Study On The Construction And Expression Of Recombinant Gene Encoding Anti-human CD19 Human-Mouse Chimeric Antibody (Hm2E8)

Hematopoietic malignancies remain the major life-threatening diseases in children. B lineage acute lymphoblastic leukemia(B- lineage ALL)is the most common type of acute leukemia in children.Improved prognosis has been achieved by combined chemotherapy with multiple anti-cancer agents.But there are still 20-30%of ALL children encountered treatment failure.The main reasons of treatment failure are multiple drug resistance(MDR)and severe side effects of chemotherapy.As compared to conventional chemotherapy,monoclonal antibody(McAb)targeting therapy is more effective with less side effects owing to its perfect selectivity and specificity.The McAbs of the cluster differentiation(CD)molecules on the surface of leukemia cells are the excellent guiders for targeting therapy.Unfortunately,most McAbs currently available with high affinity are mouse origin. The clinical application of these mouse antibodies in patients has been greatly restricted due to the occurrence of human anti-mouse antibody(HAMA)response.The efficacy decreases and severe anaphylaxis arises when HAMA occurs.Thus,it is necessary to reduce the immunogenicity of the mouse antibody to human body maximally and keep their antigen recognizing activity.As we all know,the immunogenicity of a given antibody mainly exists in its constant(Fc)region while the antigen binding activity is located at its variable(Fv)region.In order to make the mouse antibodies eligible for the clinical use,genetic engineering of the antibody molecule is necessary.One of the practical methods is to reconstruct the gene of chimeric antibody.Which includes to replace the mouse Fc gene with human's and to retain the Fab gene for its high affinity then to express the antibody protein through modern genetic engineering techniques. The successful model of chimeric antibody currently applying in clinic practice is Rituximab—a chimeric anti human CD20 monoclonal antibody.Rituximab has been accepted as one of the first line strategy of CD20+ lymphoma due to its positive efficacy.But CD20 is less expressed on the surface of progenitor B cells.This is a main obstacle of Rituximab on treating some type of lymphoma and progenitor B cell leukemia.CD19 is another specific surface marker of B cells.It is consistently and steadily expressed on almost all differentiation stages of B lineage cells. Thus the particular antigen may be an ideal target for antibody-targeting treatment of B lineage malignancy.At present,many kinds of anti-human CD19 McAbs are being studied in the world. Most results have showed that both conjugated and unconjugated McAbs are the targeting agents at various degree of efficacy to B lineage malignant cells in either cell lines or animal models.Also,there are evidences to show that not every clone of CD19 McAbs works the same way and each has its own activity and response range to B lineage malignancies.Most importantly,no product of CD19 has been approved for clinical use till now as what rituximab did.So,it is necessary to study more CD19 clones.ZCH(Zhejiang Children's Hospital)-4-2E8 or simply 2E8 belonging to murine IgM subtype was generated in our laboratory which was assigned to CD19 category by the 6^thInternational Workshop and Conference on Human Leukocyte Differentiation Antigens(HLDA6)in 1996.The purpose of this study is to insert the 2E8 Fv gene to a baculovirus shuttle vector pAc-κ-CH3 by modern genetic engineering techniques followed by the expression of the novel protein in insect cell lines.The characterization and physiological activity of the chimeric antibody was examined.Hopefully,the results of this study could provide a fundamental basis for the further engineering of this important antibody with potential of clinical targeting treatment.Baculovirus expression system is applied widely as an important eukaryotic expression system due to its great capacity of inserting genes,high yield,proper post-translation modification,easy to operate and good biological material safety.With the IgG expression cassette elements,including the authentic IgG kappa and heavy chain signal sequences,as well as light chain(kappa)and heavy chain constant region genes are integrated in a single vector and are controlled by the p10 and polyhedrin promoter respectively,the baculovirus expression vector pAc-κ-CH3 was designed specially for the expression of chimeric antibodies.It was reported that the yields of IgG were between 6 and 18 mg/L and the antigen binding function was well preserved. Thus we selected this system to express our recombinant protein.Material and Methods1.Construction of baculovirus shuttle vector pAc-κ-CH3-VH_2E8 -VL_2E8:1.1 Cloning of the VH_2E8and VL_2E8genes:The VH_2E8and VL_2E8gene were cloned from pSectag2A/ScFv_2E8which was successfully established previously in our laboratory by PCR with specific endonuclease location primer pairs and the sequences were inserted into pGEM^?-T easy vectors(TA cloning),followed by transformation to E.coli DH5α,selection, amplification and sequencing.The target sequences were confirmed by comparing with the previously cloned VH_2E8and VL_2E8gene sequences for further work.1.2 Construction of baculovirus shuttle vector pAc-κ-CH3-VH_2E8-VL_2E8:1.3 The VH_2E8and VL_2E8gene fragments were cleaved with proper endonucleases (Xhoâ… and Nheâ… for VH_2E8;Sacâ… and Hindâ…¢for VL_2E8)and followed by the sequential insertions of the target genes into the secretive baculovirus expression shuttle vector pAc-κ-CH3.After transformation to E.coli DH5α,the recombinants were selected,amplified and sequenced.The interested sequences were then compared with the previous known VH_2E8and VL_2E8gene sequences to confirm the correct insertion.2.Transfection of Sf9 cells by reconstructed baculovirus shuttle vector and formation of complete virions pAc-κ-CH3-VH_2E8-VL_2E8CV:3.2.1 Cell culture:The cell lines Sf9,2E8,Nalm-6,High Five were cultured under routine conditions.Sf9 and High Five cells were making gradually adapt to serum free medium(SFM).2.2 Transfection of Sf9 cells by reconstructed baculovirus shuttle vector pAc-κ-CH3-VH_2E8-VL_2E8:The Sf9 cells were cotransfected by the reconstructed shuttle vector pAc-κ-CH3-VH_2E8-VL_2E8and AcNPV(Autographa californica nuclear polyhedrosis virus)linearized DNA pXYIE transfection and untransfected Sf9 cells were set as positive and negative controls,respectively.Cell morphological changes were observed every day after transfection under the inverted microscope.Positive control cells expressing recombinant XyIE would turn yellow in the presence of catechol at day 4 after transfection.The supernatant of pAc-κ-CH3-VH_2E8-VL_2E8transfected Sf9 cells were harvested as primary recombinant complete virions pAc-κ-CH3-VH_2E8-VL_2E8CV (P0)for further amplifycation and the transfected Sf9 cells were collected for detection on day 7.2.3 Amplification of the recombinant baculovirus by infecting Sf9 cells with the recombinant complete virions:Preparing large stocks of virus by infecting Sf9 cells at a low MOI less than 1 (multiplicity of infection,MOI=The number of virions/The number of cells being infected)and harvesting supernatant 4-5 days post infection.Three passages were amplified and the virus stoke was to be applied to expression.2.4 Expression of recombinant antibody by Sf9 cells:For the protein expression the MOI was set between the range of 3-10 and the Sf9 cells were cultured under SFM condition.The supernatant was collected for detection 6days post infection.3.Identification of the recombinant protein:3.1 Cell immunofluorescence,SDS-PAGE,Western blot for analyzing the recombinant protein3.2 The activity analysis of the recombinant antibody by flow cytometry(FCM)3.2.1 The activity of the recombinant antibody in the supernatant was analyzed with indirect immunofluorescent assay:1x10⁶/tube of fresh Nalm-6 cells was prepared.Two tubes as tests were added with 100μl of the concentrated expression supernatant and the other four tubes as negative control were added with the same volume of concentrated regular medium or PBS (every group set 2 tubes).After 30 min of reaction,the cells were washed twice with PBS.Then FITC(fluorescence isothiocyanate)conjugated mouse anti-human IgG Fc fragment(MAH-Fc-FITC)and FITC conjugated goat anti-mouse Igκchain (GAM-κ-FITC)were added separately and reacted for 30min followed by two washes. FCM analysis was utilized to observe whether the chimeric antibody in the supernatant could bind to CD19 antigen on the Nalm-6 cell surface.3.2.2 The activity of the recombinant antibody in the supernatant was analyzed with blocking test:1x10⁶/tube of fresh Nalm-6 cells was prepared.One tube as test was added with 100μl of the concentrated expression supernatant and the other three tubes as negative controls were added with the same volume of concentrated regular medium or PBS(2 tubes). After 30 min of reaction,the cells were washed twice with PBS.The previous procedure was repeated once.Then 3 tubes were added with appropriate volumes of 2E8-FITC, one of the PBS tube was added withγ1-FITC and reacted for 30min followed by two washes.FCM analysis was utilized to observe whether the chimeric antibody in the supernatant could block the binding of 2E8-FITC to CD19 antigen on the Nalm-6 cell surface.3.2.3 The activity of the recombinant antibody in the cell lysate was analyzed with indirect immunofluorescent assay:1x10⁶/tube of fresh Nalm-6 cells was prepared.Two tubes as tests were added with 20μl of the treated infected Sf9 cell lysate and the other three tubes as negative controls were added with the same volume of uninfected Sf9 cells lysate or PBS(2 tubes).After 30 min of reaction,the cells were washed twice with PBS.Then MAH-Fc-FITC and GAM-κ-FITC were added separately and reacted for 30min followed by two washes. FCM analysis was utilized to observe whether the chimeric antibody in the infected Sf9 lysate could bind to CD19 antigen on the Nalm-6 cell surface.4.Modification of the formula for lysate to increase the dissolubility of the recombinant protein and the purification of the recombinant antibody:4.1 Breaking up the infected Sf9 cells with sterile water and ultrasound:2x10⁷ infected Sf9 cells were lysed in 1ml sterile water on ice with protease inhibitors and ultrasound with the power of 240W operated every 2 seconds for 20 times,Clear lysate from cellular debris by centrifuging at 12,000rpm for 30 min,The clear supernatant was harvested for further analysis.4.2 Dissolution,purification and refolding of the recombinant protein:2x10⁷ infected Sf9 cells were lysed in 1ml modified lysing solution on ice with protease inhibitors and ultrasound with the power of 240W operated every 2 seconds for 20 times, Clear lysate from cellular debris by centrifuging at 12,000rpm for 30 min,The clear supematant was harvested for further analysis.4.3 Purification of the recombinant protein from the lysate with protein A cartridge: Under routine conditions.5.Expression the recombinant protein with High Five cells5.1 For the protein expression the MOI was set between the range of 5-10 and the Sf9 cells were cultured under SFM condition.The supernatant was collected for detection 3days post infection.5.2 The activity analysis of the recombinant antibody by flow cytometry(FCM)5.2.1 The activity of the recombinant antibody in the supematant was analyzed with indirect immunofluorescent assay(The same as 3.2.1)5.2.2 The activity of the recombinant antibody in the supematant was analyzed with blocking test(The same as 3.2.2)Results1.Construction of pAc-κ-CH3-VH_2E8-VL_2E8recombinant baculovirus shuttle vector:1.1 Cloning and sequencing of the VH_2E8and VL_2E8genes:The 380 bps VH_2E8gene and the 330 bps VL_2E8gene were amplified and inserted into TA cloning vector followed by endonuclease digestion,sequencing and analyzing.The correct sequences were obtained which were named TA-VH_2E8and TA-VL_2E8, respectively.1.2 Construction of pAc-κ-CH3-VH_2E8-VL_2E8recombinant baculovirus shuttle vector:The TA-VH_2E8and pAc-κ-CH3 vector were digisted with endonuclease followed by the ligation with T4 DNA ligase,The recombinant was treated with the endonuclease BamH I,sequencing and the analyzing to confirm the correct insertion and orientation of the recombinant pAc-κ-CH3-VH_2E8.The VL_2E8was inserted into the pAc-κ-CH3-VH_2E8 with the same method.2.Transfection,virus amplification and expression of the recombinant antibody:2.1 The hybridoma cell line 2E8 was thawed and cultured under the regular RPMI 1640 medium supplemented with 20%of heat inactivated calf serum.The supernatant of 2E8 cells was collected for the positive control.2.2 Transfection of Sf9 cells by reconstructed baculovirus expression vector pAc-κ-CH3-VH_2E8-VL_2E8:The size of the transfected cells is bigger and the shape is irregular.The positive control cells turned yellow in the presence of catechol when harvested at day 4 after transfection which showed that the transfection was successful.The complete virion was named pAc-κ-CH3-VH_2E8-VL_2E8CV P0.2.3 Amplification of the recombinant baculovirus complete virion: Through 3 passages of amplification,high-titer virus stocks were ready for expression.2.4 Expression of recombinant antibody by Sf9 cells:According to the instruction manual,the cells were harvested at the time of 6 days post expression when about 30%of the live cells were remained3.Identification of the recombinant protein:3.1.1 Cell immunofluorescence:Since the Sf9 cells had green auto-fluorescence,the transfected Sf9 cells were checked by incubation with GAM-Fab-rhodamin,instead. 80%of cells were found positive.3.1.2 SDS-PAGE analysis: No specific protein band from the expression supernatant could be observed.3.1.3 Western blot analysis:Infected Sf9 cell lysate clearly showed the heavy and light chain bands while the uninfected Sf9 cell lysate did not.3.2 The activity analysis of the recombinant antibody by flow cytometry(FCM):3.2.1 No antibody activity could be detected by FCM in the expression supernatant of Sf9 cells.3.2.2 The expression supernatant of Sf9 cells could not block the parental antibody 2E8 binding to Nalm-6 cells.3.2.3 Nalm-6 cells incubated with handled infected Sf9 cell lysate shows 14.35% positive(VS 2.97%of the negative control)when labeled with GAM-Fab-FITC,The positive(28.67%)was even higher when labeled with MAH-Fc-FITC(VS 2.76%of the negative control).Which indicated that the functional antibody did exist in the infected Sf9 cell lysate.4.Improvement of the recombinant protein dissolubility and the purification of the recombinant antibody using the modified lysing solution formula:4.1 Breaking up the infected Sf9 cells with sterile water and ultrasound:The cell lysate had protein bands corresponding to heavy and light chains but much lighter than the conventional lysate when detected by SDS-PAGE.Western blot only showed the band of heavy chain indicating that this method was not proper to demonstrate the complete antibody.4.2 Breaking up the infected Sf9 cells with modified lysing solution and ultrasound:The infected Sf9 cells were lysed with the modified lysing solution and ultrasound.4.3 Purification of recomninant antibody with protein A cartridge: Only a small peak of the presumable protein was observed when eluted with elution buffer of pH6.0.No further peaks were seen when decreasing the pH of the elution buffer at pH5.0,pH4.0 and pH3.2.The eluted solution of the small peak was collected, concentrated and analyzed with Western blot together with the flowing through solution and the lysate.There were bands corresponding to heavy and light chains in the lysate and flowing through solution lanes but not in the elution solution lane.So the recombinant antibody was not detained by the cartridge.5.Expression of the recombinant protein with High Five cells:5.1 Expression of recombinant antibody by Sf9 cells:The size of the infected cells is bigger and the shape is irregular.The infected cells were harvested at the time of 3 days post expression.The supernantant was collected and concentrated for further detection.5.2.2 The activity analysis of the recombinant antibody by flow cytometry(FCM):5.2.1 No antibody activity could be detected by FCM in the expression supernatant of High Five cells.5.2.2 The expression supernatant of High Five cells could not block the parental antibody 2E8 binding to Nalm-6 cells.Conclusions1.The recombinant shuttle vector pAc-κ-CH3-VH_2E8-VL_2E8was successfully reconstructed.2.The complete virion was formed when the Sf9 cells were transfected with pAc-κ-CH3-VH_2E8-VL_2E8.3.The complete virions were successfully amplified to high-titer on passage 3. 4.The expression of the recombinant antibody was successfully and the functional antibody existed inside the infected Sf9 cells.5.However the recombinant antibody existed mainly inside the cells as precipitations. Secreted expression was detected from the supemantant of neither infected Sf9 nor High Five cells.Modification of the lysing solution formula and break up the cells with ultrasound did not increase the dissolubility of the novel protein from Sf9 cells.