophages, the cells were differentiated using 10 ng/ml phorbol 12-myristate 13-acetate for 24 h in 5% (v/v) CO.sub.2 at 37 C. The supernatant and unattached cells were removed by aspiration and adherent macrophages were washed twice with THP-1 culture medium before the wells were filled with 3 ml of fresh THP-1 culture medium. These were further incubated for 48 h before performing phagocytic assay.
[0276] Erythrocytes were labeled with CellTraceCFSE according to the manufacturer's instructions and opsonized with the conjugates and human plasma as described above. Labeled and opsonized erythrocytes were co-inbubated with THP-1 derived macrophages for 3 h. Erythrocytes were washed out from the wells and macrophages were then detached by trypsin treatment and subjected to flow cytometry analysis. CFSE positive THP-1 cells were regarded as cells having phagocyted at least one erythrocyte.
[0277] 1.8. Complement Activation Assay
[0278] For complement activation assays, RAJI cells were incubated with 5 g/ml of hCD20.sub.ScFv or hCD20.sub.ScFv-P18F3 in PBS 1% BSA for 1 h at 4 C. The cells were then washed twice with PBS 1% BSA and incubated for 1 h at 37 C., 5% CO.sub.2 with 10% EBV.sup.+ human plasma, in its active form of pre-inactivated by heat treatment (56 C. for 45 min). The cells were then washed three times with PBS and fixed with 4% PFA at room temperature for 15 min. The cells were then washed three times with PBS 5% BSA and blocked with PBS 5% BSA for 1 h at room temperature and then incubated with a mouse monoclonal antibody anti-C5b9 (Abcam ab66768, 1:100 in PBS 1% BSA). The cells were then washed three times with PBS 1% BSA and incubated for 30 min at room temperature with a PE-conjugated goat F(ab)2 anti-mouse IgG (Beckman Coulter, IM0855, diluted 1:100 in PBS 1% BSA). Cells were washed with PBS and subjected to flow cytometry analysis. Data was acquired using a BD FACScanto II flow cytometer (Becton-Dickinson, San Jose, Calif.) and analysis was performed using the FLOWJO 8.1 software (Tree Star Inc.). Cellular debris were excluded from the analysis by appropriated gating using the forward and side scatters. The C5-b9 deposition at the surface of RAJI cells was reflected by an increased fluorescence intensity in the PE channel.
1.9. Antibody-Dependent Cell Cytotoxicity (ADCC) Assay
[0279] For ADCC assays, RAJI cells were maintained at a density of 2.5.10.sup.5-10.sup.6 cells/ml in RPMI (Glutamax-I) supplemented with 10% FCS and 1 antibiotic-antimycotic solution (Gibco) at 37 C., 5% CO.sub.2. ADCC assays were performed using the ADCC Reporter Bioassay (Promega) according to manufacturer's instructions with slight modifications to fit our system. 300000 RAJI cells were first washed twice with PBS 2% BSA and incubated for 1 h at 4 C. with saturating concentration of conjugates (25 g/ml hCD20.sub.ScFv, hCD20.sub.ScFv-P18F3 or 50 g/ml VAR2CSA, VAR2CSA-P18F3. Cells were then washed once with PBS 2% BSA and 12500 cells (in 25 l) were introduced into the wells of white, flat bottom 96-well plates. EBV.sup.+ human plasma was inactivated/decomplemented at 56 C. for 45 min. Serial dilutions were prepared in ADCC assay medium (RPMI 1640, 4% low IgG FCS) and 25 l of diluted plasma were introduced to the wells together with the RAJI target cells. Effector cells were thawed and 75000 cells (in 25 l) were immediately distributed into the wells. Plates were incubated at 37 C., 5% CO.sub.2. After 6 h incubation, plates were removed from the incubator and let at room temperature for 15 min before addition of 75 l of Bio-Glo reagent into each well. Luminescence was measured using a VICTOR plate reader platform (PerkinElmer).
2. Results
[0280] 2.1. Expression and Purification of the Conjugates
[0281] The IMAC (Immobilized Metal ion Affinity Chromatography) purification in native conditions of the conjugates DARC.sub.VHH-EBV P18 and DARC.sub.VHH-EBV P23 (anti-DARC.sub.VHH fused to EBV P18 or EBV P23 antigen, respectively, each of said antigen being tagged in C-terminal with a poly-histidine sequence) was not successful (
[0282] N-terminal His-tagged fusions did not lead to better results.
[0283] A site-directed mutagenesis substituting the EBV P18 Cys (in amino-acid position 56) and the EBV P23 Cys (in amino-acid position 46) by serines was performed. The new conjugates DARC.sub.VHH-EBV P18 (C565)* and DARC-EBV P23 (C465)* were successfully purified by IMAC (
[0284] To overcome this problem, shorter variants pf P18 and P23 were designed. The resulting fusion proteins DARC.sub.VHH-EBV P18F2, DARC.sub.VHH-EBV P18F3, DARC.sub.VHH-EBV P18F4 considerably gained in solubility compared to DARC.sub.VHH-EBV P18(C56S)* and formed very little aggregates during the gel filtration purification step (
[0285] In order to extend the proof of concept to another conjugation system, a VHH sequence capable to recognize a P. falciparum protein present at the surface of erythrocytes of placental origin infected by P. falciparum (VAR2CSA.sub.VHH) was also fused to the EBV P18 and EBV P23 antigens. Similarly to the DARC.sub.VHH conjugates, removal of N-terminal clusters of EBV P18 and EBV P23 drastically improved conjugate solubility and stability in solution. Gel electrophoresis analysis of all the produced constructs revealed the high purity of the recombinant proteins used in the study (
[0286] The affinity of the DARC.sub.VHH-EBV P18 antigen conjugates and of the VAR2CSA.sub.VHH EBV P18 antigen conjugates for their respective molecular target was assessed by surface plasmon resonance. The specificity and affinity of the conjugates for their targets was not significantly modified in comparison to the affinity of the VHHs alone (
[0287] The proof of concept of the invention was further validated with three other conjugation systems: [0288] a scFv sequence capable of recognizing CD20 (a marker typically overexpressed by cancer cells) fused to the EBV P18F3 antigen, of which the purification was successful (
[0291] 2.2. Immune Recognition of the Conjugates
[0292] Immunoglobulin G titers, reflecting the immune recognition of the conjugates, were determined by serial dilutions of plasma from 12 healthy donors positive for EBV infection. DARC.sub.VHH-EBV P18F2 was highly recognized by the IgG present in said plasma (Table 2 and
TABLE-US-00016 TABLE 2 IgG titers (anti-DARCVHH-EBV P18F2) in plasma from 12 EBV+ donors. Donor ID IgG titers #20120621 460 #20120617 840 #20120312 230 #20120327 190 #20121029 130 #20120618 70 #20121001 440 #20120220 120 #20130313 360 #20120820 60 #20150217I 430 #20150217II 2300 Mean (SD): 470 (618)
[0293] A comparative immune recognition study performed with 22 plasma samples (dilution 1/200) showed that the C-terminal part of P18 possesses immuno-dominant epitopes. No significant differences in recognition was observed between conjugates comprises EBV P18F2 and EBV P18F3.
[0294] 2.3. Cell Surface Recognition of the Conjugates
[0295] Recognition of the native target by the conjugates was assessed by flow cytometry.
[0296] DARC.sub.VHH-EBV P18F2 was able to coat the surface of FY+ (DARC+) erythrocytes in a dose dependent manner (
[0297] The hCD20.sub.ScFv-P18F3 conjugate was able to coat CD20.sup.+ RAJI cells (
[0298] The VAR2CSA-P18F3 conjugate was able to coat CSA.sup.+ RAJI cells (
[0299] The EPCR-P18F3 conjugate was able to coat the surface of VAR19-expressing erythrocytes, in a dose dependent manner (
[0300] 2.4. Erythrocytes Opsonization by the Conjugates Targeting Said Cells
[0301] Furthermore, incubation of cell-bound conjugates with plasma samples of EBV+ donors led to FY+ (DARC+) erythrocytes opsonization, i.e. coating of cells with the donor plasma IgG (
[0302] 2.5. Opsonic Phagocytosis Assays
[0303] Phagocytic assays revealed an increased opsonic phagocytosis of erythrocytes by THP1 monocytes upon treatment with DARC.sub.VHH-EBV P18 and EBV P23 antigen fusions. Indeed, the conjugate DARC.sub.VHH-EBV P18 (C56S)* mediated an opsonic phagocytosis of FY+ red-blood cells by THP1-derived macrophages (
[0304] Incubation of cell-bound hCD20.sub.ScFv-P18F3 and VAR2CSA-P18F3 conjugates with plasma samples of EBV+ donors also led to RAJI opsonization, i.e. coating of cells with the donor plasma IgG (
[0305] Interestingly, treatment of FY+ red-blood cells with DARC.sub.VHH fused to different EBV P18 fragments revealed a link between the capability of said fragments to recruit IgG and the intensity of the elicited cellular response (
[0306] These results are the first promising in vitro data demonstrating that immune clearance of a define target cell can be achieved following treatment with binding moeity-EBV P18 or EBV P23 fusion proteins.
[0307] 2.6. Complement Activation
[0308] Complement activation assays revealed an increased activation of the complement cascade upon treatment with the hCD20.sub.ScFv-P18F3 conjugate, most likely the classical antibody dependent pathway. This is illustrated by the formation of the membrane-attack complex at the cell surface that will result in target cell lysis (
[0309] 2.7. ADCC
[0310] The ADCC assays revealed an increased activation of the NFAT pathway in competent effector cells by target cells (RAJI) pre-treated with either hCD20.sub.ScFv-P18F3 or VAR2CSA-P18F3. This reflects that opsonization of target cells occurring in presence of EBV+ plasma is leading to engagement of surface Fc receptors on effector cells and subsequent activation of intracellular pathways that will ultimately lead to target cell lysis (
3. Conclusion
[0311] Immunotherapies are being used against cancer cells, infectious diseases as well as Alzheimer's disease. Interestingly, a recent study has shown that macrophages eliminate circulating tumor cells after monoclonal antibody therapy through dependent FcRI and FcRIV phagocytosis. No cell mediated Fc effector function are driven by antibody fragment or antibody-binding fragment or alternative proteic and non-proteic moieties including, without limitation, Fab antibodies, Fab antibodies, F(ab)2 antibodies, Fv antibodies, scFv antibodies, camelid single domain antibodies (VHH), and shark single domain antibodies (VNAR). One possibility is to fuse an Fc chain to those targeting moieties. However, adding an Fc chain will result in recombinant expression difficulties as well as decrease accessibility to epitopes present in small cavities and clefts.
[0312] The present invention proposes to overcome these issues with a new and innovative immunogenic construct, which is capable to redirect an EBV-existing (or pre-existing) immune response towards an undesired target cell and/or microorganism. In order to provide the proof of concept that a targeting moiety fused to an EBV antigen is capable to promote opsonization of a defined target, and the formation of immune complexes and subsequent clearance of said target, three in vitro models were used herein. In the first model, a single domain antibody (DARC.sub.VHH) targeting a protein expressed at the surface of erythrocytes was conjugated to a couple of different EBV antigens (either EBV P18 or EBV P23). In a second model, a single domain antibody (VAR2CSA.sub.VHH) and a proteic binding moiety not derived from antibodies (EPCR), both targeting malarial proteins expressed at the surface of Plasmodium falciparum infected erythrocytes (VAR2CSA.sup.PfEMP1 and VAR19.sup.PfEMP1, respectively) were conjugated to EBV P18. In a third model, an scFv antibody (hCD20.sub.ScFv) directed towards the Cluster of Differentiation CD20 and a proteic binding moiety not derived from antibodies (VAR2CSA), targeting respectively the CD20 molecules present at the surface of B cells (including B lymphomas) or the CSA over-expressed at the surface of cancer cells were conjugated to EBV P18.
[0313] The present study demonstrates the capacity of the designed conjugates to opsonize the target cells, respectively erythrocytes, Plasmodium falciparum infected erythrocytes, cancer cells and promote their clearance by immune effector mechanisms namely opsonic phagocytosis, ADCC and CDC.
[0314] The N-terminal regions of EBV P18 and EBV P23 were identified as an obstacle for protein expression, which considerably decreased protein solubility and stability. The deletion of N-terminal segments of EBV P18 and EBV P23 allowed soluble expression and purification of the conjugates (
[0315] Importantly, surface plasmon resonance experiments showed that the affinity and specificity of the binding moieties (DARC.sub.VHH and VAR2CSA.sub.VHH) for their defined targets was not affected by their fusion to an EBV-antigen (
[0316] Immune recognition experiments performed with plasma from EBV positive individuals revealed that the conjugates comprising either EBV P18 or EBV P23 were highly recognized by circulating IgG (
[0317] Of major interest, the formation of immune complexes mediated by the conjugates led to immune effector responses. DARC.sub.VHH-EBV P18F3 treatment of DARC+ erythrocytes promoted their elimination by macrophages (
[0318] Furthermore, treatments of target cells with P18F3 conjugates also led to i) ADCC by competent effector cells (
[0319] Taken together, these results demonstrate that immunogenic constructs, such as DARC.sub.VHH-EBV P18F3, EPCR-EBV P18F3, hCD20.sub.ScFv-EBV P18F3 and VAR2CSA-EBV P18F3 are able to redirect an existing EBV immune response towards a specific target sequentially leading to the formation of immune complexes and subsequent recruitment of immune effector mechanisms such as opsonic phagocytosis, ADCC and CDC which ultimately lead to target elimination.
[0320] The types of agents, stable, easy to produce and highly efficient at opsonizing a target element are extremely valuable for clinical interventions either alone or in combination with existing therapies to fight pathogens and cancers.
REFERENCES
[0321] Aird W C, Mosnier L O, and Fairhurst R M (2014). Blood; 123(2): 163-167. [0322] Birch C M, Hou H W, Han J, Niles J C (2015). Sci Rep.; 5:11347. [0323] Chan C L, Renia L, and Tan K S (2012). PLoS One; 7(6):e38523. [0324] Chen X, Zaro J L, Shen W C (2013). Adv Drug Deliv Rev; 65(10):1357-1369. [0325] Clausen T M, Christoffersen S, Dahlback M, Langkilde A E, Jensen K E, Resende M, Agerbk M, Andersen D, Berisha B, Ditlev S B, Pinto V V, Nielsen M A, Theander T G, Larsen S, and Salanti A (2012). J Biol Chem.; 287(28):23332-45. [0326] Conrath K, Vincke C, Stijlemans B, Schymkowitz J, Decanniere K, Wyns L, Muyldermans S, and Loris R (2005). [0327] Estevez M C, Huang YF, Kang H, O'Donoghue M B, Bamrungsap S, Yan J, Chen X, Tan W (2010). Methods Mol Biol; 624:235-248. J. Mol. Biol.; 350: 112-125. [0328] Eudes R, Le Tuan K, Delettre J, Mornon J P, Callebaut I (2007). BMC Struct Biol; 7:2. [0329] Harboe M, Thorgersen E B, and Mollnes T E (2011). Adv Drug Deliv Rev; 63(12):976-987. [0330] Hu W, Li F, Yang X, Li Z, Xia H, et al. (2004). J Biotechnol; 107: 83-90. [0331] Jiang X R, Song A, Bergelson S, Arroll T, Parekh B, May K, Chung S, Strouse R, Mire-Sluis A, and Schenerman M (2011). Nat Rev Drug Discov; 10(2):101-111. [0332] Liu A Y, Robinson R R, Murray E D Jr, Ledbetter J A, Hellstrom I, and Hellstrom K E (1987). J Immunol.; 139(10):3521-6. [0333] Nunes-Silva S, Gangnard S, Vidal M, Vuchelen A, Dechavanne S, Chan S, Pardon E, Steyaert J, Ramboarina S, Chene A et al. (2014). Sci Rep; 4:7373. [0334] Olafsen T, Betting D, Kenanova V E, Salazar F B, Clarke P, Said J, Raubitschek A A, Timmerman J M, and Wu A M (2009). J Nucl Med.; 50(9):1500-8. [0335] Otz T, Grosse-Hovest L, Hofmann M, Rammensee H G, and Jung G. Leukemia; 23(1):71-7. [0336] Skrlec K, Strukelj B, and Berlec A (2015). Trends in Biotechnology; 33(7): 408-418.
[0337] Smolarek D, Hattab C, Hassanzadeh-Ghassabeh G, Cochet S, Gutierrez C, de Brevern A G, Udomsangpetch R, Picot J, Grodecka M, Wasniowska K, Muyldermans S, Colin Y, Le Van Kim C, Czerwinski M, and Bertrand O (2010). Cell Mol Life Sci.; 67(19):3371-87. [0338] Srivastava A1, Gangnard S, Dechavanne S, Amirat F, Lewit Bentley A, Bentley G A, and Gamain B (2011). PLoS One.;6(5):e20270. [0339] Stoltenburg R, Reinemann C, and Strehlitz B (2007). Biomol Eng.; 24(4):381-403. [0340] Zwicke G L, Mansoori G A, and Jeffery C J (2012). Nano Rev, 3: 18496. [0341] Wu J, Nantz M H, and Zern M A (2012). Front Biosci; 7:d717-725. [0342] Vogt A M, Barragan A, Chen Q, Kironde F, Spillmann D, and Wahlgren M (2003). Blood; 101(6):2405-2411.