Patent Application: US-52807303-A

Abstract:
the invention relates to the development of a novel technology of specific antibodies entering into the cell and their use for treatment of human diseases . such antibody would act by directly modulating the cancer - generating signals . the expected effects and principles of action of such antibodies are inactivation of intracellular proteins and thus they could be used for the treatment of diseases , where the activity of intracellular proteins must be modulated for effective treatment . above - mentioned antibody technology would also be applied elsewhere .

Description:
we have produced monoclonal antibodies against gli1 and gli3 proteins . we have conducted preliminary studies and demonstrated that antibodies coupled with cell penetrating transport peptides , are able to effectively penetrate the cell membranes and that the coupling of such peptides to antibodies does not reduce the ability of the antibodies to recognise specific antigens . polyclonal antibodies recognising the gli1 protein were obtained by immunisation of rabbits by using the gli1 ( 1 - 407 ) antigen expressed in bacteria by using standard methods . the antibodies obtained were characterised by using the western blot analysis , electrophoretic mobility shift assay ( emsa ) and immunohistochemical methods . polyclonal antibodies recognising the gli3 protein were obtained by immunisation of rabbits by using the gli3 ( 150 - 250 ) antigen expressed in bacteria and by using standard methods . the antibodies obtained were characterised by using the western blot analysis , electrophoretic mobility shift assay ( emsa ) and immunohistochemical methods . continuation of peptides entering into the cell to polyclonal gli1 antibodies the cpp ( transportan tp10 ), the shorter analogue of transportan , was synthesised in 0 . 1 mmol scale on the applied biosystem model 430a peptide synthesizer using the dicyclohexyl carbodimid / hydroxy - benzo - triazole ( dcc / hobt ) activation . peptides were cleaved from the resin according to the tfmsa cleavage protocol . resulting peptide was further purified on c 18 reversed - phase hplc column that yielded & gt ; 99 % pure product . the molecular mass of each synthetic peptide was determined by maldi - tof mass spectrometry and the obtained result was compared with the calculated molecular mass . transportan 10 ( tp10 ), the shorter analogue of transportan , was conjugated to polyclonal antibodies . fig1 shows the conjugation of cell penetrating peptides to antibodies , which was carried out as follows : 1 ) cpp was derivatized into maleimid . smcc solution ( succinimidyl 4 -( n - maleimidomethyl ) cyclohexane - 1 - carboxylate , mw 334 ; 1 / 5 molar ratio ) was added to 200 ml of peptide solution in phosphate buffer ( ph 7 . 5 ; 10 mg peptide / ml ). the mixture described above was incubated for 1 - 2 hours at room temperature . the smcc residue was removed by using the hpcl reverse - phase c 18 column . 2 ) in order to deprotect the thiol groups on the antibody , tcep ( tris ( 2 - carboxyethyl ) phosphine hydrochloride ; mw 287 ) in 1 / 5 molar ratio , was added to the antibody solution ( phosphate buffer , ph 7 . 5 ) and the reaction mixture was incubated for 15 minutes . 3 ) conjugation of maleimid - derivatized peptide to the antibody . above - mentioned maleimid - derivatized peptide and antibody solution was combined in an equimolar ratio and incubated at room temperature for 3 hours , yielding a thioester bond between the antibody and the peptide . resulting preparation was used in further experiments , estimating that the conjugative effect was 80 %. peak 7 corresponded to the calculated molecular mass of tp - 10 shown on fig1 a , as demonstrated by the maldi - tof mass spectrometry . the conjugate obtained was able to enter the eukaryotic cells in culture ( fig2 ). fig2 a shows the mouse antigli igg - tp10 conjugate incubated for 3 hours with cos - 1 cells ; anti - gli1 igg visualised with fitc conjugated mouse anti - igg antibody . fig2 b shows the mouse fitc conjugated anti - igg , which was incubated for 3 hours with cos - 1 cells . fig2 c shows the mouse anti - gli1 ( igg )- tp10 conjugate incubated for 14 hours with cos - 1 cells ; anti - gli1 igg visualised with fitc conjugated mouse anti - igg antibody . fig2 d shows the mouse fitc conjugated anti - igg , which was incubated for 14 hours with cos - 1 cells . the above - mentioned polyclonal antibodies specifically recognised the gli1 protein . monoclonal antibodies recognising the gli1 protein were obtained by immunisation of mice with gli1 ( 1 - 407 ) protein as an antigen . the protein was expressed in bacteria according to the standard protocol ( antibodies : a laboratory manual ; ed . harlow , david lane ; cold spring harbor laboratory press , isbn : 0879693142 ). the spleens from immunised mice were dissected and the spleen cells were fused with sp 2 . 0 myeloma cells by using standard methods ( antibodies : a laboratory manual ; ed . harlow , david lane ; cold spring harbor laboratory press , isbn : 0879693142 ). clones from 40 hybridomas were separated . the resulting antibodies were characterised by western blot analysis , electromobility shift assay ( emsa ) and immunohistochemical methods . monoclonal antibodies recognising the gli3 protein were obtained by immunisation of rabbits with gli3 ( 150 - 250 ) antigen . the protein was expressed in bacteria and according to standard methods ( antibodies : a laboratory manual ; ed . harlow , david lane ; cold spring harbor laboratory press , isbn : 0879693142 ). the resulting antibodies were characterised by western blot analysis , electromobility shift assay ( emsa ) and immunohistochemical methods . for obtaining a recombinant cell penetrating protein we created expression vector encoding for gst - gli3 ( 150 - 250 ) fusion protein . we used pcr based approach to add the sequences encoding for cell penetrating peptides transportan tp10 and 9arg ( 9arginine ) into previously mentioned vector . these expression constructs were sequenced . expression of these constructs showed that despite repeated efforts , it was not possible to express a recombinant fusion protein that encoded gst - gli3 ( 150 - 250 )- transportan tp10 sequence described above in e . coli system . we succeeded , though , in expressing and purifying a recombinant protein that encoded the recombinant gst - gli3 ( 150 - 250 )- 9arg cell penetrating peptide ( fig3 a ). as we have demonstrated on fig3 b , the obtained recombinant protein entered the cultured mammal cells . obtaining and characterisation of anti gli recombinant proteins entering into the cell the recombinant antibodies were obtained by inserting the sequence encoding for the 9arg peptide or transportan or transportan tp10 into the gene encoding the clones of antibodies described above . the obtained recombinant antibodies were purified using affinity chromatography and antibody titre was determined . we demonstrated that these antibodies were binding specifically to the gli1 protein . these recombinant antibodies also entered into the eukaryotic cells in culture . in order to obtain the scfv with ability to penetrate into the cell we made a construct encoding for single chain antibody , or scfv , containing the two variable domains of an antibody molecule ( the vl and the vh domain ) linked via flexible peptide linker that also contained the sequence of cpp . the rnas from the anti gli1 and 3 monoclonal antibodies were reverse transcribed and this first cdna strand was used as a template for variable regions amplification using degenerated primers : pcr products of the appropriate size ( 320 - 350 bp ) were purified and sequenced . oligonucleotide primer encoding for transportan or transportan tp10 and linker ( gly4ser ) 3 was used to construct a vl - tp - linker - vh sequence by three - step overlap extension pcr . the process was repeated for scfvfc construction with relevant vlcl and vhch1 pcr products . the final pcr products corresponding scfv and scfvfc ( both with cpp and linker encoding ) sequence were cloned into eukaryotic expression vector ( pcdna3 , pcep ) and sequenced . eucaryotic cells ( cos - 7 ) were be transfected with scfv or scfvfc constructs and according to the manufacturer &# 39 ; 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