Antibodies and fragments thereof raised against the alpha-3 domain of HLA-G protein, methods and means for their preparation, and uses thereof

Abstract

An antibody or antigen-binding fragment thereof which specifically binds the 3 domain of a HLA-G protein, in particular binds the 2-microglobulin free HLA-G protein exhibiting an 3 domain. The nucleic acid molecules encoding a human HLA-G 3 domain polypeptide, which is selected from a group of specific sequences, and vectors for the cloning and/or expression of such nucleic acid molecules, recombined cells or cell lines and compositions for use in a host in need thereof to interfere with and neutralize the immune down-regulation due to HLA-G proteins, and/or improving or treating conditions showing HLA-G+ lesions, and/or improving or treating a neoplasic condition or disease. A method of producing the antibody or antigen-binding fragment thereof, immunogenic compositions for use to elicit in a host an immune response against the 3 domain of HLA-G protein, and an in vitro method for detecting HLA-G protein in a sample are also described.

Claims

1. A method of producing an antibody or an antigen-binding fragment thereof which specifically binds the 3 domain of a HLA-G protein, the method comprising administering to a mammal a nucleic acid molecule encoding a polypeptide comprising the amino-acid sequence of SEQ ID NO: 1, or an amino-acid sequence comprising at least 99% amino acid identity over the entirety of SEQ ID NO: 1.

2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof which specifically binds the 3 domain of a HLA-G protein is a blocking antibody or antigen-binding fragment thereof.

3. The method according to claim 1, further comprising recovering antibodies elicited by said mammal from sera or plasma obtained from said mammal.

4. The method of claim 3, further comprising preparing antigen-binding fragments from the recovered antibodies.

5. The method according to claim 3, further comprising checking the recovered antibodies for specificity to the 3 domain of the HLA-G protein.

6. The method according to claim 3, further comprising preparing antigen-binding fragments from the recovered antibodies.

7. The method according to claim 1, wherein the mammal is a non-human mammal.

8. The method according to claim 1, wherein said antibody or antigen-binding fragment binds the 3 domain of a HLA-G protein when the 3 domain is in a monomeric and/or a dimeric form.

9. The method according to claim 1, wherein said antibody or antigen-binding fragment binds the 3 domain when present in the HLA-G protein.

10. The method according to claim 1, wherein said antibody or antigen-binding fragment binds the 3 domain when present in a 2-microglobulin free HLA-G protein or HLA-G protein isoform.

11. The method according to claim 1, wherein said antibody or antigen-binding fragment does not bind the 3 domain when the HLA-G protein is associated with the 2-microglobulin.

12. The method according to claim 1, wherein said antibody or antigen-binding fragment blocks binding of a HLA-G protein to at least one of LILRB1 and LILRB2 receptors.

13. The method according to claim 1, wherein said nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 6.

14. The method according to claim 1, wherein said nucleic acid molecule is administered to the mammal in the form of naked DNA.

15. The method according to claim 1, wherein said nucleic acid molecule is administered to the mammal by a delivery method selected from the group consisting of intramuscular or intradermal injection, electroporation, gene-gun delivery, needle-free delivery system, topical administration, and a combination thereof.

16. The method according to claim 1, further comprising administering an adjuvant to the mammal.

17. The method according to claim 1, wherein the nucleic acid molecule encodes a polypeptide comprising the amino-acid sequence of SEQ ID NO: 2.

18. The method according to claim 1, wherein the nucleic acid molecule encodes a polypeptide comprising the amino-acid sequence of SEQ ID NO: 1.

Description

FIGURES LEGENDS

(1) FIG. 1: HLA-G protein isoforms

(2) FIG. 2: Overall structure of the LILRB2 complex of HLA-G

(3) FIG. 3: HLA-G expression in tumor lesions

(4) FIG. 4: In vivo experiments: no tolerogenic effect of .sub.3 and (.sub.3)2 molecules after skin allograft in mice

(5) FIG. 5: Constructs details of HLA-G .sub.3 domain proteins (SEQ ID No12)

(6) FIG. 6: Luminex method principle to detect specific binding of HLA-G .sub.3 domain protein by anti-HLA-G antibodies

(7) FIG. 7: anti-HLA-G .sub.3 domain protein antibodies detected in plasmas of protein-immunized C57Bl/6J HLA-B*0702 transgenic mice after 4th boost by (A) ELISA, (B) Luminex beads and (C) slot-blot assay.

(8) FIG. 8: anti-HLA-G .sub.3 domain protein antibodies in plasmas of DNA-immunized Balb/c mice after 2nd boost (4 weeks) detected (A) by Luminex beads and (B) by Slot-blot assay

(9) FIG. 9: anti-HLA-G .sub.3 domain protein antibodies in plasmas of DNA-immunized C57Bl/6J mice after 2nd boost (3 weeks) detected (A) by Luminex beads and (B) by slot-blot assay

(10) FIG. 10: No DNA HLA-G-3 vaccination: 2.Math.10.sup.6 M8-pcDNA or M8-HLA-G1 were implanted subcutaneously on Balb/c mice (M8-pcDNA: Human Melanoma transfected with pcDNA 3.1 control plasmid; M8-HLA-G1: Human Melanoma transfected with pcDNA 3.1 plasmide containing HLA-G1 sequence). Tumor volume according to the Human Melanoma concentration is plotted. No antibodies against HLA-G-3 domain were detected in non-immunized mice monitored before tumor challenge.

(11) FIG. 11: DNA HLA-G-3 vaccination: 2.Math.10.sup.6 M8-pcDNA or M8-HLA-G1 were implanted subcutaneously on Balb/c mice (M8-pcDNA: Human Melanoma transfected with pcDNA 3.1 control plasmid; M8-HLA-G1: Human Melanoma transfected with pcDNA 3.1 plasmide containing HLA-G1 sequence). As described in the Materials and Methods section, Balb/c mice were immunized 4 times with HLA-G-3 DNA. Induction of antibodies against HLA-G-3 domain was monitored before tumor challenge: all Balb/c mice generated anti-HLA-G-3 antibodies after DNA immunization. Tumor volume according to the Human Melanoma concentration is plotted.

MATERIALS AND METHODS

(12) TABLE-US-00001 SEQIDNo1:HLA-Galpha3orHLA-G3protein sequence DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVE TRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW

(13) SEQ ID No1 is based on the disclosure found in McCluskey et al. (PNAS 2005, vol. 102, no. 9, 3360-3365 Crystal structure of HLA-G: A nonclassical MHC class I molecule expressed at the fetal-maternal interface), and/or derived from the full HLA-G human protein as found in the literature or under accession number NM_002127.5, referred to as SEQ ID No4 herein.

(14) TABLE-US-00002 SEQIDNo4: MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMG YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM NLTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALN EDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKE MLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQD VELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQS SLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD

(15) The natural, i.e. not engineered or found naturally in living organisms, nucleic acid sequence encoding a HLA-G3 domain polypeptide sequence can be found in the sequence disclosed under accession number NM_002127.5, between positions 797 and 1072 (276 pb), and is referred to as SEQ ID No 9 herein.

(16) TABLE-US-00003 SEQIDNo9 gacccccccaagacacacgtgacccaccaccctgtctttgactatgaggc caccctgaggtgctgggccctgggcttctaccctgcggagatcatactga cctggcagcgggatggggaggaccagacccaggacgtggagctcgtggag accaggcctgcaggggatggaaccttccagaagtgggcagctgtggtggt gccttctggagaggagcagagatacacgtgccatgtgcagcatgaggggc tgccggagcccctcatgctgagatgg

(17) An example of an optimized nucleic acid sequence, which is considered optimized by comparison to SEQ ID No 9 defined herein, and encoding for an HLA-G3 domain polypeptide sequence, is disclosed and referred to as SEQ ID No 10 herein.

(18) TABLE-US-00004 gacccccccaaaacccatgtgacccaccacccagtctttgactatgaa gctacactgagatgttgggccctgggcttctaccccgcagagatcatc ctgacctggcagcgcgacggagaagatcagacacaggacgtcgagctc gtggaaacccggcctgctggtgatggcacatttcagaagtgggccgcc gtggtggttccatccggtgaggaacagcgctacacttgccatgtgcag cacgagggcttgcctgagcctcttatgcttcggtgg

(19) The full human HLA-G nucleic sequence disclosed under accession number NM_002127.5 is disclosed herein as SEQ ID No 5:

(20) TABLE-US-00005 agtgtggtactttgtcttgaggagatgtcctggactcaca cggaaacttagggctacggaatgaagttctcactcccatt aggtgacaggtttttagagaagccaatcagcgtcgccgcg gtcctggttctaaagtcctcgctcacccacccggactcat tctccccagacgccaaggatggtggtcatggcgccccgaa ccctcttcctgctgctctcgggggccctgaccctgaccga gacctgggcgggctcccactccatgaggtatttcagcgcc gccgtgtcccggcccggccgcggggagccccgcttcatcg ccatgggctacgtggacgacacgcagttcgtgcggttcga cagcgactcggcgtgtccgaggatggagccgcgggcgccg tgggtggagcaggaggggccggagtattgggaagaggaga cacggaacaccaaggcccacgcacagactgacagaatgaa cctgcagaccctgcgcggctactacaaccagagcgaggcc agttctcacaccctccagtggatgattggctgcgacctgg ggtccgacggacgcctcctccgcgggtatgaacagtatgc ctacgatggcaaggattacctcgccctgaacgaggacctg cgctcctggaccgcagcggacactgcggctcagatctcca agcgcaagtgtgaggcggccaatgtggctgaacaaaggag agcctacctggagggcacgtgcgtggagtggctccacaga tacctggagaacgggaaggagatgctgcagcgcgcggacc cccccaagacacacgtgacccaccaccctgtctttgacta tgaggccaccctgaggtgctgggccctgggcttctaccct gcggagatcatactgacctggcagcgggatggggaggacc agacccaggacgtggagctcgtggagaccaggcctgcagg ggatggaaccttccagaagtgggcagctgtggtggtgcct tctggagaggagcagagatacacgtgccatgtgcagcatg aggggctgccggagcccctcatgctgagatggaagcagtc ttccctgcccaccatccccatcatgggtatcgttgctggc ctggttgtccttgcagctgtagtcactggagctgcggtcg ctgctgtgctgtggagaaagaagagctcagattgaaaagg agggagctactctcaggctgcaatgtgaaacagctgccct gtgtgggactgagtggcaagtccctttgtgacttcaagaa ccctgactcctctttgtgcagagaccagcccacccctgtg cccaccatgaccctcttcctcatgctgaactgcattcctt ccccaatcacctttcctgttccagaaaaggggctgggatg tctccgtctctgtctcaaatttgtggtccactgagctata acttacttctgtattaaaattagaatctgagtataaattt actttttcaaattatttccaagagagattgatgggttaat taaaggagaagattcctgaaatttgagagacaaaataaat ggaagacatgagaacttt
Immunizing Agents
Protein Immunization

(21) The HLA-G .sub.3 domain used for protein immunization was produced by chemical synthesis (Patent: WO 2010/150233). This protein is made up of 108 amino acids, of molecular weight 11957 Da, and is composed by the following protein sequence (SEQ ID No2), for which the first 12 amino acids contain a linker sequence (SEQ ID No 3):

(22) TABLE-US-00006 GCGGGGSGGGGSRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQ RDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPE PLMLRWKQ SEQIDNo3: GCGGGGSGGGGS
DNA Immunization

(23) For DNA immunization, the sequence of the .sub.3 domain was fused to the simian virus 5 V5 protein tag sequence, the ensemble was cloned into the pcDNA 3.1(+) plasmid vector (Invitrogen) by the HindIII and XhoI restriction sites (underlined). The nucleotide sequence used is (SEQ ID No 6):

(24) TABLE-US-00007 5'AAGCTTGCCGCCAtggtcgttatggcacccaggaccttgttcctcct gctctctggagcactgacccttactgagacatgggcc agagccga cccccccaaaacccatgtgacccaccacccagtctttgactatgaagcta cactgagatgttgggccctgggcttctaccccgcagagatcatcctgacc tggcagcgcgacggagaagatcagacacaggacgtcgagctcgtggaaac ccggcctgctggtgatggcacatttcagaagtgggccgccgtggtggttc catccggtgaggaacagcgctacacttgccatgtgcagcacgagggcttg cctgagcctcttatgcttcggtggaagcagtcatccctgccaactattcc catcatgggcattgtggccggactggtggttctggcagctgtggtgactg gcgctgccgtcgccgctgtcctctggaggaaaaagagcagc ggca agccaattcctaatccattgctgggcctggactcaacttgaTGATAACTC GAG3'
Legend for SEQ ID No 6:
(SEQ ID No7) GCCGCCAtg at 5 extremity: 5 Kozak sequence
Signal Peptide as Found in Exon 1 of HLA-G
Nucleotides encoding added amino-acids
Nucleotides found at the end of the 2 (alpha2) portion of HLA-G 3 (alpha3) domain (corresponding to SEQ ID No10)
Transmembrane-Anchor Protein Encoding Portion
V5-tag sequence
(SEQ ID No8) tgaTGATAA at 3 extremity: 3 Stop codon

(25) The sequence cloned into the pcDNA 3.1(+) plasmid vector between GCCGCCA (SEQ ID No7) and tgaTGATAA (SEQ ID No8) has therefore a length of 534 pb.

(26) The HindIII and Xho sites are underlined while the V5 sequence corresponding to the simian virus 5 V5 tag is shown in italics, according to the legend provided above. The gene was synthesized by GeneGust after codon optimization with respect to the natural nucleic acid sequence to eliminate low abundance codons.

(27) For illustration purposes, the protein sequence (SEQ ID No 11) corresponding to, i.e. encoded by, the nucleic acid sequence elaborated for DNA immunization is given below. Domains 1 (alpha1) and 2 (alpha2) were deleted leaving a signal peptide (also found in the exon1 portion of HLA-G), two amino-acids of the end of the 2 (alpha2) domain, the 3 (alpha3) domain and a transmembrane anchor sequence, as annotated below.

(28) TABLE-US-00008 SEQIDNo11 MVVMAPRTLFLLLSGALTLTETWASRADPPKTHVTHHPVFDYEATLRCWA LGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQ RYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAA VLWRKKSSDGKPIPNPLLGLDST
Legend for SEQ ID No11:
Signal Peptide as Found in Exon 1 of HLA-G
Added Amino-Acids
Amino-acids found at the end of the 2 (alpha2) portion of HLA-G 3 (alpha3) domain (corresponding to SEQ ID No1)
Transmembrane-Anchor Protein Encoding Portion
V5-tag sequence

(29) The nucleic acid sequence disclosed under SEQ ID No6 is not the same as that in the database under NM_002127.5 (SEQ ID No5), especially between positions 797 and 1072 corresponding to a portion encoding a polypeptide encompassing the 3 domain of HLA-G protein, as well as in the portion encoding a peptide signal and a transmembrane anchor. Most of the codons have been changed with 12 codons not being used at all. This is called codon optimization, according to conventional methods. The present rule set eliminates rare tRNAs and codons that are used infrequently in the human genome, infrequently being defined as <10%. Consequently the following codons were eliminated: Leu TTA, Leu CTA, Ile ATA, Val GTA, Ser TCG, Pro CCG, Thr ACG, Ala GCG, Gln CAA, Arg CGT, Arg CGA and Gly GGG.

(30) The correspondence between the natural nucleic sequence of HLA-G and the optimized nucleic acid sequence used for experiments herein is provided below: optimized SEQ ID No6 is depicted with interlineated nucleotide bases, which are the nucleotide bases found in the natural nucleic sequence of HLA-G.

(31) TABLE-US-00009 AAGCTTGCCGCC Atggtcgttatggcacccaggaccttgttcctcctgctctctggagcactgacccttactga gcgcaccgggcgc gacatgggcc agagccgacccccccaaaacccatgtgacccaccacccagtctttgact cgcgcgcggact atgaagctacactgagatgttgggccctgggcttctaccccgcagagatcatcctgacctgg gccgctga cagcgcgacggagaagatcagacacaggacgtcgagctcgtggaaacccggcctgctggtga gtggccggaag tggcacatttcagaagtgggccgccgtggtggttccatccggtgaggaacagcgctacactt accatgttagagag gccatgtgcagcacgagggcttgcctgagcctcttatgcttcggtggaagcagtcatccctg tgcgccgaat ccaactattcccatcatgggcattgtggccggactggtggttctggcagctgtggtgactgg ccctcttctctac cgctgccgtcgccgctgtcctctggaggaaaaagagcagc ggcaagccaattcctaatc agtggagtca cattgctgggcctggactcaacttgaTGATAACTCGAG
Legend:
HindIII and XhoI Restriction Sites
(SEQ ID No7) GCCGCCAtg at 5 extremity: 5 Kozak sequence
Signal Peptide as Found in Exon 1 of HLA-G
Added Amino-Acids
Nucleotides found at the end of the 2 (alpha2) portion of HLA-G 3 (alpha3) domain (corresponding to SEQ ID No10)
Transmembrane-Anchor Protein Encoding Portion
V5-tag sequence
(SEQ ID No8) tgaTGATAA at 3 extremity: 3 Stop codon

(32) With respect to the nucleotide sequence encoding for the 3 (alpha3) domain, 46 nucleotides were modified over the 276 nucleotides of said domain.

(33) As previously stated, the coding sequence was bounded at 5 extremity by a good Kozak initiation sequence (GCCGCCATG (SEQ ID No7), initiator codon in bold face) and a pair of stop codons at 3 extremity (TGATAA (SEQ ID No8) in bold face), the ensemble being flanked by 5 HindIII and 3 XhoI restriction sites (underlined). The DNA insert was synthesized by GeneGust and cloned into the expression vector pcDNA3.1+ by way of the HindIII and XhoI sites.

(34) The resulting sequence is disclosed under SEQ ID No6, which encodes a HLAG signal peptide, the 3 domain of human HLA-G, a HLA-G transmembrane spanning sequence fused to the simian virus 5 V5 protein tag sequence, with a 5 Kozak initiation sequence and a 3 pair of stop condons, and HindIII and XhoI restriction sites.

(35) Plasmid production, transfection and electroporation were performed using standard techniques.

(36) Mice

(37) C57Bl/6J HLA-B*0702 transgenic mice bred in Institut Pasteur's animal facility were used for protein immunization (8 weeks of age). Balb/c and C57Bl/6J mice (8 weeks of age) were obtained from Janvier Laboratories and used for DNA immunization. Mice were anesthetized prior to immunization by intra-peritoneal (IP) route with a mix solution of xylazine 2% (Bayer AG) and ketamine 8% (Imalgen 1000) PBS according to individual animal weight. All animals were housed at Institut Pasteur's animal facility and were handled in agreement with good animal practice and according to the Ethics Charter of Institut Pasteur.

(38) HLA-G .sub.3 Domain Protein and DNA Immunization Protocols

(39) The .sub.3 domain protein (0.1 mg/ml) was dissolved in PBS1x and emulsified in equal volumes of Complete Freund's adjuvant (CFA, Sigma) for the prime immunization and with Incomplete Freund's adjuvant (IFA, Sigma) for subsequent immunization boosts. C57Bl/6J HLA-B*0702 transgenic mice were immunized every two weeks performing intra-dermic (ID) administration of the protein in the dorsal area after shaving, using specific insulin needles U-100. The inventors used non-dimerized forms of SEQ ID No2 for administration. DNA immunization was performed by non-invasive intra-muscular (IM) electroporation. Animals were shaved and aseptically swabbed prior to the immunization. The plasmid was administered in the inferior tibialis cranialis muscle, injecting 50 l of the plasmid DNA (0.1 mg/ml dissolved in PBS buffer, Invitrogen) in each leg. DNA immunizations were performed as followed; prime DNA immunization was followed 2 weeks after by DNA immunization boosts made every week during 3 weeks. Immunized mice were bleeded at the time of immunization boosts to monitor antibodies presence within plasmas. Immediately following the inoculation, legs were covered with ultrasound gel, and the non-invasive plate electrodes for the Agilepulse electroporator (BTX) delivery system applied. A single pulse of 560 V per cm of electrode spacing for 50 ms with a frequency of 1 Hz was applied, and the plate electrodes were 0.5 cm apart.

(40) Sample Analysis

(41) Specific anti-HLA-G .sub.3 domain antibodies were determined on sera or plasma samples. Sera were obtained by orbital sinus bleeding and plasmas were obtained by tail vain bleeding techniques for protein and DNA immunizations respectively. 20-50 l of blood samples were harvested in heparin microtubes.

(42) Enzyme-Linked Immunosorbent Assay (ELISA)

(43) The presence of anti-HLA-G .sub.3 domain antibodies within the plasmas of protein-immunized mice was analyzed by an indirect colorimetric ELISA. Microtiter plates were coated with synthetic proteins. Sera were serially diluted (from 1/500 to 1/32,000) and added to the plate after blocking and washing. Bound antigen-specific antibodies were detected using 0.1 g/ml mouse-specific antibodies horse-radish conjugated anti IgG antibodies, incubated in the wells for 1 hour at 37 C. Plates were developed with OPD and read at 405 nm (OD).

(44) Flow Cytometry Analysis (Luminex)

(45) Anti-HLA-G .sub.3 domain antibodies screening in sera and plasma of protein and DNA immunized mice respectively, were performed with Bio-Plex System Bead Coupling (Bio-Rad). One million beads (80 l) were activated and coupled with 30 g/ml of HLA-G .sub.3 domain synthetic protein, as previously described [42] and outlined in FIG. 7. Coupled microspheres were counted by a cytometer Epics XL cytometer (Beckman Coulter) to evaluate the final number of beads. For antibody screening, 10 l of serum sample were added to a 96-well filter microplate (Millipore, Billerica, Mass., USA) and 2 l of HLA-G .sub.3 domain coated beads were added and incubated in the dark for 30 min at RT. After washing thoroughly with PBS buffer, 50 l of 20 g/ml phycoerythrin-conjugated goat anti-mouse IgG Ab (GAM-PE, R&D Systems) secondary antibody was added to the beads and samples were again conjugated for 30 min in the dark at RT. Then, the microspheres were washed by vacuum filtration. All samples were analyzed in duplicate by flow cytometry. The results were presented in median fluorescence intensity (MFI) and the cut-off of positive reactions was defined using sera of non-immunized mice of each cohort.

(46) Slot-Blot Analysis

(47) HLA-G .sub.3 domain protein was blotted onto a nitrocellulose membrane after separation in a 12% SDS-PAGE electrophoresis (GE Healthcare). Membranes were blocked by incubation with PBS containing 0.2% Tween 20 and 5% nonfat dry milk for 1 hour at room temperature (RT). The membranes were then probed with plasmas from protein and plasmid DNA immunized mice, diluted at 1/20 and incubated in a Mini-PROTEAN II Multiscreen apparatus (Bio-Rad) overnight at 4 C. After washing with PBS containing 0.2% Tween 20, membranes were blocked again with PBS containing 0.2% Tween 20 and 5% nonfat dry milk for 20 min. The membranes were subsequently incubated for 1 hour at room temperature with peroxidase-conjugated goat anti-mouse IgG Ab (Sigma), washed thoroughly, stained with enhanced chemiluminescence reagent ECL (GE Healthcare), and exposed to X-ray film.

(48) Results

(49) Anti-HLA-G .sub.3 Domain Protein Antibodies Produced in HLA-B*0702 Transgenic Mice after Protein Immunization.

(50) For protein immunization 4 C57Bl/6J HLA-B*0702 transgenic mice were first immunized with the HLA-G .sub.3 domain protein in CFA, and injected every two weeks with the protein in IFA. Sera were tested by ELISA every two weeks after the second immunization. After 8 weeks of immunization, specific antibodies for HLA-G .sub.3 domain protein were detected in one mouse, and another mouse responded after 12 weeks of immunization (the equivalent of 6 boosts), as shown in FIG. 7 (A).

(51) Afterwards, two other methods were performed to monitor antibody secretion, in order to confirm these results. In the first place, a Luminex bead assay (set up and validated for HLA-G by this/SRHI laboratory), was carried out to evaluate the presence of conformational antibodies. As shown in FIG. 7 (B), the plasmas of the C57Bl/6J HLA-B*0702 transgenic mice 4 and 5 were positively bound to the HLA-G .sub.3 domain-coated beads, in accordance with the ELISA results. Interestingly, we observed that the plasma of the C57Bl/6J HLA-B*0702 transgenic mouse 5 by the 8.sup.th week of immunization was already positive for the Luminex beads assay, and was not positive for ELISA until the 12.sup.th week. Finally, the presence of HLA-G .sub.3 domain specific antibodies against denatured epitopes was confirmed by a slot-blot assay. Results shown in FIG. 7 (C) indicate a positive result only for plasma of the mouse 4.

(52) Anti-HLA-G .sub.3 Domain Protein Antibodies Produced in Balb/c, C57Bl/6J and HLA-B*0702 Transgenic Mice Following DNA Immunization

(53) The second strategy of immunization was set up using DNA coding for the .sub.3 domain of the HLA-G molecules. Three murine strains were immunized with the specific sequence, which was delivered by EGT (Electro-Gene Transfer) in muscular cells. In the case of Balb/c mice, three immunized animals were positive for the Luminex beads assay as well as for the slot-blotting analysis, shown in FIGS. 8 (A) and (B). For the C57Bl/6J strain, three out of four immunized animals were positive as well, for both Luminex beads and Slot-blot assays as shown in FIGS. 9 (A) and (B).

(54) Comparative Experiences with Respect to DNA HLA-G-3 Vaccination

(55) The inventors also investigated whether DNA-HLA-G a3 domain electroporation could inhibit the immunosuppressive effect of the complete HLA-G molecule. For this purpose, Balb/c mice were electroporated or not with DNA-HLA-G a3 domain and circulating antibodies raised against HLA-G-a3 domain, as described herein, were generated. Non-electroporated and DNA-HLA-G a3 electroporated Balb/c mice were inoculated subcutaneously with 2.Math.10.sup.6 M8 melanoma cells transfected or not with HLA-G1 molecules and respectively referred as M8-HLA-G1 and M8-pcDNA cells [38]. As shown on FIG. 10, without DNA-HLA-G 3-electroporation, M8-HLA-G1 tumor growth was higher than M8-pcDNA cells as previously reported [17]. However, after DNA-HLA-G 3 electroporation, tumor growth was similar between subcutaneously implanted M8-HLA-G1 and M8-pcDNA tumor cells (FIG. 11). Thus, HLA-G1 protective effect was prevented in electroporated mice. The inventors further showed that suppression of HLA-G protective effect on transfected M8-HLA-G1 cells was correlated with the presence of circulating antibodies raised against HLA-G 3 domains in mice sera (data not shown).

(56) Discussion

(57) The inventors have produced anti-HLA-G antibodies by using the HLA-G .sub.3 domain by protein and DNA immunization. DNA immunization proved to be the efficient way to raise antibodies against the .sub.3 domain of HLA-G protein, as illustrated in mice. This strategy was considered relevant to the generation of anti-HLA-G antibodies because the .sub.3 domain of HLA-G contains an unique motif of the molecule, present in no other HLA molecule. According to a particular embodiment, generated anti-HLA-G antibodies of the invention recognize a conformational epitope on HLA-G proteins, as present in HLA-G proteins naturally expressed by the cellular machinery. Furthermore, the use of the HLA-G .sub.3 domain was anticipated to be particularly relevant to the generation of blocking antibodies, because the function of HLA-G is dependent on the association between LILRB molecules and the .sub.3 domain of HLA-G, even though additional binding to 2M is required for the LILRB1 receptor. However, as discussed above, all attempts to target said HLA-G .sub.3 domain by producing anti-HLA .sub.3 domain antibodies have failed so far. Finally, this strategy represents a significant advance because the antibody produced will be able to recognize 2M-associated, 2M-free, and truncated HLA-G isoforms, which all contain the .sub.3 domain and are all immune-inhibitory and pathologically relevant.

(58) Two methods were implemented, which allowed efficient immunization of most HLA-G isoforms using a DNA sequence and an engineered protein corresponding to the HLA-G .sub.3 domain. These innovative immunization strategies allowed the development and production of anti-HLA-G antibodies, which are fundamental to anti-HLA-G-tumor based therapies and HLA-G monitoring.

(59) The inventors have demonstrated by three different detection methods, that it was possible to produce new anti HLA-G antibodies by immunization of HLA-B*0702 transgenic mice with the .sub.3 domain of HLA-G. The difference observed amongst the ELISA, Luminex beads and the slot-blot assays can be explained by the fact that the last method utilizes denatured epitopes, for which some of the clones producing antibodies, detected in the other assays, do not recognize such epitope. Hence, some polyclonal antibodies could bind to denatured epitopes while others could bind to conformational ones.

(60) A different strategy was also performed with the purpose of producing specific anti-HLA-G 3 domain antibodies through DNA immunization. DNA immunization yielded 3/6 for the strain Balb/c and 3/4 positive results for the C57Bl/6J strain, detected by both Luminex and slot-blot assays.

(61) In DNA HLA-G-3 vaccination experiments, the inventors have proved that Balb/c mice generated anti-HLA-G-3 antibodies after DNA immunization, with a strong effect on the tumor growth.

REFERENCES

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