EP2212353A1 - Fragments d'anticorps inhibiteurs de la proteine nef du vih - Google Patents
Fragments d'anticorps inhibiteurs de la proteine nef du vihInfo
- Publication number
- EP2212353A1 EP2212353A1 EP08852180A EP08852180A EP2212353A1 EP 2212353 A1 EP2212353 A1 EP 2212353A1 EP 08852180 A EP08852180 A EP 08852180A EP 08852180 A EP08852180 A EP 08852180A EP 2212353 A1 EP2212353 A1 EP 2212353A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sdab
- nef
- sdabs
- fragments
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the invention relates to single domain antibody fragments (sdAb) capable of inhibiting the Nef protein of HIV and to their immunological applications, more particularly in immunotherapy for the treatment of AIDS.
- sdAb single domain antibody fragments
- AIDS Acquired Immunodeficiency Syndrome
- the target may be a protein of human immunodeficiency type 1 or 2 (HIV-I and HIV-2).
- VHH variable domains specifically recognizing one type of antigen, were selected from an immunized animal and were produced from plasmid constructs. As shown in the examples, these antibody fragments have been shown to specifically target regions of the Nef protein (negative regulatory factor) of HIV involved in the development of Acquired Immunodeficiency Syndrome (AIDS).
- Nef protein negative regulatory factor
- the invention is directed to their immunotherapeutic applications.
- the sdAbs fragments of the invention are characterized in that they are HIV Nef antiprotein fragments corresponding to all or part of the VHH domains of camelids, in particular llamas. According to an aspect of great interest, these fragments have a high stability and can be obtained in high quantities in soluble forms in bacteria, yeasts or any other production system from prokaryotic or eukaryotic cells.
- the invention is directed to anti-Nef antibody fragments having an amino acid sequence as encoded by a nucleotide sequence selected from the group consisting of SEQ ID Nos. 1 to 6.
- the invention thus more specifically targets the anti-Nef antibody fragments having an amino acid sequence chosen from the group comprising the sequences SEQ ID Nos. 7 to 12.
- the invention also relates to the CDRs of these sdAbs fragments.
- Nucleic acids capable of encoding said fragments are also within the scope of the invention.
- the invention aims, in particular, as new products, the nucleic acids corresponding to the sequences SEQ ID Nos. 1 to 6.
- the invention also relates to a method for producing the anti-Nef antibody fragments defined above.
- the Nef protein used for immunization is devoid of its first 56 amino acids.
- the construction of the library advantageously comprises: extraction of the total RNAs of the B lymphocytes, reverse transcription of the RNAs to obtain the corresponding cDNAs, amplification by PCR of the genes coding for the variable regions of the single anti-Nef heavy chain antibodies, the ligation of VHH DNA fragments, obtained by cleavage by enzymes of the amplified DNAs, with a phagemid.
- the sdAbs are isolated from the libraries by the phage display technique and purified.
- the selected sdAbs genes were then introduced into expression vectors, including plasmids, to produce different anti-Nef sdAbs capable of binding to Nef in HIV-infected cells.
- the invention relates more specifically to expression vectors, in particular plasmids, containing, between two unique sites of restriction enzymes, the promoters, the signal sequences, the nucleotide sequences capable of encoding the sdAbs fragments defined above, or the CDR regions of sdAbs.
- vectors in particular these plasmids, are capable of expressing the fragments of the invention in high quantities, in soluble forms, for example in bacteria.
- the invention thus relates to the plasmids pET14bNefl3, pET14bNefW12, pET14bNefW10, pHen ⁇ HisGS, pHenPhoA ⁇ His, pHen-sdAb Nefl, pHen-sdAb Nef2, pHen-sdAb Nef5, pHensDAb Nefl2, pHen-sdAb Nefl9, pHen-sdAb Nef20, pET- sdAb Nef1, pET-sdAb Nef2, pET-sdAb Nef5, pET-sdAb Nef1 2, pET-sdAb Nef1 9, pET-sdAb Nef20, pcDNA-sdAb Nef1 of sequences, respectively, SEQ ID Nos. 13 to 30.
- the genes encoding sdAbs are introduced between single restriction enzyme sites in the different plasmids.
- the plasmids according to the invention are capable of expressing in high amounts the sdAbs defined above, in soluble forms, for example in bacteria. Regions encoding sdAbs can be easily transferred to other prokaryotic or eukaryotic expression systems or transferred to plasmids for transfection into eukaryotic cells.
- the identification according to the invention of a new target of intervention represented by a direct inhibition of the functions of the viral protein Nef during natural infection with HIV, constitutes an original approach for the development of antiviral molecules. capable of disrupting HIV replication in the target cell, but also improving the immune response of infected patients.
- the invention therefore aims to exploit the immunological properties of antibody fragments in immunotherapy.
- the invention more specifically targets the antibody fragments defined above, optionally vectorized, for use as medicaments.
- compositions of the invention are then characterized in that they contain an effective amount of at least one sdAb fragment as defined above in association with a pharmaceutically acceptable vehicle.
- compositions can be used as antiviral drugs.
- sdAbs fragments are vectorized to cross the cell membrane and be released within the infected cell.
- the vector for example a peptide sequence, can be conjugated to the sequence of the fragments.
- the vector is combined with sdAbs fragments and corresponds for example to lipid compounds.
- compositions of the invention are used in immunotherapy to inhibit the Nef molecules released into the plasma medium.
- compositions are advantageously in forms suitable for oral or injectable administration.
- the invention relates to a gene therapy medicinal product consisting of a transfection vector comprising a nucleic acid as defined above coding for a sdAb fragment of the invention.
- Vectors useful for gene therapy purposes include adenoviruses, adenovirus (AA) associated viruses, retroviruses.
- AA adenovirus
- These drugs are used for intracellular immunization by transfection of infected cells.
- FIGS. 1 to 10 which represent, respectively,
- FIG. 1 (A) the phage-sdAb Nefl, Nef2, Nef5 and Nef 19 phage titration curves on the Nef protein adsorbed in the wells of a microplate and (B) the competition curves of the phage-binding. sdAb Nef5 and Nefl 9 on the Nef W10 protein adsorbed in the wells of a microplate by the soluble Nef W10 protein,
- FIG. 2 an SDS-PAGE gel showing the fractions of the sdAb Nefl 9 protein purified on TALON
- FIG. 3 (A) the titration curves of the Nef5 and Nefl9 sdAbs on the Nef W10 protein adsorbed in the wells of a microplate, (B) the Nef5 sdAb titration curve after amplification of the signal and (C) the competition curve of the binding of sdAb Nefl 9 on the Nef W10 protein adsorbed in the wells of a microplate by the soluble Nef W10 protein,
- FIG. 4 the table of affinity constants of sdAb Nefl 9 obtained by Biacore
- FIG. 5 co-localization analyzed by immunofluorescence of sdAb Nef 19 with the Nef protein in HeLa cells
- FIG. 6 (A) flow cytometric analysis of sdAb Nef 19 inhibition of the effect of Nef on CD4 expression level at the surface of HPB-ALL and (B) T cells. ) the flow cytometric analysis of the sdAb Nef 19 inhibition of the effect of Nef on the CD4 expression level at the HeLa cell surface,
- FIG. 7 (A) flow cytometric analysis of sdAb Nef 19 inhibition of Nef's ability to interact with the cellular machinery of the endocytotic pathway when expressed in the form of a CD8-Nef fusion in HeLa cells and (B) immunofluorescence analysis of sdAb Nef 19 inhibition of Nef's ability to interact with the cellular machinery of the endocytosis pathway when expressed in the form of a CD8-Nef fusion in HeLa cells,
- FIG. 8 analysis by coimmunoprecipitation experiments of the interaction of sdAb Nefl 9 with the Nef protein in 293T cells;
- FIG. 9 analysis of the inhibition by the Nefl 9 sdAb; Infectivity status of HIV-I during a single replication cycle measured on HeLa-CD4 and
- B T HPB-ALL cells
- Example 1 Construction of the different expression vectors to produce the truncated Nef recombinant proteins in E. coli and for the selection of sdAbs from phage-sdAB libraries.
- Oligonucleotides used 5 'Nef-Ncol-pET SEQ ID NO: 34
- PCR Conditions One ⁇ l (5 ng) of plasmid pNef-GST (gene coding for amino acids 57 to 205 of the Nef protein introduced into the plasmid pGEX-2T, (GE Healthcare)), 5 ⁇ l of Tp 1 OX Deep- Wind, 1 ⁇ l of 100 mM MgSO 4 , 4 ⁇ l of 2.5 mM dNTP mix, 10 ⁇ M of each oligonucleotide (5 'Nef-Nco-pET and 3' Nef-Blpl-pET), 0.5 U of Deep Vent, in final 50 ⁇ l (94 ° C. for 3 min, 94 ° C. for 1 min, 55 ° C. for 1 min, 72 ° C. for 1 min, 30 cycles then 72 ° C. for 10 min).
- the PCR products are purified from 2% agarose gel (Qiagen gel extraction kit, final volume 30 ⁇ l).
- the ligation is carried out with 5 .mu.l of the fragment, 0.5 .mu.l of the vector and 3 U Weiss of T4 DNA ligase (Biolabs) in a final volume of 10 .mu.l for 2H at room temperature.
- Competent BL21 (DE3) bacteria (CaCl 2 technique) are transformed with 5 .mu.l of the ligation product.
- the plasmid pET14bNefl3 whose nucleotide sequence is given in the appendix (SEQ ID No. 13), is thus obtained, which makes it possible to produce the clone Nef 13, the amino acid sequence of which is given in the appendix (SEQ ID No. 31).
- Oligonucleotides Used: 5'Nef.Ncol.W SEQ ID NO: 36: CTTTAAGAAGGAGATATACCATGGGCCACCACCATCATCATCACGGATCCGCCTG GCTAGAAGCACAAGAGGAGGAGGAG 3 'Nef-Blpl-pET SEQ ID NO: 37: GGGGTTATGCTAGTTATTGCTCAGCGTTCTTGAAGTACTCCGGATG PCR conditions on pET14bNefl3:
- the ligation is carried out with 5 .mu.l of the fragment, 0.5 .mu.l of the vector and 3 U Weiss of T4 DNA ligase (Biolabs) in a final volume of 10 .mu.l for 2H at room temperature.
- Competent BL21 (DE3) bacteria (CaC12 technique) are transformed with 5 .mu.l of the ligation product.
- the plasmid pET14bNefW12 whose nucleotide sequence is given in the appendix (SEQ ID No. 14), is thus obtained, which enables the production of the Nef W12 clone whose amino acid sequence is given in SEQ ID No. 32.
- SEQ ID NO: 38 TTAAGAAGGAGATATACCATGGGCTGGCTNGARGCNCARGARGAGGAGGAGGT
- PCR products are purified from 2% agarose gel (Qiagen gel extraction kit, final volume 50 ⁇ l).
- the ligation is carried out with 5 .mu.l of the fragment, 0.5 .mu.l of the vector and 3 U Weiss of T4 DNA ligase (Biolabs) in a final volume of 10 .mu.l for 2H at room temperature.
- Competent BL21 (DE3) bacteria (CaC12 technique) are transformed with 5 .mu.l of the ligation product.
- the plasmid pET14bNefW10 whose nucleotide sequence is given in the appendix (SEQ ID No. 15), is thus obtained, which allows the production of the Nef W10 clone whose amino acid sequence is given in the appendix (SEQ ID No. 33).
- Oligonucleotides used Sup-6HisGS / P3 SEQ ID NO: 42:
- PCR1 and PCR2 Conditions 1 ⁇ l pHen1 at 50 ng / ⁇ l, 10 ⁇ l Tp 1 OX Dynazyme (Biolabs), 2 ⁇ l 100 nM dNTP mix, 2 ⁇ l 5 'oligonucleotide at 10 pmoles / ⁇ l, 2 ⁇ l 3' to 10 pmol oligonucleotide / ⁇ l (Primer pairs used: PCR 1, Amont-Hind3 and Inf-6HisGS / cmyc, PCR 2: Sup-6HisGS / P3 and Aval-Bsml), 0.7 ⁇ l of Taq polymerase Dynazyme (Biolabs), 82 ⁇ l H 2 O.
- PCR program used 95 ° C, 3 min; 95 ° C, 45 s; 50 0 C, 45 s; 72 ° C, 45 s; 72 ° C, 3 min; 30 cycles.
- the size of the fragments of PCR1 and PCR2 is checked on 1% agarose gel and the fragments are purified using the "Qiaquick gel extraction" kit (Qiagen). These 2 fragments are then used for the overlap PCR3.
- PCR program used 95 ° C, 3 min; 95 ° C, 45 s; 50 0 C, 45 s; 72 ° C, 45 s; 72 ° C, 3 min; 30 cycles.
- the size of the PCR3 fragment is checked on 1% agarose gel and then the fragments are purified using the "Qiaquick gel extraction” kit (Qiagen). This fragment is then used for cloning. Analysis of the PCR3 product on 1% agarose gel is as expected (424 bp). This fragment was purified using the "Qiaquick gel extraction" kit (Qiagen) and then cloned.
- the PCR3 product is purified and cut in a volume of 50 .mu.l with 20 units of restriction enzyme HindIII in the presence of BSA at 37.degree. C. for 4 hours. Twenty units of restriction enzyme Bsml are then added, the sample is incubated at 65 ° C, 4 h. The Bsml enzyme is denatured at 80 ° C. for 20 min.
- the cleavage products are analyzed on 0.7% agarose gel to control the cut.
- the PCR3 product and the pHen1 cut with HindIII and Bsml are purified on 0.7% gel using the "Qiaquick gel extraction" kit (Qiagen).
- the PCR fragment is then cloned into the phagemid pHenl between the HindIII and Bsml sites (molar ratio DNA insert / phagemid, 1/5, 2000 units of T4 DNA ligase (Biolabs), 2 h at 20 ° C.).
- the ligase is denatured at 65 ° C. for 15 minutes.
- Competent TG1 bacteria are transformed with 10 .mu.l of ligation product. Phagemid preparation was then performed from an isolated colony and sequencing was performed. The expression of the p3 protein of pHen ⁇ HisGS was verified by Western blotting using an antibody directed against the p3 protein. The sequence proved to be as expected. The nucleotide sequence of pHen ⁇ HisGS is given in the appendix (SEQ ID No. 16).
- the new pHen ⁇ HisGS vector can be directly used for the construction of the naive bank. It is advantageous to improve it to facilitate the evaluation of cloning efficiency. Indeed, the isolation of VHH (or sdAb) of good specificity and in large numbers requires to obtain banks of great diversity. A very good cloning efficiency is therefore necessary during the construction of the banks.
- the phoA gene coding for alkaline phosphatase is inserted into the phagemid pHen ⁇ HisGS upstream of the gene coding for the c-myc tag.
- This gene introduced in the good reading phase allows the synthesis of a fusion protein "PhoA-cmyc-6HisGS-p3" having phosphatase activity.
- a colorimetric selection thus makes it possible to distinguish the closed vectors on themselves (blue colonies) from the vectors having inserted, in place of the PhoA gene, the genes coding for the VHH or sdAb (white colonies).
- PCR conditions One ⁇ l p55PhoA6HisGS / NAB- at 50 ng / ⁇ , 10 ⁇ l 10X Tp Dynazyme (Biolabs), 2 ⁇ l 100 nM dNTP mix, 2 ⁇ l 5 'PhoA / pHen oligonucleotide at 10 pmol / ⁇ l, 2 ⁇ l oligonucleotide 3 'PhoA / pHen at 10 pmol / ⁇ l, 0.7 ⁇ l of Taq polymerase Dynazyme (Biolabs), 82 ⁇ l H 2 O.
- PCR program used 95 ° C, 3 min; 95 ° C, 1 min; 60 ° C, 1 min, 72 ° C, 1 min; 72 ° C, 10 min; 35 cycles.
- the PCR product is analyzed on 1% agarose gel.
- the PCR product purified using the "Qiaquick gel extraction" kit (Qiagen) is cut in a volume of 50 ⁇ l with 20 units of the restriction enzyme SfiI in the presence of BSA, at 50 ° C., for 16 hours. Twenty units of the NotI restriction enzyme are then added, the sample is incubated at 37 ° C, 4 h.
- pHen ⁇ HisGS Ten ⁇ g of pHen ⁇ HisGS are cut in a volume of 50 ⁇ l with 20 units of the restriction enzyme SfiI in the presence of BSA at 50 ° C. for 4 hours. Twenty units of the NotI restriction enzyme are then added, the sample is incubated at 37 ° C, 4 h.
- the PCR product and pHen ⁇ HisGS cut with Sfil and NotI are purified on 0.7% gel using the "Qiaquick gel extraction" kit (Qiagen).
- the PCR fragment is then cloned into the pHen ⁇ HisGS phagemid between the SfiI and NotI sites.
- the ligase is denatured at 65 ° C. for 15 minutes.
- Competent TG1 bacteria are transformed with 10 ⁇ l of ligation product and then plated on LB / ampicillin medium 100 ⁇ g / ml / BCIP 30 ⁇ g / ml.
- a preparation of the phagemid was then made from a blue colony. Expression of the PhoA-cmyc-6HisGS-p3 fusion protein was verified as well as the efficacy of the phagemid for infection.
- the nucleotide sequence of the phagemid pHenPhoA ⁇ His is given in the appendix (SEQ ID No. 17).
- a male llama was immunized with region 57-205 of the recombinant Nef protein (Nef57-205) of HIV-1.
- the animal was immunized every month, for 3 months, with 500 ⁇ g of Nef57-205.
- One hundred ml of blood was taken 15 days after each immunization.
- a titration of sera and purified antibodies (IgG1, 2 and 3) was performed to detect the presence of antibodies against the Nef57-205 immunogen.
- the B lymphocytes were then purified on a Ficoll gradient (histopaque-1077, Sigma-Aldrich) and then washed twice with PBS.
- phage-sdAb libraries purification of total RNAs, reverse transcription, PCRl, PCR2 and cloning in phagemids pHen ⁇ HisGS and pHenPhoA ⁇ His
- Total B-cell RNAs are extracted by the method using guanidium isothiocyanate (Chomczynski and Sacchi, 1987). After phenol / chloroform extractions in an acidic medium, the total RNAs are precipitated with ethanol. The quality of the RNAs and their quantification are evaluated on 1% agarose gel. They are then converted to cDNA by reverse transcription.
- SEQ ID NO: 52 CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTC
- SEQ ID NO: 53 CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTCACCTTGAAGGAGTC
- RNA Five ⁇ g of total RNA are hybridized with 1 pmole of CH2FORTA4 (Arbabi Ghahroudi et al., 1997) or CH2-2-specific oligonucleotide of the CH2 domain of lamb retrospenous heavy chain IgG retranscribed with 150 U superscript II (BRL ) for 30 min at 50 ° C.
- the single-strand cDNAs are purified on beads (BioMagR Carboxyl Terminator, Polyscience Inc.) and eluted with 17 ⁇ l of 10 mM Tris-acetate pH 7.8.
- Three DNA fragments are amplified: a fragment of approximately 900 bp encoding the VH-CH1-CH2 domains of IgG1; and 2 fragments of approximately 600 bp coding for the VHH-CH2 domains of IgG2 and 3.
- the 600 bp fragments are purified on 1% agarose gel ("Qiaquick gel extraction" kit, Qiagen) and then amplified by PCR with 1 U of Deep Vent (Biolabs) and 10 pmoles of the 4 domain-specific 5 ⁇ H1-4 Sfi primers. VH human IgG and 10 pmol of the 3 'VHH-NotI primer.
- the approximately 400 bp fragments encoding the VHH are purified on 1% agarose gel ("Qiaquick gel extraction" kit, Qiagen) pooled and precipitated with ethanol. They are then cut with restriction enzymes Ncol and NotI, or BglII and NotI (Biolabs) to be cloned into the phagemid pHenlhisPhoA at the NcoI and NotI or SfiI and NotI sites.
- phagemid pHen ⁇ HisGS (or pHen ⁇ HisPhoA for the "naive" library): Twenty ⁇ g of phagemid pHen ⁇ HisGS are cut in a volume of 300 ⁇ l with 50 U of SfiI in the presence of BSA, at 50 ° C., 16 h; or with 50 U Ncol in the presence of BSA, at 37 ° C, 16 h.
- the linearized phagemid is purified on 0.7% agarose gel ("Qiaquick gel extraction" kit, Qiagen).
- the eluted DNA is then cut with 50 U of NotI at 37 ° C in a volume of 200 ⁇ l, 16 h.
- the enzyme is destroyed by heat for 15 min at 65 ° C and the DNA is phenol / chloroform extracted and ethanol precipitated.
- the cut pHen ⁇ HisGS is controlled on 0.7% agarose gel, quantified and adjusted to 200 ng / ⁇ l.
- VHH fragments Five ⁇ g of VHH fragments are cut in a volume of 300 ⁇ l with 50 U of BgII and NotI in the presence of BSA at 37 ° C. for 16 hours; or with 50 U Ncol and NotI in the presence of BSA, at 37 ° C, 16 h.
- the enzymes are denatured at 65 ° C, 15 min; then the DNAs are extracted with phenol / chloroform and precipitated with ethanol in the presence of 10 ⁇ g of glycogen (Roche).
- the VHH fragments cut with NcoI and NotI are purified on 1% agarose gel, then checked on 2% agarose gel, quantified and adjusted to 100 ng / ⁇ l.
- the ligase is inactivated at 65 ° C, 15 min, and the ligation product is cleaved with 20 U Xho I (Biolabs) to remove the remaining unligated vector, 37 ° C, 4 h. Six ligatures are thus realized.
- the ligation products are then combined into 2 tubes and extracted with phenol / chloroform, precipitated in the presence of 10 ⁇ g of glycogen and taken up in 2 ⁇ 18 ⁇ l H 2 O ultrapure. Two ⁇ l are used by electroporation. Colonies of different electroporations are collected.
- the male llama phage-sdAb library represents 4.1-10 4 clones.
- the blood (about 2400 ml) was taken from about sixty non-immune lamas from four different farms.
- the "naive" phage-sdAb library represents 3,107 clones.
- the different sdAbs were isolated by the phage-display technique (Smith, 1985, Hoogenboom et al., 1991) regardless of the bank used.
- Ten ⁇ l of the stock of the library (TG1 cells transformed with phagemids) are inoculated into 50 ml of (2TY, 100 ⁇ g / ml of ampicillin, 2% glucose) and incubated at 37 ° C. until an OD600 equal to 0 5.
- Five ml of the culture are then infected with 5 ml of M13KO7 at 13 pfu / ml and incubated for 30 min at 37 without shaking. After centrifugation, the phage pellet is taken up in 25 ml of (2TY, 100 ⁇ g / ml of ampicillin, 25 ⁇ g / ml kanamycin). The culture is incubated for 16 h at 30 ° C. with stirring. The phages are then precipitated with 1/5 vol of 2.5 M NaCl / 20% PEG 6000 and concentrated 25 times in PBS.
- the beads are compacted with a magnet, suspended in 250 ⁇ l of 2% milk / PBS and incubated with 200 ⁇ l of biotinylated antigen for 30 min at room temperature on a wheel. 150, 75 and 25 nM final biotinylated antigen are used on the 1st, 2nd and 3rd round, respectively.
- the 500 .mu.l of phages are added for 3 hours at room temperature with stirring on one wheel.
- the mixture of beads / antigen-biotin / phage is washed 5 times with 800 ⁇ l of 4% -PBS milk and then transferred to a new Eppendorf tube.
- Five further washings are carried out with 800 .mu.l of 0.1% PBS-Tween, and the mixture is then transferred to a new Eppendorf tube.
- 5 washes are carried out with 800 .mu.l of PBS.
- the antibody phage bound to the beads / antigen-biotin are suspended in 200 .mu.l of PBS and incubated for 30 min at 37.degree. C., without shaking, with 1 ml of TG1 made competent for fixing pili phages (competent cells: from a culture of TG1 in 2YT on the night, a 1/100 dilution is carried out and 50 ml of 2YT are inoculated at 37 ° C. with stirring until an OD600 close to 0.5). At each selection, phages are counted and amplified for a new round of selection.
- Dilutions were performed with transfected cells TGI phage (see above) 10 February to 10 May with 2YT.
- TGI phage transfected cells
- One, 10 and 100 ⁇ l of each dilution are spread on a petri dish (2YT / ampicillin 100 ⁇ g / ml / glucose 2%).
- the dishes are incubated for 16 h at 30 ° C. . f4 - Spreading of selection for colony isolation:
- transfected TG1 The 5 ml of transfected TG1 are centrifuged for 10 min at 3000 g to concentrate the cells and the pellet is taken up with 1 ml of 2YT. Two hundred and fifty ⁇ l are spread per Petri dish (12 cm ⁇ 12 cm) (2TY / ampicillin 100 ⁇ g / ml / 2% glucose) and incubated for 16 h at 30 ° C.
- sdAb Nefl9 SED ID No. 1 and 7
- sdAb Nef20 SEQ ID Nos. 2 and 8
- sdABs specific for the Nef protein were isolated by this method: sdAb Nefl (SED ID Nos. 3 and 9), sdAb Nef2 (SEQ ID Nos. 4 and 10), sdAb Nef5 (SEQ ID Nos. 5 and 11) and sdAb Nefl2 (SEQ ID Nos. 6 and 12).
- TG1 containing the phagemid corresponding to the phage-sdAb selected The culture is incubated at 37 ° C with stirring to a DO ⁇ OOnm close to 0.5. Five ml of this culture are infected with 5 to 10 ⁇ l of helper phage M13KO7 (10 13 pfu / ml) and incubated for 30 min at 37 ° C. in a water bath (without agitation). The culture is centrifuged for 10 min at 3000 g and the supernatant is removed. The pellet is taken up with 25 ml of 2TY (Ampicillin 100 ⁇ g / ml, Kanamycin 25 ⁇ g / ml). The culture is incubated at 30 ° C.
- the container is put in ice for 10 min.
- the culture is then centrifuged for 20 min at 3000 g, 4 ° C.
- the supernatant is removed and precipitated by adding 1/5 vol of 2.5 M NaCl / 20% PEG 6000 for 1 h in ice.
- the solution is centrifuged for 20 min at 3000 g, 4 ° C.
- the pellet is taken up with 1 ml of PBS and transferred to a silicone Eppendorf tube. Rapid precipitation is carried out by adding 200 ⁇ l of NaCl / PEG and then centrifuging at 13,000 rpm.
- the pellet is taken up with 1 ml of PBS and centrifuged for 1 minute. 000 rpm.
- the supernatant is filtered through 0.45 ⁇ m and transferred to a silicone Eppendorf tube and then stored at 4 ° C.
- TG1 cells are cultured in 2YT at 37 ° C. Successive dilutions (from 10 to 10) of sdAb phage are carried out in silicone Eppendorf tubes containing 500 ⁇ l of 2YT. When the TG1 cells are at a OD 50 nm of 0.5, 500 ⁇ l of TGl are added, and then the cells are left for 30 minutes at 37 ° C. without stirring. One hundred ⁇ l of each tube are plated on Petri dishes 2YT (Ampicillin 100 ⁇ g / ml, glucose 2%). The dishes are incubated for 16 hours at 30 ° C. or 37 ° C. Colonies are counted to determine the number of sdAb phage in the starting solution. This solution will be used to characterize sdAb phages by ELISA.
- biotinylated antigen (Nef W10) are fixed on a streptavidin plate (BioBind Assembly Streptavidin Coated, ThermoLabsystems) previously saturated with 2% -PBS milk. Different dilutions of sdAb-phages are brought into contact with the antigen. The antigen / antibody binding is detected by means of an ELISA composed of a monoclonal antibody directed against phage protein P8 (HRP / anti-M13 monoclonal conjugate, Pharmacia).
- ABTS diammonium salt 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) to 20 ml revealing buffer (18 ml PBS, ImI citric acid 1 M, 1 ml 1 M sodium citrate, 10 ml H 2 O 2 30%), allows the reaction to be read at 405 nm (Tecan).
- FIG. 1A shows the results obtained with Nef, Nef2 and Nef5-sdAb phages obtained with the "naive” library and the sdAb Nefl 9 phage obtained from the "immune” library.
- a decrease in the measurement of the interaction between sdAb-phages and the biotinylated Nef W10 protein fixed in streptavidin-coated wells of a microplate is observed in all titration curves when the amount of sdAb-phage decreases.
- competitive ELISAs were performed.
- FIG. 1B shows that the binding of Nef5 and Nefl9 phage-sdAb on the biotinylated Nef W10 decreases when Nef W10 is increased. biotinylated in the test. This decay proves the specificity of the interaction of phages-sdAb for Nef W10 protein. Equivalent results are obtained with the other phage-sdAb selected.
- An isolated colony is inoculated into 3 ml of 2YT / ampicillin 100 ⁇ g / ml / 2% glucose and incubated at 37 ° C with shaking. Fifty ml of 2YT / ampicillin 100 ⁇ g / ml / 2% glucose are then inoculated with a dilution of the preceding culture and incubated for 16 h at 30 ° C. with stirring. Four hundred ml of 2YT / ampicillin 100 ⁇ g / ml are inoculated with the equivalent of 0.1 unit OD ⁇ OOnm, and incubated at 30 ° C. with shaking, to a DO ⁇ OOnm of 0.5 to 0.7. The culture is then induced with IPTG (isopropyl- ⁇ -D-thiogalactopyranoside, 0.1 mM final) and cultured at 30 ° C. for 16 h.
- IPTG isopropyl- ⁇ -D-thiogalactopyranoside, 0.1
- the cultures from which the sdAbs are produced are centrifuged at 4200 g, 4 ° C, 40 min.
- the pellet is taken up in 4 ml of ice-cold TES (0.2 M Tris-HCl pH 8.0, 0.5 mM EDTA, 0.5 M sucrose). 160 ⁇ l of lysozyme (10 mg / ml in TES, freshly prepared) are then added, followed by 24 ml of cold TES diluted 1/2 in H 2 O. The mixture is incubated for 30 min in ice.
- TES Tris-HCl pH 8.0, 0.5 mM EDTA, 0.5 M sucrose
- the column (BD TALON TM Metal Affinity, BD Biosciences Clontech) is equilibrated with the equilibrium buffer (50mM sodium acetate, 0.1M NaCl, pH 7.0). The periplasmic fraction is deposited on the column. After washing the column with 5 volumes of equilibration buffer, the sdAb is eluted by pH or imidazole gradient (gradient between pH 7.0 equilibration buffer and 50mM sodium acetate pH 5.0 or the imidazole solution from 0 to 200 mM). Each fraction is checked on an SDS / PAGE gel (15% acrylamide) after staining with Coomassie blue. The fractions of interest are pooled and dialysed against PBS.
- the SDAB is membrane-based (Amicon Ultra 5000MWCO, Millipore) and assayed by Lowry's colorimetric method using the Biorad Protein Assay kit.
- Figure 2 shows a purification profile (C: load, NR: fraction not retained on the column, L wash in charge buffer).
- the sdAb Nefl9 is eluted (fractions 9 to 56) by a pH gradient of 7 to 5.
- biotinylated antigen Five ⁇ g / ml of biotinylated antigen (Nef W10) are fixed on a streptavidin plate (BioBind Assembly Streptavidin Coated, ThermoLabsystems) previously saturated with 2% -PBS milk. Each sdAb (range of 0.001 ⁇ g / ml to 1 ⁇ g / ml) is linked to the antigen adsorbed in the microwells. The binding is revealed with a monoclonal antibody 9E10 directed against the c-myc tag (Santa Cruz Biotechnology, Inc.) diluted 1/1000 and a goat polyclonal antibody against mouse IgG coupled to peroxidase diluted 1/5000. (ref 55556, ICN) in the presence of ABTS (diammonium salt of 2,2'-azino-di- (3-ethylbenzthiazoline sulfonate) Roche).
- ABTS diammonium salt of 2,2
- FIG. 3A shows the results obtained with the Nef5 and Nef 19 sdAb.
- a microplate when the amount of sdAb decreases. Since sdAb Nef 5 was less so that sdAb Nefl 9, signal amplification (FIG. 3B) was obtained by preincubating sdAB Nef5 with mAb 9E10 for 1 h at 25 ° C prior to deposition in the microplate wells. As a control, mAB 9E10 was used in the absence of SDAB.
- phages-SDAB competitive ELISAs were performed. For this, a constant amount of sdAb (5 ⁇ g / ml) was preincubated with different amounts of non-biotinylated Nef W10 protein 16 h at 4 ° C. The ELISAs are then made as previously described.
- Figure 3C shows that binding of sdAb Nefl 9 to biotinylated Nef WlO decreases when increasing the amount of non-biotinylated Nef W10 protein in the assay. This decay proves the specificity of the interaction of sdAb Nefl 9 for Nef W10 protein. Equivalent results are obtained with other SDABs.
- sdAb Nefl 9 in the plasmid pET14bNefW10 10 ⁇ l of the vector pET14bNefW10 and 5 ⁇ l of the vector pHen-sdAb Nefl9 are cut with 1OU Ncol and Notl for 16H at 37 ° C.
- the fragments are purified on 1% agarose (gel extraction kit Qiagen final volume 50 ⁇ l for the vector pET14bNefW10 and 20 ⁇ l for the fragment corresponding to the sequence of the sdAb Nef 19)
- the ligation is carried out with 10 .mu.l of fragment and 1 .mu.l of vector in a final volume of 15 .mu.l in the presence of 3 Weiss U of T4 DNA ligase (Biolabs) for 2 hours at room temperature.
- Competent BL21 (DE3) bacteria (CaC12 technique) are transformed with 7.5 ⁇ l of the ligation product.
- the plasmid pET-sdAb Nef 19 (SEQ ID No. 28) is obtained, the sequence of which is indicated in the appendix.
- BIACORE uses the principle of surface plasmon resonance (SPR) to monitor in real time interactions between molecules without labeling them.
- SPR surface plasmon resonance
- One of the interaction partners is immobilized covalently on one biosensor while the other is injected into a continuous flow.
- the principle of detection by SPR makes it possible to follow the changes in mass at the surface of the biosensor due to the formation and then the dissociation of the molecular complexes.
- the response, quantified in resonance unit (RU) is a direct indication of the rate of fixation of the analyte by measuring the variation of the refractive index.
- Nef W10 and sdAb Nef 19 produced either from the periplasm (sdAb Nef 19P) or from the cytoplam (sdAb Nef 19C) of bacteria were studied on a BIACORE 3000 fitted with a CM5 biosensor on which 1089 RU of Nef W10 were covalently immobilized following the standard amine coupling procedure proposed by BIACORE (NHS / EDC activation).
- the sdAbs Nefl9P or Nefl9C (in buffer: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) are then injected.
- Nefl9P sdAb and Nefl9C of SEQ ID Nos. 1 and 7 are indicated in FIG. 4 (It should be noted that Nefl9P and Nefl9C sdAbs have the same amino acid sequences).
- Oligonucleotides used SEQ ID NO: 57:
- pHen-sdAb Nefl9 at 50 ng / ⁇ l, 10 ⁇ l Tp 10X Dynazyme (Biolabs), 2 ⁇ l 100 nM dNTP mix, 2 ⁇ l 5 'oligonucleotide at 10 pmoles / ⁇ l, 2 ⁇ l 3' oligonucleotide at 10 pmoles / ⁇ l (Pair of primers used: ANefEcoK5p and ANefXho3p), 0.7 ⁇ l of Taq polymerase Dynazyme (Biolabs), 82 ⁇ l H 2 O.
- PCR program used 95 ° C, 3 min; 95 ° C, 45 s; 50 0 C, 45 s; 72 ° C, 45 s; 72 ° C, 3 min; 30 cycles.
- the size of the PCR fragment is checked on 1% agarose gel, then the fragments are purified using the "Qiaquick gel extraction" kit (Qiagen).
- ⁇ l of the purified PCR product are cut in a volume of 100 ⁇ l with 1 OR of restriction enzyme EcoRI and 10u of restriction enzyme Xhol in the presence of BSA at 37 ° C. for 12 hours. The enzymes are then denatured at 65 ° C for 20 min.
- the pcDNA3.1 + vector (Invitrogen) was used for the expression of sdAb Nef 19 in mammalian cells. 2.5 ⁇ g of pcDNA3.1 + are cut in a volume of 100 ⁇ l with 10 units of restriction enzyme EcoRI and 10 units of restriction enzyme XhoI in the presence of BSA at 37 ° C. for 12 hours. The enzymes are then denatured at 65 ° C for 20 min.
- the digestion products are analyzed on 0.7% agarose gel to control digestion.
- the PCR product and the pHen-sdAb Nef 19 cut with EcoRI and HindIII are purified on 0.7% gel using the "Qiaquick gel extraction" kit (Qiagen).
- the ligation is carried out with 5 .mu.l of the PCR fragment, 0.5 .mu.l of the vector and 3 U Weiss of T4 DNA ligase (Biolabs) in a final volume of 10 .mu.l for 2 hours at room temperature.
- Competent TG1 bacteria are transformed with 10 .mu.l of ligation product. Plasmid preparation was then performed from an isolated colony and sequencing was performed. The resulting plasmid called pcDNA-sdAB Nef 19 allows the production of sdAb Nef 19 in eukaryotic cells transfected with this plasmid.
- the sequence of pcDNA-sdAb Nef 19 is given in the appendix (SEQ ID No. 30).
- the intracellular distribution of sdAb Nef 19 was analyzed by indirect immunofluorescence on HeLa cells transiently expressing the Nef-GFP fusion protein or the GFP control protein, the expression vectors of which have previously been described (Burtey et al. 2007).
- the cells (4 x 10 5 ) were transfected by lipofection technique with Lipofectamine 2000 (Invitrogen) following the procedure recommended by the manufacturer.
- the cells were fixed for 20 min at 4 ° C with a solution of PBS-paraformaldehyde (PFA) at 4%, and then permeabilized with a solution of PBS-Triton X100 at 0.1% for 10 min.
- PFA PBS-paraformaldehyde
- the sdAb was then detected by an antibody (Ac) directed against the c-myc epitope (9E10, Roche) in PBS-BSA at 0.1%, and then a second mouse anti-IgG antibody coupled to Alexa594 (Jackson Laboratories).
- Ac antibody directed against the c-myc epitope
- Alexa594 Jackson Laboratories
- sdAb Nef 19 In order to explore the potential effects of sdAb Nef 19 on the functional properties of Nef, its ability to modulate CD4 receptor expression on the surface of CD4 + T cells was first analyzed in cells expressing sdAb.
- the expression level of CD4 at the cell surface was analyzed on cells expressing Nef-GFP or GFP by flow cytometry using a Cytomics FC500 device after labeling for 1 h at 4 ° C with an anti-CD4 Ab directly coupled to ⁇ -hycoerythrin CY5 (RPA-T4, Beckton-Dickinson), then fixing the cells with a solution of formaldehyde at 3.7%.
- sdAb does not induce a nonspecific effect, since this one, even at the highest dose, does not modify the level of CD4 present on the surface of the cells expressing the control protein GFP (white bars).
- This inhibitory effect of sdAb Nef 19 is also observed on non-lymphoid cells stably expressing the CD4 receptor.
- HeLa cells stably expressing CD4 (HeLa-CD4) were co-transfected as previously by the lipofection technique with 1, 2 or 3 ⁇ g of the expression vector of sdAb Nef 19 and 1 ⁇ g of the Nef-expression vector. GFP or GFP control (Coleman et al., 2006).
- the level of CD4 surface expression was analyzed as previously by flow cytometry on cells expressing Nef-GFP or GFP.
- CD8-Nef Multi-team use of a CD8-Nef fusion protein in which the extracellular and membrane regions of CD8 are fused to the N-terminus of Nef (CD8-Nef) has shown that the Nef sequence contains determinants allowing it to interact directly with vesicular protein transport machinery at the level of the endocytosis pathway.
- the CD8-Nef membrane construct has the property, as does the native myristilled Nef protein, of modulating the surface expression of the CD4 receptor in trans, but also of modulating in cis its own level of expression on the cell surface, thus reflecting its ability to connect directly to the cellular machinery of the endocytic pathway.
- the cells are co-transfected by lipofection with 1, 2 or 3 ⁇ g of the expression vector of sdAb Nefl 9, 0.7 ⁇ g of the CD8-Nef expression vector or the CD8-control. Stop corresponding to the CD8 receptor devoid of cytoplasmic domain, and 0.3 ⁇ g of the expression vector of GFP. 24 h after transfection, the cells are fixed for 20 min by a 4% PBS-PFA solution, and the level of expression of the CD8-Nef chimera on the surface of the GFP-expressing cells was evaluated using anti-CD8 Ab (SKl, Becton-Dickinson) coupled to phycoerythin-Cy5.
- anti-CD8 Ab SKl, Becton-Dickinson
- the cells were transfected with 1 ⁇ g of the CD8-Nef or CD8-Stop expression vector and 1 ⁇ g of the sdAb expression vector. 24 h after transfection, the cells are fixed for 20 min by a solution of PBS-PFA at 4% and then permeabilized with a solution of PBS-Triton X100 0.1%. The sdAb was detected as before (see FIG. 5), whereas the CD8-Nef fusion is detected by a FITC-coupled anti-CD8 Ab (SFCI, Coulter).
- SFCI FITC-coupled anti-CD8 Ab
- Part A corresponds to the results of the cytometric analysis; the top panel represents a representative experience while the bottom panel represents the averages of 3 independent experiments.
- the level of expression of the CD8-Nef chimera is approximately five times lower than that of the CD8-Stop control protein (white bars).
- the expression of increasing amounts of sdAb results in a progressive accumulation of the CD8-Nef protein at the cell surface (black bars).
- the expression of sdAb has no effect on the expression level of the CD8-Stop control (white bars).
- the results of FIG. 8 suggest that the zone recognized by the sdAb is located at the C-terminus of Nef, on a region between residues 190 to 206 of the protein.
- the inhibitory activity of sdAb Nef 19 on the contribution of Nef to the infectious properties of viral particles was analyzed in an experimental system for evaluating the infectivity of HIV-I during a single replication cycle (Madrid et al. ., 2005).
- the recombinant viral particles carrying the GFP reporter gene were produced by co- transfection of 293T cells as previously described (Basmaciogullari et al., 2006) with 8 ⁇ g of the vector allowing the expression of the proteins derived from the gag and pol genes of HIV-1 (isolate NL43) (Owens et al., 2003), 8 ⁇ g of the transgene expression vector GFP, 2 ⁇ g of the vector allowing the expression of the envelope of HIV-1 (isolate 89.6) or VSV (VSV-G), 1 ⁇ g of the expression vector of the Nef protein labeled at its C-terminus by the HA epitope (Dorfman et al., 2002), and 8 ⁇ g of the sdAb expression vector.
- Virus particles pseudotyped by the HIV-I envelope or that of the VSV were recovered in the culture supernatant 48 h after transfection and stored at -80 ° C.
- the viral stocks were titrated by measuring the reverse transcriptase activity. (RT), and then used to infect HeLa-CD4 cells or T cells of the HPB-ALL line.
- RT reverse transcriptase activity
- 3 x 10 4 HeLa-CD4 cells were infected in 24-well plates with 500 ⁇ l of a 5 x 10 5 dilution and 5 x 10 4 arbitrary units of RT / ml pseudotyped viral stocks, respectively, by the envelope of HIV-I or VSV.
- the results corresponding to the averages of 3 independent experiments are reported in FIG. 9.
- the top panel (A) corresponds to the infectivity of the viral particles measured on the HeLa-CD4 cells
- the bottom panel (B) corresponds to the infectivity. measured on HPB-ALL T cells.
- the results are reported according to the infectivity of virus particles pseudotyped by the envelope of HIV-I (blue bars) or VSV (burgundy bars) and produced in the absence of Nef.
- the expression of Nef in the producer cells results in a marked increase in the infectivity of viral particles expressing the HIV-1 envelope (14 times and 3 times, respectively, in the cells).
- Nefl9 causes a significant and specific inhibition, of the order of 75%, of the effect of Nef on the infectivity of the viral particles, regardless of the cell type used. In contrast, expression of sdAb does not affect the infectiousness of viral particles expressing VSV G protein, whether the particles are produced in the absence or presence of Nef.
- the viral particles thus purified were then taken up in Laemli buffer and analyzed by immunoblotting using anti-HA (3F10, Roche), anti-c-myc (9E10, Roche) and anti-p24 (obtained from NIH AIDS Research and Reference Reagent Program "); the lysates of the producer cells were also analyzed by immunoblotting.
- the immunoprecipitated material was then analyzed by immunoblotting using an Ab specifically directed against Nef protein (Ac a56 obtained from the NIH AIDS Research and Reference Reagent Program) and anti-c-myc Ab (9E10). ).
- Ad specifically directed against Nef protein Ac a56 obtained from the NIH AIDS Research and Reference Reagent Program
- anti-c-myc Ab (9E10).
- sdAb is detected only in the immunoprecipitate of cells expressing Nef-HA, but is not detected in material precipitated from cells transfected with the control vector.
- Nef-Stop left panels
- allowing the expression of a polyptic corresponding to the first 46 residues of the Nef protein, even if the sdAb is well expressed in these cells right panels).
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US20140235492A1 (en) | 2011-09-20 | 2014-08-21 | Institut National De La Sante Et De La Recherche Medicate (Inserm) | Methods for preparing single domain antibody microarrays |
EP2857415A1 (fr) | 2013-10-02 | 2015-04-08 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Structure cristallographique du complexe Nef/sdAb19/HckSH3 et son utilisation |
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Title |
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OVOD V ET AL: "IMMUNOLOGICAL VARIATION AND IMMUNOHISTOCHEMICAL LOCALIZATION OF HIV-1 NEF DEMONSTRATED WITH MONOCLONAL ANTIBODIES", AIDS, LONDON, GB, vol. 6, no. 1, 1 January 1992 (1992-01-01), pages 25 - 34, XP009076670, ISSN: 0269-9370, DOI: 10.1097/00002030-199201000-00003 * |
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