Anti hla-g specific antibodies
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example 1
n of Anti-HLA-G Antibodies in Mice
[0267]Design of the Immunogen
[0268]The inventors designed a highly HLA-G specific peptide corresponding to the α3 amino acid region 189-203 of HLA-G, referenced as “Peptide Constrained: PC-1” (FIG. 1A; in bold and underlined), and used it to immunize mice.
[0269]PC-1 sequence: VTHHPVFDYEATLRC (SEQ ID NO: 56)
[0270]PC-1 immunogen is expected to generate therapy-suitable anti-HLA-G monoclonal antibodies specific for HLA-G α3-containing isoforms independently of β2M association.
[0271]Immunization, Hybridoma Generation and scFv Production
[0272]The immunization protocol using the PC-1 peptide coupled to KLH is described in Materials and Methods section. C57BL / 6 and BALB / c mice were used for peptide immunization.
[0273]Briefly, mice were intraperitoneally primed with PC-1-KLH conjugate in CFA and boosted with 4 IP in IFA. Sera from immunized mice were collected at different time points along the immunization procedure and were tested using Bioplex-HLA-G5, HL...
example 2
haracterization of the Mouse Monoclonal Antibody 15E7 and scFv R4C-C3
[0278]The cDNA sequences encoding the light and heavy chain variable regions of the monoclonal antibody 15E7 and the R4C-C3 scFv were obtained using standard PCR and DNA sequencing methods.
[0279]Monoclonal Antibody 15E7
[0280]The nucleotide and amino acid sequences of the light chain variable region of 15E7 are shown in FIG. 2A.
[0281]The nucleotide and amino acid sequences of the heavy chain variable region of 15E7 are shown in FIG. 3A.
[0282]The greatest variability in the light and heavy chains is located mainly within the hypervariable regions called Complementarity-Determining Regions (CDRs) which define the specificity of the antibody. Analysis of the 15E7 VL and VH sequences led to the CDR1, CDR2 and CDR3 regions delineation of the light and heavy chains respectively as shown in FIGS. 2A and 3A.
[0283]The sequence of the 15E7 κ light chain was compared to the known mouse germline immunoglobulin light chain seque...
example 3
ization of Anti-HLA-G Monoclonal Antibody 15E7
[0291]Protein Analysis
[0292]The 15E7 monoclonal antibody was analyzed by SDS-PAGE gel electrophoresis (FIG. 6A). The heavy chain molecular weight is around 50 kDa and light chain around 25 kDa. Having two copies of each, the molecular weight of 15E7 is estimated to 150 kDa confirming that the monoclonal antibody 15E7 belongs to the murine IgG 2a class.
[0293]Isotyping and Affinity Determination
[0294]The isotype of 15E7 was assessed by ELISA. 15E7 isotype was determined to be IgG2a. The affinity of 15E7 was assessed by the BLITZ technology as described in the Materials and Methods section. Representative data are shown in FIG. 6B. Various concentrations of the PC-1 peptide conjugated to BSA ranging from 5 to 600 nM were incubated with the 15E7 coupled to the biosensor chip. For each concentration, the association (ka) and dissociation (kd) rates were measured and used to calculate the affinity constant KD (ka / kd), evaluated here as 1.57 nM...
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