Fig 1.
Antigen validation and quantification of eluted phages thorough rounds of selection A) Antigen validation, the purity and integrity of human recombinant TK1s produced in E. coli cells and Expi293F cells were assessed in SDS-PAGE and validated using Western blot with the anti-TK1 antibody ab91651. B) Enrichment of TK1 binders through 5 rounds of selection. The number of binders was estimated based on titrations of eluted phages used to infect TG1 bacteria. C) A representative image of a viral titration to determine the number of eluted phages. TG1 bacteria were infected with serial dilutions of the eluted phages after each biopan. The infected TG1 bacteria were then plated on TYE amp plates.
Table 1.
Enrichment of anti-TK1-sdAb phages through 5 rounds of selection.
Fig 2.
Selection and expression of anti-TK1-sdAb phages.
A) Analysis of 80 clones using monoclonal phage ELISA was performed after each round of selection. B) Polyclonal phage ELISA using the total purified phages per round of selection. In both A and B the overall signal and number of positive clones increased after each round of selection. C) Detection of anti-TK1-sdAb phages using dot blot with an anti-VSV-G-HRP antibody. Also, verification of packaging of the library from PEG purified phages after initial amplification of the sdAb library. D) Representative image of monoclonal phage ELISAs during the rounds of selection.
Fig 3.
Confirmation of positive clones.
To verify the positive clones found during the initial screening, their capacity to reproduce a positive signal was tested. The strongest positive clones were selected through the different rounds of selection. Bacteria corresponding to each of these clones were streaked for a second time on TYE amp plates. New cultures were grown from a single colony and infected with KM13 helper phage to induce the production of their respective anti-TK1 phage-sdAbs. A) Positive anti-TK1 phage-sdAbs tested in monoclonal phage ELISA. The capacity to bind TK1 and the stability of each clone was confirmed. B) Representative image showing the color development generated by the positive clones and plate layout indicating the position of each clone that was tested.
Fig 4.
Testing of the anti-TK1 sdAbs with dose response curves.
A) Dose response curves corresponding to anti-TK1 sdAb fragments obtained through various rounds of selection. The data was Log transformed and the curves were analyzed using a 4-parameter non-linear regression. B) A representative image of the colorimetric reaction showing that the signal of each anti-TK1 sdAb fragment is proportional to the concentration of TK1 protein. Negligible or no significant signals were produced in the blanks.
Fig 5.
Sensitivity and specificity of the anti-TK1-sdAb fragments.
A) The 14 anti-TK1 sdAb fragments were tested against a minimal fixed concentration of 23 ng/ml of human TK1 produced in human cells (H-TK1). The sdAbs 4-H-TK1_A1 and 4-H-TK1_D1 produced the highest signals. B) Dilution curves with H-TK1 and the best clones. Concentrations ranged from 500 ng/ml to 3.9 ng/ml, the fragments kept their binding properties after being expressed as sdAb fragments C) siRNA TK1 knock down validation. TK1 was knocked down in A549 cells. The knockdown was validated using the commercial anti-TK1 antibody ab91651. D) Validation of the best 2 anti-TK1-sdAbs. The binding capacity of the sdAb fragments was tested against 3 different sources of human recombinant TK1, cell lysate from the cancer cell line A549 and cell lysate from A549 TK1 knockdown. It can be seen that both fragments bind to all recombinant TK1 proteins. A significant difference can be seen in the signal coming from the normal cell lysate in comparison with the TK1 knockdown cell lysate.
Fig 6.
Amplification and ligation of anti-TK1-dAb fragments into the pET-scFv-T expression vector.
A) a representative image of a PCR showing amplification of anti-TK1 dAb fragments. B) Restriction enzyme analysis with NcoI and NotI enzymes of pET-anti-TK1-dAb constructs. C) Map of a pET-anti-TK1-dAb construct. Fragments are ligated into the pET-scFv plasmid using NcoI and NotI restriction sites. D) Alignment of the 4-H-TK1_A1 and 4-H-TK1_D1 sdAb sequences shows that the differences of the sdAbs are in their CDRs.
Table 2.
The deduced amino acid sequences of the best two anti-TK1 sdAb fragments isolated through phage display.
Fig 7.
The 3D structure of the two best anti-TK1-sdAb fragments based on their deduced amino acid sequences.
The anti-TK1-sdAb fragments were modeled using the GalaxyWeb TBM server. The most stable structures were then visualized using the VMD 1.9.3 software. CDRs were mapped by analyzing the anti-TK1 sdAb amino acid sequences with the IgBlast tool from NCBI. A) H-4-TK1_A1 sdAb. B) H-4-TK1_D1 sdAb. C) High ambiguity driven protein-protein docking analysis using the HADDOCK 2.4 web server. The most stable structures of the H-4-TK1-A1 sdAb fragment and human TK1 protein monomer were analyzed to predict their protein-protein interactions. The analysis shows that in the most stable TK1-TK1sdAb complex, the TK1-sdAb would bind to TK1 through its CDRs. The CDRs would interact with the α1-ribbon towards the N-terminus and two regions close to the β-ribbons towards the c-terminus of the TK1 molecule. D) Docking between the anti-TK1 sdAb H-4-TK1_A1 and the monomer of human TK1 from the 1XBT crystal structure.
Fig 8.
Expression and purification of anti-TK1 sdAbs.
A) Detection of anti-TK1 sdAb fragments with anti-His-HRP antibody using Western blot and SDS-PAGE analysis. B) Quantification of the His-tag purified sdAb fragments with BCA assay. The protein yields were between 1–4 mg/ml of purified sdAb. C) Western blot and SDS-PAGE analyses of protein A purified anti-TK1 sdAb H-4-TK1_A1. The antibody fragments can alternatively be purified with protein A purification. D) Quantification of protein A purified anti-TK1 sdAb H-4-TK1_A1.
Fig 9.
sdAb ELISA and Western blot analyses.
A) Purified recombinant anti-TK1 sdAbs kept their binding properties to H-TK1 after being expressed in E. coli and His-tag purified as observed using sdAb ELISA. B) The anti-TK1 sdAbs showed binding to recombinant human TK1 produced in both E. coli (E-TK1) and human cells (H-TK1). The fragments were validated by comparing their binding to TK1 in cell lysates of A549 cells (100 μg) and A549 TK1 knockdown (100 μg). C) Detection of TK1 in cell lysates of 4 different cancer cell lines and normal MNCs (20 μg each). The fragments were able to detect the tetrameric form of TK1, controls included commercial anti-TK1 abcam91651 and GAPDH.
Fig 10.
Detection of mTK1 on cancer cells and healthy MNCs using anti-TK1 sdAb fragments.
A) NCI-H460 cells were stained with the purified phage-anti-TK1 sdAb fragments, a shift in the population could be observed using both phage-sdAb fragments. B-E) Expressed and purified anti-TK1-sdAb_A1 and D His-tagged and VSV-G tagged were used to stain NCI-H460 (lung), HT-29 (colon), HCC1806 (Triple negative breast cancer) and healthy lymphocytes. The TK1 levels detected using the anti-TK1 sdAb fragments were comparable to the levels detected by commercial anti-TK1 antibody. The highest levels of mTK1 were detected on NCI-H460, followed by HT-29 and then HCC1806. No significant binding was detected on normal MNCs with the anti-TK1 sdAb fragments nor with the commercial anti-TK1 antibody. F) The expression levels of mTK1 on cancer cells and the binding of anti-TK1 sdAb fragments. Only A1-His sdAb showed consistent binding similar to commercial antibody ab91651.
Fig 11.
Expression and testing of the Anti-TK1 sDab-IgG1 antibodies.
A) Construction of an anti-TK1 sdAb fragment. Restriction analysis of pFUSE-anti-TK1-sdAb plasmids and their respective maps. B) Expression and detection of anti-TK1-sdAb-IgG1 antibodies. As can be observed sdAb-IgG1 fusions produced higher molecular weight bands compared to IgG1-no sdAb constructs. No human IgG was detected in non-transfected CHO.K1 cells. C) After fusing to an Fc the anti-TK1-sdAb antibodies were able to detect mTK1 on NCI-H460 cells. IgG1-no sdAb control did not bind to NCI-H460 cells.
Fig 12.
Anti TK1-sdAb-Fc antibodies elicit ADCC responses against NCI-H460 cells expressing mTK1.
A) Optimization of several antibody concentrations. The decrease in GFP+ NCI-H460 cells co-cultured with MNC over time is proportional to the concentration of anti-TK1-sdAb-IgG1/ml used. B) A significant ADCC response is elicited by MNCs against NCI-H460 cells when anti-TK1-sdAb-IgG1 antibodies are added compared to controls. After 96 hours the cell killing can visually be appreciated to be more severe in the cells treated with the anti-TK1-sdAb-IgG1 antibodies. C) Percentage of cell killing calculated every 24 hours. The percentage of cell killing was significantly higher after 72 and 96 hours when anti-TK1-sdAbs were added in comparison to IgG1 isotype control and effector only-no sdAb control.