Figure 1: Overview of our engineering cycle.

Gibson Assembly

Figure 2: Principle of Gibson assembly. Primers are designed so that they carry extensions complementary to each other to clone based on base pairing between backbone and insert.

We opted to use Gibson assembly as our method of choice when it comes to cloning. Similar to classical restriction cloning this method relies on complementary DNA overhangs shared between the backbone and the inserted sequence, however the major difference is that in Gibson cloning overhangs are attached to the primers used for PCR-amplification of both the backbone and insert (Figure 2) 1,2. Primers in general were designed to have a melting temperature of 60-65°C and overhangs to show a melting temperature of 60°C. These components were then used to run Q5 PCRs which turned out to need optimization in some cases. Therefore, we tweaked the conditions to find more optimal settings. It turned out that mainly the annealing temperature was influencing the PCR efficiency (Figure 3) 3.

Figure 3: Improvement of PCR conditions. An example is shown here for the Q5 PCR of the backbones of constructs C, P, Q, U and V. Under standard conditions hardly any amplicon was observable. Upon increasing annealing time and temperature the expected amplicon was significantly more detectable.

This however seemed to be only one source of errors. Additionally, some of our originally designed primers were not well suited when it comes to self- and cross-dimerization or formation of stable secondary structures (Figure 4).

Figure 4: Example of primer redesigning. An example is shown for primers with a relatively high tendency to form hairpins. Therefore, we redesigned our intended primers as well as their overhangs to allow for more efficient PCR.

After all we managed to successfully clone most of our constructs (Figure 5).

Figure 5: Example of cloning success. An example is shown for sequencing data of construct B which confirmed successful cloning analyzed using the alignment function in Geneious.

Antibody production and purification

Figure 6: Workflow for Antibody production in Expi293 cells. Here Expi293 cells were transfected using lipofection with our cloned constructs. Supernatant was harvested after five days of incubation followed by Protein A purification. Created with BioRender.com.

To test our designed and cloned constructs we transfected them into Expi293 cells to check antibody production. Here, we used a cationic, lipid-based transfection reagent to transiently introduce DNA into these cells.4 After five days the supernatant was harvested and dialyzed to prepare for Antibody purification via Protein A columns (Figure 6). Here some constructs yielded more protein than others (Figure 7).

Figure 7: Example results of the Protein A purification for construct F.

Antibody testing

Since we decided to look into multiple targets it was necessary to develop up with assays for analyzing the functionality of our modified antibodies.

ELISA - HBsAG

Figure 8: Principle of an indirect ELISA. For an indirect ELISA the antigen of interest is coated onto 96-well plates and incubated with a primary antibody specific for the antigen. Afterwards a detection antibody is applied which binds to the constant region of IgGs and allows for a color reaction as a readout for successful antigen binding. Created with BioRender.com.

For our Antibodies targeting the surface antigen of hepatitis B we decided on an indirect ELISA to show functionality. A 96-well plate was coated with HBsAG followed by an incubation with our produced Antibody. Afterwards this plate was incubated with a secondary antibody specific for human IgGs which is coupled to HRP to allow a colorimetric readout, thus allowing to determine weather the primary antibody is functional (Figure 8) 5.

Immunofluorescence – CAIX-expressing cancer

Figure 9: Principle of fluorescence microscopy. In immunofluorescence microscopy cells are tagged with a primary antibody against an antigen of interest, followed by a fluorophore-coupled antibody specific for the constant region of IgGs, allowing imaging in a fluorescence microscope. Created with BioRender.com.

Similar to the ELISA strategy we imagine that we could validate functionality of our anti-CAIX Antibodies via labeling CAIX-expressing cells with the antibody followed by a secondary antibody, again specific for human IgGs fluorescently labeled to allow visualization of our construct binding by immunofluorescence (Figure 9) 6.

Neutralization assay hemolysin

Figure 10: Principle of neutralization assays. Here the toxin is incubated with neutralizing antibodies, allowing for hemolysis as a readout for efficient neutralization. Created with BioRender.com.

We also wanted to test a different functional assay, demonstrating the neutralization capacity of our modified antibodies, allowing us to draw conclusions whether our constructs remain functional regardless of the added domains. Our assay of choice was a classical hemolysis assay, where hemolysin of S. aureus is mixed with different anti-hemolysin antibody derivatives and added to blood agar 7,8. Successful neutralization would be demonstrated by less lysis of red blood cells (Figure 10). Ideally all our constructs show similar potential to neutralize the toxin, thus indicating that the function of antibody is unaffected by addition of targeting domains.

Transcytosis assay

Our final target is the blood brain barrier (BBB), which poses a great hurdle when it comes to shuttling therapeutics into the brain. Thus, we decided to try and attach domains to our anti-hemolysin antibodies binding the transferrin-receptor (TfR) on brain microvascular endothelial cells. Therefore, we first needed testing of general functionality of these constructs (see neutralization-assay) and validation of successful targeting of the TfR. The simplest way of testing and quantifying transcytosis is a Transwell-System covered in HUVEC cells which are commonly used as a simple BBB-endothel model 9,10. One simply added the modified antibodies into the Transwell, incubated for a day and analyzed the antibody amount in the lower well. Ideally our TfR-targeting Anti-Hemolysin Antibodies (both scFv-anti-TfR & Melanotransferrin) show larger amounts of ABs than the ones without additional domain (Figure 11).

Figure 11: Principle of Transwell-Assays. This setup is a simple model of the BBB often used to analyze cell migration. In this case it can be used to show antibody transport across endothelial cells which can be influenced by modifications on antibodies.

One could also take the set-up even further and use a perfused 3D-system simulating the actual BBB environment more closely, allowing for more accurate predictions of TfR-targeting efficiency.

Stable integration

Figure 12: Design of our homology template. It consists of homology arms on either site of the gene cassette, allowing for locus specific stable integration. The cassette itself contains a selection marker in the form of truncated EGFR and the antibody of choice.

Since B-cell engineering relies on the introduction of a desired antibody into the endogenous IgH locus of B-cells, we planned to insert this sequence via homologous recombination (HR) orthotopically. HR is a mechanism to repair DNA double strand breaks, using a homology template, naturally this is role is taken by the sister chromosome and play an important role during meiosis, allowing for crossing over events to happen 11,12. This can be exploited and used to stably integrate DNA directed into a specific locus without the usage of viral delivery systems which commonly insert the template randomly 13. To make use of HR in a bioengineering setting, two conditions are prerequisites, DSBs and a donor that shows homology. DSBs can be easily introduced using CRISPR/Cas9 equipped with guides specific for the desired locus. The donor-homology is also simply achievable by adding ~500 bp of the DNA sequence upstream and downstream of the Cas9-cutting site to the homology template (LHA & RHA), depending on the size of the insert 14. Our designed homology template first contains a strong polyA signal to terminate any transcription that could be going on and allowing initiation of a new transcript under the control of the endogenous IgH promoter 15,16. To allow selection of successfully transfected clones truncated EGFR was introduced which already serves as a selection marker in CAR T-cells and in adoptive T-cell therapy 17. This is followed by our antibody construct of choice (Figure 12). In summary this approach allows for stable integration of a gene cassette into the endogenous locus without using viruses at a reasonable efficiency 13.

Footnotes

  1. Biolabs NE. How Gibson Assembly® is Changing Synthetic Biology | NEB; 2023 [cited 2023 October 11] Available from: URL: https://www.neb.com/en/tools-and-resources/feature-articles/gibson-assembly-building-a-synthetic-biology-toolset. ↩

  2. Team:Freiburg/Project/Classic vs Gibson - 2015.igem.org; 2022 [cited 2023 October 11] Available from: URL: https://2015.igem.org/Team:Freiburg/Project/Classic_vs_Gibson. ↩

  3. Biolabs NE. PCR Troubleshooting Guide | NEB; 2023 [cited 2023 October 11] Available from: URL: https://www.neb.com/en/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide. ↩

  4. Thermo Fisher Scientific (2 September 2020). Expi293 Expression System User Guide (Pub. No. MAN0019402 B.0). ↩

  5. Mandy Alhajj, Muhammad Zubair, Aisha Farhana. Enzyme Linked Immunosorbent Assay. In: Alhajj M, Zubair M, Farhana A, editors. StatPearls [Internet] StatPearls Publishing 2023. ↩

  6. Im K, Mareninov S, Diaz MFP, Yong WH. An Introduction to Performing Immunofluorescence Staining. Methods in molecular biology (Clifton, N.J.) 2019; 1897: 299–311 [https://doi.org/10.1007/978-1-4939-8935-5_26][PMID: 30539454] ↩

  7. Sæbø IP, Bjørås M, Franzyk H, Helgesen E, Booth JA. Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity. International Journal of Molecular Sciences 2023; 24(3) [https://doi.org/10.3390/ijms24032914][PMID: 36769243] ↩

  8. Ridder MJ, Daly SM, Hall PR, Bose JL. Quantitative Hemolysis Assays. In: Staphylococcus aureus. Humana, New York, NY 2021; 25–30. ↩

  9. Zhao N, Guo Z, Kulkarni S, et al. Engineering the human blood-brain barrier at the capillary scale using a double-templating technique. Advanced Functional Materials 2022; 32(30): 2110289 [https://doi.org/10.1002/adfm.202110289][PMID: 36312050] ↩

  10. Sade H, Baumgartner C, Hugenmatter A, Moessner E, Freskgård P-O, Niewoehner J. A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding. PLOS ONE 2014; 9(4): e96340 [https://doi.org/10.1371/journal.pone.0096340][PMID: 24788759] ↩

  11. Li X, Heyer W-D. Homologous recombination in DNA repair and DNA damage tolerance. Cell Res 2008; 18(1): 99–113 [https://doi.org/10.1038/cr.2008.1][PMID: 18166982] ↩

  12. Xue C, Greene EC. DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing. Trends in genetics : TIG 2021; 37(7): 639–56 [https://doi.org/10.1016/j.tig.2021.02.008][PMID: 33896583] ↩

  13. PubMed Central (PMC). Principles of Genetic Engineering; 2023 [cited 2023 October 11] Available from: URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140808/. ↩ ↩2

  14. How to Design Homologous Recombination Template for CRISPR; 2023 [cited 2023 October 11] Available from: URL: https://www.benchling.com/blog/how-to-design-homologous-recombination-template-for-crispr. ↩

  15. Nahmad AD, Raviv Y, Horovitz-Fried M, et al. Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion. Nature Communications 2020; 11(1): 5851 [https://doi.org/10.1038/s41467-020-19649-1][PMID: 33203857] ↩

  16. Nahmad AD, Lazzarotto CR, Zelikson N, et al. In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice. Nature biotechnology 2022; 40(8): 1241–9 [https://doi.org/10.1038/s41587-022-01328-9][PMID: 35681059] ↩

  17. Paszkiewicz PJ, Fräßle SP, Srivastava S, et al. Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia. The Journal of Clinical Investigation 2016; 126(11): 4262–72 [https://doi.org/10.1172/JCI84813][PMID: 27760047] ↩