Preparation And Identification Of Anti-CD44 Nanobodies

ObjectiveAcute myeloid leukemia(AML)is a blood heterogeneous cancer,the treatments of AML currently are mainly traditional chemotherapy and hematopoietic stem cell transplantation(HST).However,the cure rate was not high for low specificity of chemotherapy and low success rate of matched marrow donor.Noticeably,cure rate and overall survival rate of AML patients can be improved by targeting therapy for AML cells based on new generation targeted drug such as low molecular drug and immune therapy.For the past few years,antibody treatment has gained widespread social recognition and many antibody drugs have appeared on the market,such as anti-CD33 antibody,which applied for AML treatment.Additionally,other targets for AML treatment have been investigated.There are many studies show that CD44 molecule can express on AML cells' surface abundantly,and CD44 molecule also play a significant role in the occurrence and development of AML stem cells.However,traditional mAb is difficult to prepare and the process of humanized is too complex,so it is hard to transfer from theory research to practical application.In this study,CD44 was selected as the target,and nanobody was selected as research tool for its high stability and easy humanized,so that we can provide basis for research and development of new generation therapeutic drugs for AML.Method1.Preparation for CD44 antigen.Preparation of large amounts of CD44 antigen by expressing in E.coli and identification by SDS-PAGE and ELISA.2.Construction of phage library of nanobody against CD44.Lymphocyte cells were separated from immunized camel that immunized with CD44 antigen,and then using PCR technology to amplify nucleotide sequence of nanobody.Hereafter,phage display technology was used to construct phage library of anti-CD44 nanobody.3.Bio-panning of anti-CD44 nanobody.The constructed phage library was applied for biopanning through four cycles' “bind-elute-bind” with CD44 antigen before that PE-ELISA and sequence analysis,after then specific anti-CD44 nanobody was acquired.4.Expression and purification of anti-CD44 nanobody and primary identification involved with specificity and activity.Construct the expression vector of anti-CD44 nanobody before transformed into BL21 and then purify anti-CD44 nanobody through high affinity Ni-NTA column and gel filtration chromatography.In the end,specificity and activity of anti-CD44 nanobody was primary identification by ELISA.Results1.Preparation for CD44 antigen.Large amounts of CD44 antigen without tag was acquired by renaturation process and verified the antigenicity through ELISA.The purity of CD44 antigen reached over 95% by SDS-PAGE.2.Construction of phage library of nanobody against CD44.After the final immunization,the total titer IgG titer of immunized camel serum reached 1:10000,which indicated that the camel has immunoreaction to CD44 antigen.A large and diversified phage library of anti-CD44 nanobody was constructed finally.3.Bio-panning of anti-CD44 nanobody.After four rounds of bio-panning against CD44 antigen and PE-ELISA,twenty-nine colonies were selected to sequence.In the end,four nanobodies have different sequence by sequence alignment.4.Expression and purification of anti-CD44 nanobody and primary identification involved with specific and activity.The recombined vector of four nanobodies were constructed and transformed into WK6 to expression,and purity of purified nanobodies reached over 95% by SDS-PAGE.Additionally,the ELISA results indicated that four nanobodies have specific for CD44 and have better thermostability compared with antibody of anti-CD44.ConclusionThe results showed the success of phage library construction of anti-CD44 nanobody.Four nanobodies that specific to CD44 were screened from this phage library and have better thermostability.Therefore,four nanobodies can be further used to research of targeting AML cells and have the potential to become generation targeted drug.