A 4-year-old female alpaca introduced from China was subcutaneously injected with 100 mug purified human TIM-3 protein (Sino Biological, Inc.) plus adjuvant FAMA (Gerbu, Germany) once a week. Peripheral blood was collected from jugular vein before the first injection and 7 days after the last injection, and serum was isolated by Sepmate tubes for comparison of the antibody titer by ELISA. A 96-well microplate well (Nunc, Danmark) was coated with 100 muL 1 mug/mL TIM-3 protein, and blank wells were coated with PBS. After washing and blocking, the preimmunization and postimmunization serum was added at serial dilutions. HRP-conjugated goat against Llama antibody (Thermo) was added as the II antibody, followed by the addition of ABTS reagent for the development of reaction. The measurement of OD (405 nm) was performed with the use of a microplate reader (Bio-Rad). All animal experiments were carried out with the approval of the relevant ethics committee and in strict accordance with the relevant regulations on the care and use of experimental animals in the international code of ethics and national health guidelines.
Using Sepmate tubes (STEMCELL) and Lymphoprep (STEMCELL), 100 mL peripheral blood was drawn to isolate peripheral blood mononuclear cells (PBMCs) 7 days after the last immunization. Trizol (Invitrogen)-separated total RNA from PBMCs was subjected to cDNA synthesis by SuperScript II reverse transcriptase (TAKARA) using random 6 primer. Then, via nested PCR, VHH gene amplification was done using primers CALL001 (5'-GTCCTGGCTGCTCTT CTACAAGG-3') and CALL002 (5'-GGTACGTGCTGTTGAACTGTTCC-3'). The heavy chain antibody fragments (700 bp) were subjected to agarose gel (1%) electrophoresis and extraction via Quick Gel Extraction (Thermo) and then used as the template for the second PCR via primer VHH-sense (5'-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3') and VHH-antisense (5'-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3'). These primers, designed for framework regions 1 and 4, involved restriction sites Pst 1 and Eco91I. The second PCR product was electrophoresed and purified by PCR Purification Combo Kit (Thermo). The phagemid vector pMES4 was digested with Pst 1, XbaI, and Eco91I restriction enzyme, while purified PCR product was digested with Pst 1 and Eco91I. The triple-digested pMES4 and double-digested PCR product was ligased with T4 DNA ligase enzyme (Invitrogen). After transfecting with the recombinant vector, chemically competent E. coli TG1 cells were subcultured on ampicilin-containing LB agar plates. Twenty colonies were randomly picked, and colony PCR was perform by vector primers GIII (5'-CCACAGACAGCCCTCATAG-3') and MP57 (5'-TTATGCTTCCGGCTCGTATG-3') to ensure that VHH was inserted into most of the transformants.
Following infection of transformed TG1 with M13K07 helper phage (Invitrogen), the VHH library was displayed on phage. Infected bacteria were cultured overnight in 2x TY medium comprising ampicillin (AMP; 100 mug/mL) and kanamycin (50 mug/mL) while shaking (200 rpm). The medium was centrifuged for 15 min at 3200 g in 4 C. The supernatant was mixed with 20% ice cold PEG6000/NaCl and incubated for 30 min on ice. After centrifuged for 10 min at 3200 g in 4 C, such resuspended the phage pellet in ice cold PBS for biopanning.
Three rounds of biopanning were performed on microplate wells coated with 100 muL 1 mug/mL TIM-3 protein in PBS to screen anti-TIM-3 phage displaying nanobodies. After blocking with 5% milk in PBS and 5 PBST rinses (250 muL; 0.05% (V/V) Tween 20 in PBS), over 1011 p.f.u phage library preincubated in 0.1% milk in PBS was added into both well coated with TIM3 protein and PBS. Wells were washed with PBST for 15 times and eluted by adding 100 muL 0.25 mg/mL trypsin solution per well for 30 min. The eluted phage was transferred into 100 muL 10%BSA-PBS to stop protease reaction. The infecting log phase E. coli TG1 cells were obtained by serial dilution of 10 muL phage eluted from wells coated with TIM-3 and PBS. 5 muL drops of TG1 infected by dilutions of phage were pipetted on LB agar plate comprising AMP. The enrichment of phages carrying TIM-3-specific VHHs was identified by comparing the titers between phages eluted from antigen-coated wells and those from PBS wells. The rest of the eluted phages were applied to infecting log phage E. coli TG1 and cultured overnight to amplificate phage library as mentioned above for next round of biopanning.
 E. coli TG1 cells infected by second and third round of phage sublibrary were subjected to overnight cultivation (37 C) on AMP-containing LB agar plates. 66 colonies from second rounds and 132 colonies from third-round sublibrary were picked randomly and cultured in TB medium containing AMP at 200 rpm in 37 C. Log phase E. coli TG1 were induced with 1 mM IPTG (Thermo) overnight (28 C). Via osmotic shock, the periplasmic proteins were extracted for ELISA using anti-HIS antibody (1 : 1000, Thermo), following by HRP-conjugated anti-mouse antibody(1 : 2000, Thermo) and TMB reagent. Anti-TIM3 (1 : 1000, Cell Signaling Technology) and HRP-conjugated anti-rabbit (1 : 2000, Cell Signaling Technology) antibodies were also added successively as the positive control. Positive clones were indicated if the OD450 in antigen-coated wells was three times higher than OD450 in PBS-coated wells. Clones considered positive were chosen for fragment sequencing and were classified according to CDR3 region.
The TIM3-specific nanobody gene sequence was inserted into pHEN6c plasmid and transfected into E. coli WK6. His-tagged recombinant nanobodies were expressed in 1 L TB medium through 1 mM IPTG inducement and extracted via osmotic shock, followed by immobilized metal affinity chromatography (IMAC) purification with the use of a Ni-NTA column. The buffer of nanobody was exchanged from imidazole elution buffer to PBS by dialyzing.
Measurements of nanobody expression and purity were made by Coomassie brilliant blue stained 4%-20% SDS-PAGE and Western blot. The nitrocellulose membranes were transferred and blocked with 5% milk in TBST, and detected using anti-HIS (1 : 1000, Cell Signaling Technology) and HRP-conjugated anti-mouse (1 : 2000, Cell Signaling Technology) antibodies.
Affinity of TIM3-specific nanobody was firstly determined by ELISA. Briefly, a 96-well plate coated by TIM3 protein or PBS overnight in 4 C and blocked with 5% milk PBS. A serial 10-fold dilution of nanobodies in 0.1% milk PBS was added. Antibodies anti-HIS (1 : 1000, Cell Signaling Technology) and HRP-conjugated anti-mouse (1 : 2000, Cell Signaling Technology) as well as TMB reagent were added into wells after washing, successively. The OD450 was measured, and the combination curve was drawn by GraphPad prism.
All statistical analysis was performed with SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). All data are reported as the mean +- standard deviation (SD). Paired t test was used to compare the results in two matched groups. One-way ANOVA were applied to compare the differences between groups. P < 0.05 was considered as statistically significant.