Patent Application: US-32214202-A

Abstract:
an isolated cdr - derived polypeptide from monoclonal antibody 13b8 . 2 , a pharmaceutical composition including a pharmaceutically acceptable carrier and at least a peptide , and a method for treating a subject suffering from an autoimmune disorder , including administering to the subject a therapeutically effective amount of a pharmaceutical composition , a method for treating a subject suffering from a transplant rejection including administering to the subject a therapeutically effective amount of a pharmaceutical composition , a method for treating a subject suffering from an hiv immunodeficiency disorder including administering to the subject a therapeutically effective amount of a pharmaceutical composition and a method for treating a subject suffering from a tumoral disorder including administering to the subject a therapeutically effective amount of a pharmaceutical composition .

Description:
we developed the concept of paratope - derived peptides ( pdps ) which correspond to short amino acid sequences derived from antibody variable regions and which display antigen binding and biological activities [ 35 - 38 ]. these small molecules are screened from a systematic exploration or antibody variable domain sequences by the spot method [ 39 , 40 ]. given the pharmaceutical interest of 13b8 . 2 mab , it appeared to us attractive to design such anti - cd4 pdps . to this end , we chimerized the 13b8 . 2 mab as a recombinant fab fragment expressed in the baculovirus / insect cell system . the recombinant chimeric fab 13b8 . 2 displays similar cd4 - binding and immunosuppresive properties as the parental mouse mab . these functional effects of chimeric fab 13b8 . 2 make it a good candidate for therapeutic purposes . therapies of particular interest include , but are not limited to , autoimmune disorder , transplant rejection , hiv immunodeficiency disorder and tumoral disorder . preferred autoimmune disorders include , for example , psoriasis , rheumatoid arthritis and lupus arythematosus . specifically , we identified nine pdps from the 13b8 . 2 variable regions by using the spot method . all the selected pdps , prepared in a soluble cyclic form , were able to bind histidine - tagged recombinant cd4 ( his 6 - scd4 ) expressed in baculovirus . mab 13b8 . 2 specifically displaced the binding of his 6 - scd4 to pdps cb1 and cb8 , indicating that anti - cd4 pdps recognize an epitope on the cd4 molecule closely related or similar to that identified for the 13b8 . 2 parental mab . pdp cb1 displayed biological properties very similar to those of the parental 13b8 . 2 mab , inhibiting in vitro antigen presentation and hiv - 1 promoter activation . taken together , we believe that the bioactive pdp cb1 , derived from the cdr - h1 region of the anti - cd4 13b8 . 2 mab , is valuable for anti - cd4 peptidomimetics . recombinant human soluble cd4 ( rhcd4 ) was obtained from repligen ( needham , mass ., usa ). rhcd4 was biotinylated using a commercial reagent ( amersham pharmacia biotech , cleveland , ohio , usa ) according to the manufacturer &# 39 ; s instructions and stored in pbs at − 20 ° c . until use . 13b8 . 2 mab [ 19 , 31 ] was obtained from immunotech - coulter ( marseille , france ). the murine hybridoma cell line that produces 13b8 . 2 mab ( igg1 / κ [ 19 ]) was provided by dr . d . olive and dr . c . mawas ( inserm u119 , marseille , france ). the pmv7 - t4 plasmid , encoding the full - length cd4 - cdna sequence [ 41 , 42 ], was provided by dr . q . j . sattentau ( centre d &# 39 ; immunologie de marseille - luminy , marseille , france ). the human lymphoblastoid b cell line ebv - lu , expressing the hla dr5 , 6 , drb52 , dq6 , 7 , and a2 molecules , and the murine t cell pdb10f , expressing human cd4 and pep24 ( pagfailkcnnktfny )- specific chimeric tcr , have been previously characterized [ 43 , 44 ] were provided by p . deberardinis ( consiglio nazionale delle ricerche , napoly , italy ). the hela p4 hiv - 1 ltr β - galactosidase indicator cell line [ 45 ] was provided by dr . o . schwartz ( institut pasteur , paris , france ). the nucleotide sequence of soluble cd4 ( d1 - d4 ) was sorted by pcr from the pmv7 - t4 plasmid , then cloned into the p119l baculovirus transfert vector to allow the expression of cd4 under the p10 promoter , as described elsewhere except that no histidine tag was inserted in the construction . transfection of sf9 cells and further expression of cd4 in baculovirus supernatant was performed [ 94 , 95 ]. an enriched - cd4 fraction was prepared following 80 % ammonium sulphate precipitation of the baculovirus supernatant and subsequent dialysis in a 0 . 1 × 160 mm pbs solution . construction of recombinant baculovirus producing the chimeric mouse / human anti - cd4 fab 13b8 . 2 the general procedures concerning the cloning and sequencing of 13b8 . 2 mab variable regions have been described [ 99 ]. two plasmid cassette - transfer vectors pbhuck and pbhufdγ1 were constructed that contain the human cκ gene [ 94 ] and the first domain of human cγ 1 ( fdγ 1 ), allowing the insertion and expression of variable heavy ( vh and kappa light ( vκ ) chains of the anti - cd4 mab 13b8 . 2 under the control of the polyhedrin and p10 promoter : pbhufdγ 1 was obtained by using the same procedure as that described by poul et al . [ 94 ] for the construction of the pbhucγ 1 plasmid vector except that a stop codon was inserted at the end of the gene encoding for the first domain of the cγ 1 constant region . a two - step recombination procedure [ 94 , 95 ] was carried out to construct the recombinant baculovirus , named 5756 , expressing both heavy and light chains of the chimeric fab 13b8 . 2 . a 400 ml - supernatant of spodoptera frugiperda sf9 cells ( atcc crl 1711 ) infected with the recombinant baculovirus 5756 in a spinner culture 10 6 cells / ml ) was recovered 96 - h post infection and precipitated with a saturated ammonium sulphate solution until 80 % saturation . after centrifugation at 10 , 000 g for 30 min , the pellet was dissolved in 160 mm pbs , ph 7 . 2 , and extensively dialyzed against pbs . the antibody solution , diluted v / v with 100 mm sodium acetate buffer , ph 5 . 0 , was filtered ( 0 . 22 μm ) and applied to a protein - g column )( pierce , rockford , ill .) which has been equilibrated with 100 mm sodium acetate buffer , ph 5 . 0 . bound recombinant fab 13b8 . 2 was eluted with 50 mm glyciner - hcl buffer , ph 2 . 5 , and immediately neutralized to ph7 with a 0 . 2 m tris solution , ph 10 . 5 . the protein content was monitored at 280 nm and purification fractions were checked for anti - cd4 activity by elisa as described below . samples were analyzed under reducing and non - reducing conditions on 12 . 5 % polyacrylamide gel , according to the laemlli procedure [ 100 ]. proteins were subsequently transferred to a nitrocellulose membrane ( hybond ecl , amersham pharmacia biotech ) and detected with a peroxidase - conjugated anti - human kappa chain ( sigma ) and a sheep anti - human fdγ 1 ( the binding site , birmingham , uk ) by using the ecl detection kit ( amersham pharmacia biotech ). peptide synthesis on cellulose membrane covering the d1 domain of cd4 the general protocol has been described previously [ 101 ]. by the spot method , we synthesized 98 overlapping dodecapeptides frameshifted by one residue , representing the d1 domain of the cd4 molecule , on a cellulose membrane . assay for recombinant fab 13b8 . 2 interaction with cellulose - bound peptides covering the d1 domain of cd4 the saturated membrane was incubated either with a 6 . 25 nm solution of mouse mab 13b8 . 2 or with a 50 nm solution of recombinant fab 13b8 . 2 for 2 h at 37 ° c . bound antibodies were detected with a 1 : 500 solution of either peroxidase - labeled anti - mouse igg conjugate ( sigma , saint louis , mo .) or peroxidase - labeled anti - human kappa chain conjugate ( sigma ), followed by ecl revelation ( amersham pharmacia biotech ). a 1 : 500 dilution of the enriched - cd4 fraction in 0 . 1 m carbonate / bicarbonate buffer , ph9 . 6 , was coated overnight at 4 ° c . onto 96 - well enzyme immuno assay plates ( nunc , paisley , united kingdom ). four washes with 160 mm pbs , ph 7 . 2 , containing 0 . 1 % tween 20 ( pbs - t ) were performed before and after saturating plates in 1 % nonfat powdered milk in pbs - t for 1 h at 37 ° c . thereafter , 100 μl of two - fold serial dilutions of the antibody solution was added to each well . following incubation of 2 h and four washes in pbs - t , bound antibodies were detected by addition of 100 μl of a 1 : 1000 solution of peroxidase - conjugate anti - mouse igg ( sigma ) or peroxidase - conjugated anti - human kappa chain ( sigma ), followed by subsequent addition of peroxidase substrate . absorbance was measured at 490 nm ( a490 ). for the inhibition of cd4 binding to mouse mab 13b8 . 2 by recombinant fab 13b8 . 2 , a similar elisa method was performed except that 13b8 . 2 mab , at a 6 . 25 nm concentration giving an a490 of 1 . 0 , was co - incubated with 2 - fold serial dilutions of recombinant fab 13b8 . 2 . three replicates were tested for each dilution with an initial fab concentration of 1 μm . cd4 / 13b8 . 2 mab residual binding was evaluated as described above . the kinetic parameters of the binding of cd4 to 13b8 . 2 antibody were determined by surface plasmon resonance analysis using a biacore instrument ( bi - acore ab , uppsala , sweden ). in an initial experiment , cd4 was immobilized on a cm5 sensorchip and 100 nm mab 13b8 . 2 or recombinant fab in hbs buffer ( 10 mm hepes ph 7 . 6 , 150 nm nacl ) were then injected . in a second experiment , 400 nm recombinant fab were immobilized on a cm5 sensorchip by using the anti - human fdγ 1 conjugate ( the binding site ). the binding kinetics were determined by injecting various concentrations of cd4 in hbs buffer . the kinetic parameters were calculated by using the bia evaluation 3 . 0 software and the so - called “ global ” method [ 102 ]. the ebv - lu antigen - presenting cells ( apc ) were maintained in rpmi medium ( biowhittaker , walkersville , md .) supplemented with 10 % fcs , 2 mm glutamine and 100 μg / ml penicillin / streptomycin ( sigma ). responder pdb10f t cells were maintained in dmem medium ( gibco , paisley , united kingdom ) supplemented with 10 % fetal calf serum ( fcs ), 50 nm 2 - mercaptoethanol , 10 nm hepes , 2 mm glutamine , 100 μg / ml penicillin / streptomycin and kept under selection with 400 nm methotrexate and 900 ng / ml puromicine ( sigma ). evb - lu cells ( 10 6 cells / ml ) were pulsed overnight at 37 ° c . with the pep24 stimulator peptide ( 75 μm ) from hiv gp120 [ 97 ]. cells were washed in pbs buffer without ca 2 + and mg 2 +( biowhittaker ) and plated at 10 5 cells / well . pdb10f reporter cells were washed with the same pbs buffer , diluted in dmem medium without methotrexate and puromycin to a final concentration of 4 × 10 5 cells / ml . 50 μl of cell suspension were plated onto ebv - lu cells . fifty microliters of inhibitor antibodies were then added to cells and antigen presentation was performed for 24 h at 37 ° c . thereafter , 100 μl of supernatant was harvested and tested for il - 2 secretion using a commercial elisa kit ( pharmingen , san diego , calif .). a positive control assay for il - 2 secretion was performed as described above except that the pdb10f cells were activated using a murine anti - cd3 antibody ( 0 . 6 nm , pharmingen ). hela p4 indicator cells ( 8 × 10 4 cells / ml ) were cultured in medium supplemented or not with infectious hiv - 1 lai in the presence or absence of antibodies for three days , harvested and lysed . β - galactosidase activity was then determined as previously described by measuring the absorbance at 410 nm [ 98 ]. expression , secretion and purification of the recombinant fab 13b8 . 2 from baculovirus - infected insect cells the nucleotide sequences of the vh and vl domains of 13b8 . 2 mab ( accession numbers aj279001 and 279000 ) have been previously established according to the general procedure described by chardè et al . [ 99 ]. genetic analysis of these sequences [ 99 ] showed that the vh region of 13b8 . 2 mab resulted from the rearrangement of vh2 - dq52 - jh3 genes and that the vl region resulted from a vκ12 / 13 - jκ2 gene rearrangement . in the recombinant baculovirus designated 5756 , the chimeric cκ - vκ 13b8 . 2 and fdγ 1 - vh13b8 . 2 genes are under the control of the very late polyhedrin and p10 promoters , respectively . recombinant fab 13b8 . 2 was protein - g immunopurified from 400 ml of supernatant , obtained 96 h post infection of insect cells with 5756 baculovirus ( fig1 ). the wash fractions 1 to 10 revealed a decrease in protein content with no detectable anti - cd4 activity , whereas eluted fractions 14 to 18 showed strong anti - cd4 activity in correlation with an increase in protein content . these fractions were further pooled for antibody analysis by coomassie blue sds - polyacrylamide gel electrophoresis ( sds - page ) and western blotting ( fig1 inset ). coomassie blue sds - page revealed a single band at 50 kda , corresponding to the expected size of a correctly - processed fab under non - reducing conditions ( fig1 lane 1 ). no other band was observed by coomassie blue staining , demonstrating the quality of the protein - g immuno purification . the identity of the 50 - kda band was confirmed by western blotting using anti - human k chain ( fig1 lane 2 ) or anti - human fdγ 1 chain ( fig1 lane 4 ). individually - expressed heavy and light chains were not detected by western blotting following non - reduced sds - page . reduction of the purified recombinant fab 13b8 . 2 generated one band of approximately 25 kda , detected by using anti - human κ chain ( fib . 1 , lane 3 ), and one band around 28 kda , detected by using anti - human fdγ 1 chain ( fig1 lane 5 ). taken together these results indicate that the anti - cd4 recombinant fab 13b8 . 2 produced in the baculovirus / insect cell system is correctly assembled and secreted in the supernarant as a dimeric hl complex . the yield of purified baculovirus - expressed fab 13b8 . 2 was about 5 mg / l . the ability of recombinant fab 13b8 . 2 to bind cd4 was assessed by an elisa method ( fig2 ) and by bia - core analysis ( fig3 ). by elisa , cd4 binding activity was demonstrated for a chimeric fab concentration in the 10 - 1000 nm range ( fig2 inset ), whereas no binding was obtained with the irrelevant baculovirus - expressed fab 1c10 ( data not shown ). control mab 13b8 . 2 displayed cd4 binding in the 1 - 100 nm range ( fig2 inset ). by using biacore technology ( fig3 ), cd4 / fab interaction was confirmed with a k d value of 3 . 3 nm , whereas the affinity of the parental mab was about 2 . 5 nm . this fab interaction was cd4 dose - dependent ( fig3 inset ). no measurable finding was obtained with the irrelevant fab 1c10 ( data not shown ). finally , the recombinant fab 13b8 . 2 was able to displace the binding the parental mab to cd4 in a dose - dependent manner ( fig2 ). a 50 % inhibition of binding was obtained for a fab concentration of 80 nm . these results indicated that the baculovirus - expressed fab 13b8 . 2 finds the cd4 molecule on the same region as the parental antibody . furthermore , the same cdr3 - like region 87edqkeevqllvfglta102 on the d1 domain of cd4 was identified as the binding region of the fragment and the intact antibody products , as shown by spot analysis ( fig4 ), definitively demonstrating that a similar epitope is recognized by the mouse parental antibody and the recombinant fab . no spot reactivity was observed with dodecapeptides covering the other regions on the d1 domain of cd4 . the stimulation of pdb10f responder t cells by pep24 - pulsed ebv - lu apc leads to the lymphocyte secretion of il2 [ 97 ]. this t cell activation model was found to be specific since no il - 2 secretion occurred when ebv - lu antigen - presenting cells were pulsed with a non - stimulator pep23 antigen . the viability of pdb10f responder cell line was checked by activating cells with a murine anti - cd3 mab which induced il - 2 secretion . as shown in fig5 ( a ), 7 . 8 nm of the irrelevant anti - digoxin mab 1c10 showed no inhibitory activity of il2 secretion in contrast to the same concentration of anti - cd4 13b8 . 2 mab which blocked the il2 production ( 95 . 7 ± 0 . 7 % inhibition ). as compared with the irrelevant recombinant fab 1c10 showing no inhibition , 200 nm of 13b8 . 2 fab displayed 61 . 3 ± 4 . 6 % inhibitory activity of il2 secretion . taken together , these results indicate that , as already demonstrated for other anti - cd4 mabs [ 103 ], the baculovirus - expressed fab 13b8 . 2 is able to inhibit the antigen - presenting function , a biological property also demonstrated for the 13b8 . 2 parental mab . in order to verify the ability of recombinant fab to inhibit hiv - 1 promoter activity , as the parental 13b8 . 2 mab does , we measured the β - galactosidase reporter gene expression after infection of the indicator cell line hela p4 cultured for three days in the presence of products . as shown in fig5 ( b ), 7 . 8 nm of 1c10 mab did not display any inhibitory activity in contrast to the same concentration of aprental 13b8 . 2 mab which inhibited hiv promoter activation ( 60 . 3 ± 5 . 0 % inhibition ). culturing infected hela p4 cells with irrelevant baculovirus - expressed fab 1c10 did not affect the β - galactosidase expression , whereas significant inhibition ( 61 . 2 ± 4 . 5 %) was found with 200 nm of the anti - cd4 recombinant fab 13b8 . 2 . these results indicate that the baculovirus - expressed fab 13b8 . 2 showed anti - viral property , as already demonstrated for the parental anti - cd4 mab 13b8 . 2 [ 85 , 86 ]. we prepared a chimeric recombinant anti - cd4 fab expressed in baculovirus that mimics the biological properties of the parental 13b8 . 2 antibody . such a functional anti - cd4 antibody provides for use of the recombinant fab in mammals . the chimeric mouse - human fab 13b8 . 2 was able to recognize a cdr3 - like region in the d1 domain of cd4 , comprising glu87 and asp88 residues previously described by site - directed mutagenesis as involved in the 13b8 . 2 epitope [ 83 , 104 ]. il2 secretion of activated t cells upon antigen presentation can be inhibited by the chimeric fab , indicating that the baculovirus - expressed recombinant molecule showed immunosuppressive property classically observed for anti - cd4 antibodies [ 103 ]. as previously reported for the mouse mab 13b8 . 2 [ 85 ], the recombinant anti - cd4 fab was able to prevent hiv - 1 promoter activation . this activity was probably related through the inhibition of the erk / mek signaling pathway as already demonstrated for the 13b8 . 2 mab [ 105 ]. taken together , these results indicate that the fab has retained a major part of the parental 13b8 . 2 mab properties and can be used without the side - effects of a mouse mab . this is especially beneficial in pharmaceutical compositions . the mab 13b8 . 2 was shown to block gp120 binding to cd4 , inhibit hiv - induced cell fusion and prevent viral production by infected cells [ 82 ]. inhibition of viral gene transcription in hiv - cell culture and blockade of viral production from chronically - infected cells have been demonstrated following in vitro treatment with 13b8 . 2 antibody , these activities being obtained with hiv - 1 and hiv - 2 virus isolates [ 85 ]. in addition , the 13b8 . 2 mab was able to elicit gp120 - specific idiotypic antibody response in rabbits , thereby inhibiting syncitium formation [ 106 ]. these results led to experiments using mouse 13b8 . 2 antibody for hib - 1 - infected patients [ 87 - 89 ]. although these experiments led to clinical benefit for the patients , i . e ., disappearance of circulating p24 together [ 87 , 89 ] with negativation of the reverse transcriptase assay [ 87 ] or generation of serum antibodies to gp120 and hiv - 1 neutralizing antibodies [ 89 ], adverse effects such as an anti - allotype or - isotype response to the foreign antibody [ 87 , 89 ] or cd4 + clearance by the fc portion of the antibody [ 87 , 89 ] have also been documented . in sharp contrast , the chimeric fab of this invention , showing antiviral activity like 13b8 . 2 mab [ 83 , 85 , 86 ], is a valuable tool to overcome such problems . some anti - cd4 antibodies have already been used in the treatment of various autoimmune diseases or allograft rejection [ 107 - 110 ]. early clinical trials using murine anti - cd4 mabs [ 111 ] have often been discouraging , with mild tolerance of the antibody preparation and a systematic human anti - mouse response . this latter side - effect leads to the clearing of the infused mab and induction of anaphylactic responses [ 112 ]. one way to overcome these undesirable effects has been the generation of human mab from human immunoglobulin transgenic mice [ 113 ]. several strategies using dna technology have also been described , such as primatization in which anti - cd4 variable regions ( v - regions ) from antibody generated in macaques are fused to human constant regions [ 114 ], humanization by grafting of murine anti - cd4 cdrs inside human antibodies [ 92 ], chimerization in which murine anti - ocd4 v - regions are co - expressed with human constant domains [ 91 ]. these re - designed recombinant anti - cd4 molecules demonstrated efficient immunosuppressive activity for treatment of autoimmune diseases or transplant rejection and were shown to be devoid of the adverse side - effects of mouse antibodies . in a similar manner , the engineered chimeric anti - cd4 fab 13b8 . 2 , which inhibits antigen presentation , is a potent immunosuppressive agent that can be used against autoimmune diseases or for allograft rejection . furthermore , the fab format could overcome prolonged cd4 + cell depletion , a negative effect often described following in vivo treatment with whole anti - cd4 antibody [ 115 , 116 ]. this effect has been attributed to synergy between complement binding capacity and fe receptor binding on phagocytic cells [ 114 ]. since a fab molecule lacks the second and third domains of the heavy chain constant region which bear complement and fc binding abilities , the chimeric fab fragment might be devoid of cell - depleting potential , as is also the case for f ( ab )′ 2 fragments which block immune response to coadministered antigens and prevent the development of spontaneous autoimmune conditions [ 117 ]. our anti - cd4 recombinant fab probably acts as a pure receptor antagonist , either blocking cd4 receptor function or modulating the cd4 molecule on the t lymphocytes . the baculovirus / insect cell expression system is an interesting way to produce antibodies for therapeutic purposes [ 118 ] because of its capability for a high production level [ 119 ] and the absence of known intrinsic or secreted molecules toxic for man . this is in contrast with e . coli which can release endotoxins or plants which contain toxic or allergenic compounds [ 119 ]. in addition , the ability of insect cells to grow without serum avoids the presence of mammalian contaminants [ 119 ], leading to safe and secure antibody preparation . furthermore , the development of baculovirus surface display [ 120 - 124 ] coupled with a non - lytic insect cell expression system [ 125 ] might increase the applicability of this alternative eukariotic source for the generation of human antibodies . the baculovirus / insect cell expression system allowed the production and purification of active recombinant fab directed against the cdr3 - like region of the d1 domain of cd4 . this chimeric molecule retained the cd4 binding activity and the immunosuppressive properties of the parental mab 13b8 . 2 , thereby demonstrating its usefulness for pharmaceutical compositions . cloning and sequencing of 13b8 . 2 mab v h and v l genes the general procedures concerning the cloning and sequencing of 13b8 . 2 mab variable regions have been described [ 46 ]. 202 overlapping dodecapeptides frameshifted by one residue representing the v h and v l sequences of 13b8 . 2 mab on a cellulose membrane were synthesized according to previously described protocols [ 37 ]. the saturated membrane was incubated with a 20 nm solution of biotinylated - rhcd4 for 2 h at 37 ° c . bound biotinylated - rhcd4 was detected by incubating the membrane for 1 h at 37 ° with a 1 : 3000 solution of alkaline phosphatase - conjugated streptavidin ( sigma , st . louis , mo ., usa ) and subsequent addition of 5 - bromo - 4 - chloro - 3 - indolyl phosphate substrate . inhibition of biotinylated - rhcd4 binding was performed as described above , except that biotinylated - rhcd4 ( 20 nm ) was pre - incubated for 18 h at 4 ° c . with 13b8 . 2 mab ( 6 . 25 μm ). in all cases , the reactivity of the spots was evaluated by scanning the membrane and measuring the intensities of the spots with the nih image 1 . 61 software [ 39 ]. the nine selected pdps , named cb1 to cb9 ( fig6 right panel for sequences ), a scrambled form of pdp cm9 ( sccm9 : gsdqwnkmqyyp ) [ 35 ], a scrambled form of the cd4 - derived cdr3 - like peptide ( sccdr3 - like : keeicevedqty ), and an unrelated peptide ( dig97c : fgdyyclqyass , derived from the cdr - l3 region of the anti - digoxin 1c10 mab ), with lys - cys residues added to both carboxyl - and amino - termini of all peptides , were synthesized by fmoc solid - phase synthesis on an ams422 robot ( abimed , langelfeld , germany ), cyclized and purified as described previously [ 35 ]. lys and cys residues were added , respectively , to improve the solubility and permit cyclization of the peptides . the peptides showed homogeneity in high performance liquid chromatography at the expected monomeric molecular weight . thereafter , the peptides were resuspended in deionized water , except for the sccdr3 - like peptide which was suspended in a 10 % acetonitrile solution and the cb8 pdp in a 20 % acetonitrile solution . the nucleotide sequence of soluble cd4 ( d1 - d4 ) was sorted by polymerase chain reaction from the pmv7 - t4 plasmid by using the sense primer scd4fb ( 5 ′- gaagatctatgaaccggggagtcc ), which matches codons 1 to 6 , and the anti - sense primer scd4rtb ( 5 ′- gaagatcttcaatggtgatggtggtggtgacctaatgcggccattggctgcaccggg ), which contains the reverse complement of codons 367 to 372 of cd4 and encodes the his 6 - tag and a bglii restriction site . following sub - cloning into the pgem - t vector ( promega , madisoin , wis ., usa ), the scd4 sequence was verified by using the dideoxy termination method with the t7 sequencing kit ( pharmacia , uppsala , sweden ). the bglii - linearized his 6 - scd4 fragment was cloned into the p119l baculovirus transfer vector to allow the expression of his 6 - scd4 under the p10 promoter . after transfection of spodoptera frugiperda sf9 cells ( atcc crl 1711 ), recombinant baculoviruses were further purified by using a plaque assay and propagated in sf9 cells [ 47 , 48 ]. supernatant of sf9 cells infected with the his 6 - scd4 recombinant baculovirus in a spinner culture ( 10 6 cells / ml ) were harvested 6 days post - infection and clarified by centrifugation at 1000 × g for 5 min . purification of the his 6 - scd4 product was carried out by using ni - nta agarose beads ( qiagen , chatswroth , calif ., usa ) according to the manufacturer &# 39 ; s procedure with minor modifications . briefly , the clarified baculovirus supernatant was dialyzed against washing buffer ( 50 mm nah 2 po 4 , 500 mm nacl , 5 mm imidazole ) for 24 h at 4 ° c . ni - nta agarose beads were then added to a final concentration of 5 %, and the binding of his 6 - scd4 was performed for 18 h at 4 ° c . beads were washed with 8 volumes of washing buffer and his 6 - scd4 was eluted as 1 ml fractions with 3 volumes of elution buffer ( 50 mm nah 2 po 4 , 500 mm nacl , 300 mm imidazole ). the ni - nta agarose beads were regenerated with 1 m imidazole . all purification fractions were checked for the presence of his 6 - scd4 by enzyme - linked immunosorbent assay ( elisa ) and western blot using the anti - cd4 13b8 . 2 mab as detection reagent . three replicates corresponding to 10 - fold serial dilutions of the nine cyclic pdps ( cb1 - cb9 ) were coated overnight at 4 ° c . onto 96 - well elisa plates ( nunc . paisley , uk ) with an initial peptide concentration of 50 μm . four washes in 160 mm pbs , ph 7 . 2 , containing 0 . 1 % tween 20 ( pbs - t ) were performed before and after saturating the wells with 1 % non - fat dry milk in pbt - t for 1 h at 37 ° c . thereafter , 100 μl of 20 nm his 6 - scd4 was added to each well . following a 2 h incubation and four washes in pbs - t , bound his 6 - scd4 was detected by addition of 100 μl of a 1 : 2000 solution of peroxidase - conjugated anti - his 6 mab ( sigma ) and subsequent addition of peroxidase substrate . absorbance was measured at 490 nm . inhibition of his 6 - scd4 binding to pdps was performed by an elisa method with pdps cb1 and cb8 coated at 12 . 5 and 2 . 5 μm , respectively , as capture reagent . a 20 nm solution of his 6 - scd4 showing an absorbance at 490 nm of 1 . 0 was co - incubated with two - fold serial dilutions of 13b8 . 2 mab . three replicates were tested for each dilution with an initial mab concentration of 3 μm . his 6 - scd4 binding was evaluated as described above . ebv - lu antigen - presenting cells ( 10 6 cells / ml ), overnight pulsed with the pep24 stimulator peptide ( 75 μm ) from hiv gp120 [ 44 ], were co - cultured with pdb10f responder cells ( 4 × 10 5 cells / ml ) in the presence or absence of inhibitor pdps or mabs for 24 h . thereafter , 100 μl of supernatant was harvested and tested for il - 2 secretion using an elisa commercial kit ( pharmingen , san diego , calif ., usa ). a positive control for il - 2 secretion was performed as described above except that activation of pdb10f cells was done using a murine anti - cd3 antibody at a concentration of 0 . 6 nm ( pharmingen ) [ 49 ]. hela p4 indicator cells ( 8 × 10 4 cells / ml ) were cultured in medium supplemented or not with 1000 tcid 50 of infectious hiv - 1 lai in the presence or absence of peptides or mab for 3 days , harvested and lysed . the β - galactosidase activities were then determined as previously described [ 45 ] by measuring the absorbance at 410 nm . characterization of nine peptides from 13b8 . 2 mab demonstrating cd4 binding ability the nucleotide sequences of vh and vl domains of 13b8 . 2 mab were established according to the general procedure described by chardès et al . [ 46 ] and made available from the embl database under the accession numbers aj279001 and aj27900 , respectively . the complete amino acid sequences of both chains are given in fig6 with somatic mutations indicated . genetic analysis of these sequences showed that the v h region of 13b8 . 2 mab resulted from the rearrangement of v h 2 - dq52 - j h 3 genes and that the v l region resulted from a v κ 12 / 13 - j κ 2 gene rearrangement . more precisely , computer - assisted comparison of these sequences showed that the v h and v l genes of 13b8 . 2 displayed significant homologies with ox2 [ 50 ] and k2 [ 51 ] germline genes , respectively ( fig6 ). it is worth noting that no significant homology was found between 13b8 . 2 mab variable sequences and other anti - cd4 variable domains . 202 overlapping dodecapeptides frameshifted by one residue , corresponding to the deduced amino acid sequence of v h and v l from 13b8 . 2 mab , were synthesized on a cellulose membrane by using the spot method . the anti - cd4 immunoreactivity of these peptides was assessed by incubating the membrane with biotinylated - rhcd4 . the results are quantitatively expressed in fig7 ( left panel ) in which the reactivity of peptides that comprise at least one residue from the cdrs are boxed . anti - cd4 reactivity was observed for peptides including amino acids from five of the six cdrs of 13b8 . 2 mab ( peptides 20 , 22 , 28 - 35 ; 46 , 48 - 52 , and 93 - 97 for cdr - h1 , cdr - h2 and cdr - h3 , respectively , and 22 , 23 , 29 - 34 ; 83 - 89 and 91 for cdr - l1 and cdr - l3 , respectively ). anti - cd4 activity was also obtained for peptides containing residues from the framework , mainly flanking the cdrs ( peptides 18 , 36 - 38 and 66 - 71 for v h , and 35 - 38 and 57 - 61 for v l ) but the majority of peptides comprising only framework residues did not display any significant binding activity . this reactivity was drastically decreased when biotinylated - rhcd4 was pre - incubated with an excess of 13b8 . 2 parental mab . nine peptides ( 29 , 30 , 48 , 52 , 90 and 94 for v h and 22 , 29 and 86 for v l ), named cb1 to cb9 ( fig2 right panel ), were selected for further study in a soluble form . these peptides , except for 22 and 29 from cdr - l1 , showed the highest anti - cd4 activity and comprised at least 50 % of residues belonging to cdrs . since the most reactive peptides derived from the cdr - l1 showed less than 50 % of residues from the cdr ( peptides 31 and 34 ), we selected two adjacent reactive peptides , namely , 22 and 29 . except for pdp cb4 , that exclusively comprised amino acids from the cdr - h2 , all selected pdps comprised amino acids from both cdr and framework sequences . peptides selected according to the spot results were synthesized and n - to c - terminus cyclized through cysteine oxidation . cyclization has already been demonstrated as a useful tool to improve antigen binding [ 36 ]. the ability of recombinant his 6 - scd4 expressed in baculovirus to specifically bind cyclic pdps was assessed by elisa ( fig8 ). the selected pdps , except cb3 from the cdr - h2 , showed a dose - dependent cd4 binding activity . pdps cb1 and cb2 derived from the 13b8 . 2 cdr - h1 region , and pdp cb8 derived from the 13b8 . 2 cdr - l1 region , displayed high binding activity in a 0 . 5 - 50 μm concentration range . the non - reactivity of irrelevant sccdr3 - like and sccm9 peptides indicated that the addition of lysine and cysteine residues for solubilization and cyclization of peptides had no effect on cd4 binding . since pdps cb1 and cb2 only differ by one amino acid residue , we focused our attention on pdp cb1 , derived from the cdr - h1 , and pdp cb8 , derived from the cdr - l1 of 13b8 . 2 mab for further specificity studies . pdps cb1 and cb8 specifically bind to cd4 on the same region as that of parental 13b8 . 2 mab the ability of the parental mab 13b8 . 2 to displace the binding of pdps cb 1 and cb8 to his 6 - scd4 was studied by using an elisa inhibition assay . the absorbance of residual his 6 - scd4 binding to pdps was measured at 490 nm and expressed as percent inhibition of the binding ( fig9 ). we found that 13b8 . 2 mab was able to displace the binding of his 6 - scd4 ( 20 nm ), in a dose - dependent manner , to both coated pdps cb1 ( 12 . 5 μm ) and cb8 ( 2 . 5 μm ) with similar efficiencies . a 50 % inhibition of binding pdps cb1 and cb8 to cd4 was obtained for 13b8 . 2 mab concentrations of 20 and 8 nm , respectively . no inhibition was found when using the igg1 isotype - unrelated anti - digoxin mab 1c10 , demonstrating the specificity of the competition studies . similar results were obtained in a symmetric experiment in which the binding of his 6 - scd4 ( 20 nm ) to 13b8 . 2 mab ( 0 . 3 nm ) was inhibited by various concentrations of pdps cb1 and cb8 . a 50 % inhibition of binding of his 6 - scd4 to 13b8 . 2 mab wase obtained for concentrations of pdps cb1 and cb8 up to 75 and 125 μm , respectively . taken together , these data are consistent with the hypothesis that 13b8 . 2 mab , cb1 , and cb8 peptides recognized the same antigenic region on the cd4 molecule . pdp cb1 is able to inhibit il - 2 secretion following antigen presentation the stimulation of pdb10f responder t cells by pep24 - pulsed ebv - lu apc leads to the lymphocyte secretion of il2 [ 43 , 44 ]. this t cell activation model is specific since no il - 2 secretion occurs when ebv - lu antigen - presenting cells are pulsed with a non - stimulator pep23 antigen . we checked the viability of pdb10f responder cell line by activating cells with a murine anti - cd3 mab which induced il - 2 secretion ( data now shown ). as shown in table 1 , the irrelevant anti - digoxin mab 1c10 showed no inhibitory activity of il2 secretion in contrast to the anti - cd4 13b8 . 2 mab which blocked the il2 production ( 99 . 6 ± 0 . 2 % inhibition ). as compared to the irrelevant sccm9 peptide showing no inhibition , pdp cb1 displayed inhibitory activity of il2 secretion in a 125 - 250 μm concentration range . the biological activity of pdp cb2 was found to be very moderate since no activity was found at a concentration lower than 250 μm . the lack of activity for pdp cb8 was found to be consecutive to cell death , probably due to the presence of 20 % acetonitrile in the buffer used to solubilize the peptide . the other selected pdps demonstrated no or extremely low blocking of il2 secretion . taken together , these results indicate that , as already demonstrated for other anti - cd4 mabs [ 52 , 53 ], the cdr - h1 - derived pdp cb1 is able to inhibit the antigen - presenting function , a biological property also demonstrated for the 13b8 . 2 parental mab . pdp cb1 displays a strong capacity to inhibit hiv - 1 lai ltr - driven β - galactosidase reporter gene expression the parental mab 13b8 . 2 has been previously demonstrated to be an inhibitor of viral particle production by cells infected with hiv - 1 lai , hiv - 1 eli , hiv - 1 sf2 , hiv - 1 ger and hiv - 2 rod strains [ 13 , 54 ]. in addition , viremia negativation has been observed for hiv - infected patients treated with 13b8 . 2 antibody , demonstrating its efficiency towards primary clinical isolates [ 31 , 32 ]. in order to assess the ability of selected 13b8 . 2 pdps to inhibit hiv - 1 promoter activity , we measured the β - galactosidase reporter gene expression after infection of the indicator cell line hela p4 cultured for 3 days in the presence of peptides . as shown in fig1 a , the 1c10 mab did not display any inhibitory activity in contrast to the parental 13b8 . 2 mab which inhibited hiv promoter activation . culturing infected hela p4 cells with irrelevant dig97c or sccm9 peptides did not affect β - galactosidase expression , whereas a significant inhibition was found when the cells were cultured with a 50 μm solution of 13b8 . 2 pdps cb1 , cb2 and cb5 . the lack of activity for pdp cb8 was found , in this assay , also to be consecutive to cell death caused by the presence of 20 % acetonitrile in the buffer used to solubilize the peptide since no inhibition was observed with the pdp cb1 diluted in the same 20 % acetonitrile buffer . in fig1 b , we demonstrated that the inhibitory effect of cb1 is dose - dependent , with an ic50 for cb1 of about 15 μm , whereas the ic50 of the parental mab was found to be 5 nm . these results indicate that the cdr - h1 - derived dodecapeptide cb1 has anti - viral activity , as already demonstrated for the parental anti - cd4 13b8 . 2 mab . the cd4 molecule plays a key role both in the mhc class ii - restricted immune response and the human immunodeficiency virus infection process by acting as a receptor either for the tcr - antigen engagement complex or for the envelope glycoprotein gp120 of hiv [ 7 , 9 , 55 , 56 ]. in these two cases , cd4 has been demonstrated to induce signal transduction leading to t cell activation [ 12 , 19 , 21 , 22 ]. both of these mechanisms can be inhibited by treatment with anti - cd4 mabs including murine 13b8 . 2 mab [ 28 - 30 ]. such inhibitory properties have lead to the use of 13b8 . 2 mab in hiv - infected patients [ 31 - 33 ]. to avoid problems encountered when using mabs in therapeutic approaches , such as immunogenicity and low tissue diffusion , we designed and synthesized pdps from the 13b8 . 2 anti - cd4 mab . we demonstrated that the cdr - h1 - derived pdp cb1 displays significant and specific biological properties mimicking those of the parental anti - cd4 13b8 . 2 mab . first , in an in vitro model of mhc class ii - restricted immune response , we demonstrated that anti - cd4 pdp cb1 , as well as parental mab 13b8 . 2 , inhibits il2 secretion by activated t cells following antigen presentation . this inhibitory effect , classically observed for anti - cd4 mabs [ 52 , 53 ], was dose - dependent . since anti - cd4 mabs have been described to prevent t cells from il2 - induced proliferation and b cell adhesion through inhibition of ca 2 + and p21 ras signaling pathways [ 57 , 58 ], it remains to be assessed whether our anti - cd4 pdps could interfere with such mechanisms . the effect of cb1 in an in vivo model of immune disorder remains to be investigated , as it was done for anti - cd4 cdr3 - like - derived analogs in a murine experimental allergic encephalomyelitis model [ 59 ]. second , we found that the pdp cb1 inhibited hiv - 1 promoter activation , as 13b8 . 2 mab does [ 29 ]. the mechanism by which 13b8 . 2 mab exerts this anti - hiv property has been related to inhibition of signal transduction , involving the extracellular regulated kinase / mitogen - activated protein kinase kinase signaling pathway [ 13 ]. we also believe that cb1 acts by disrupting the same signal transduction machinery , thereby preventing hiv pro - virus transcription . we found that peptides cb1 and cb8 displayed specific anti - cd4 binding activity . a relatively high concentration of peptides is preferred to achieve efficient biological activity with regard to the efficient antibody dose . to explain this difference , we measured the binding of cd4 to pdp cb1 and observed a 50 % decrease in binding when following pre - incubation of the peptide for 25 min at 37 ° in a buffer containing 10 % fetal calf serum ( data not shown ). this finding strongly suggests that the high concentrations of cb1 which achieve a biological effect reflects degradation of the peptide in the culture medium . we believe that use of d - amino acids for peptide synthesis would improve their metabolic stability [ 60 ]. we believe that a longer sequence of pdp cb8 including hydrophilic flanking residues will improve the solubility of the peptide and avoid the use of organic solvent . this is of great interest because of a high sequence / position homology between this cdr - l1 - derived pdp cb8 and a previously characterized cdr - l1 - derived pdp , the cm9 peptide , derived from the anti - cd4 st40 mab that inhibits hiv transcription [ 35 ]. comparison of alanine - scanning analysis of these two peptides indicated that similar residues located at the same positions , i . e . tyr 32 , trp 35 , tyr 36 , lys 39 , contribute to cd4 binding [ 36 ]. this may help us understand the structure - function relationship in these series of anti - cd4 mab - derived bioactive molecules . the systematic exploration of the 13b8 . 2 mab paratope has led us to the characterization of pdps cb1 and cb8 , displaying high anti - cd4 reactivity and including residues from the cdrs and from the framework flanking these cdrs . the role of residues outside the cdrs ( i . e . trp 36 and arg 38 in the cb1 sequence , and tyr 36 in the cb8 sequence ) has already been described as being important in structuring the active cdr loops in the full mab paratopes [ 61 , 62 ]. moreover , park et al . [ 63 ] demonstrated the relevant importance of adding aromatic residues to improve the efficiency of their cdr - h3 - derived anti - her2 / neu peptide mimetics . the addition of aromatic residues has been found to be a valuable strategy for enhancing stability , folding , and avidity of peptidomimetics [ 25 , 64 - 66 ]. this could explain the high reactivity obtained for pdp cb1 , in which the natural trp 36 residue may have such function . we demonstrated differences , both in anti - cd4 binding activity and in biological properties , between pdps cb1 and cb2 , that are frameshifted by only one residue . two key points may explain these differences . first , pdp cb2 contains the pro 41 residue which may constrain the peptide in an unfavorable conformation . second , in pdp cb2 the leu 29 residue is absent . the leu 29 residue is part of the vernier zone [ 67 ], already described to stabilize ag / ab interactions . these two factors may contribute to twisting pdp cb2 into a less favorable conformation for cd4 binding , thereby explaining the decreased bio - logical properties of cb2 . taken together , these data confirm the capacity of the spot method in defining antigen - specific peptides derived from a mab paratope that present paratope - derived residues in an environment compatible for antigen binding . the cdr - 3 - like loop of domain 1 of cd4 , and more precisely the negatively charged residues glu 91 and glu 92 , has been shown to play a role in activating t cell signal transduction [ 20 , 21 ]; glu 91 and glu 92 are also involved in the 13b8 . 2 paratope [ 16 ]. inhibition studies demonstrated that pdp cb1 and 13b8 . 2 mab specifically compete for cd4 binging on the same region of the molecule . alanine scanning of the cb1 sequence showed that the main contributor residues are positively charged ( i . e ., his 35 and arg 38 , unpublished data ), thereby possibly interacting with its negatively charged epitope on the cd4 molecule . the cdr3 - like loop from the d1 domain of cd4 has been reported to be involved in on of the two potential cd4 dimerization sites [ 20 , 21 , 68 , 69 ]. the dimerization / oligomerization processes have been shown to be necessary for optimal activation of cd4 + t lymphocytes [ 16 , 26 , 70 - 72 ]. it is , therefore , possible that the inhibitory effects of pdp cb1 on t cell stimulation and hiv - 1 promoter activation can result from a cd4 dimerization / obligomerization disruption , thereby uncoupling the cd4 molecule from the signal transduction machinery . even if this molecular mechanism remains to be established , our approach has led to the characterization of the anti - cd4 pdp cb1 . this kind of small bioactive molecule is useful for more potent and stable molecules of pharmaceutical interest . 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