Considering the unique advantages of microbiological therapy in treating cancer, we selected cancer as one of our application and built a double-status switch in our platform to solve the problem of biocontainment and biosafety, which is used to lower the off-target effect simultaneously.
In our project, we used CD47 nanobody as a drug molecule to block CD47 on the surface of cancer cells, so that they could be killed by microphages.
Take advantage of the natural targeting property of certain microorganisms to approach the microoxygen area near the tumor tissue, we put CD47 nanobody gene under an anaerobic induced promoter so that we can primarily lower the off-target effect.
To make sure the release of CD47 nanobody could not hurt healthy tissues, we put a heat-inducible switch before the lysis gene, then heat the cancer tissues with our amazing hardware.
To further make sure the release of CD47 nanobody could not hurt healthy tissues, we used our thermo-sensitive switches to construct a biosafety module besides original biocontainment module, in which the cold-inducible ON-switch for the toxin gene and the heat-inducible ON-switch for the drug releasing gene.
Cancer is a generic term for a large group of diseases characterized by the growth of abnormal cells beyond their usual boundaries that can then invade adjoining parts of the body and/or spread to other organs. According to the data from WHO, cancer is the second leading cause of death globally among all noncommunicable diseases (NCDs) and is estimated to account for 9.6 million death in 2018. It indicted that cancer is still one of the most difficult diseases waiting for human to solve.
Figure 1. Top 4 causes of death among all noncommunicable diseases (NCDs) worldwide in 2018
It's worth noting that among all cancers, colorectum cancer, one of the intestinal cancers, accounts for 10%, which is a major kind of all cancers.
Figure 2. The number and proportion of different types of cancer worldwide in 2018 (both sexes, all ages)
There are many treatment strategies for cancer, in which molecular-based therapeutics is one of the major therapeutics in clinical applications. Recently, nanobodies have also been applied to cancer treatment.
In our project, we chose CD47 nanobody as the anticancer drug protein. CD47 is a common anti-phagocytic receptor that is overexpressed in several human cancer types. The CD47 nanobody can bind to the CD47 protein on the surface of cancer cells, which makes it unable to form an anti phagocytic pathway, thus enhancing the macrophage's ability to clear tumor cells.
Figure 3. CD47 nanobody interacts with CD47 on cancer cells
However, because most molecular drugs, including nanobodies, are not able to target cancer cells with high specificity, systemic receptor blockade can result in severe side effects to human body. For example, CD47 not only expresses on cancer cells, but has high expression on red blood cells and platelets so that systemic CD47 blockade can result in anemia and thrombocytopenia. Therefore, targeting cancer cells with high specificity and efficacy is the most urgent problem in the treatment of cancer.
Microbial therapeutics have irreplaceable role and natural advantages in solving off-target problem.
Because the oxygen concentration around the cancer tissue is generally low, some anaerobes, like Bifidobacterium, tend to colonize tumor tissue. Based on this character, we connected the gene of CD47 nanobody behind the anaerobic induced promoter, PfnrS, to ensure its expression is only near the cancer tissue.
Figure 4. CD47nanobody under the anaerobic induced promoter, PfnrS
The combination of microbial therapeutics and our platform
To achieve lower off-target effect, we used our thermo-sensitive switches to construct a biosafety module besides original biocontainment module. Combining both of them together, we built a double-status switch.
As shown in Figure 5, the biocontainment module consists of parts in cold-inducible ON-switches, including TEVts, thermo-sensitive mutants of TEV protease, and TFts, thermo-sensitive mutants of transcription factors. This module can open the expression of toxin gene when temperature transforms to 25℃ from 37℃ to prevent engineered microorganism escaping to environment.
Another is biosafety module that consists of part in heat-inducible ON-switches, TCI42, thermo-sensitive mutant of transcription factors CI. This module can open the expression of lysis gene and produce lysis protein to make engineered bacterial release CD47nb when temperature rises to 42℃ from 37℃ using our hardware to heat on cancer tissues. This module can prevent engineered bacterial release drug molecules on healthy tissues.
See more information of our hardware from here.
Figure 5. The combination of double-status switch and the circuit of microbial therapeutics
Combining this double-status switch with the circuit of microbial therapeutics, we can lower the off-target effect significantly and make full use of unique advantage of microbial therapeutics.
We have also characterized this double-status switch using mRFP and GFP as reporters.
Based on the temperature response curve of cold-inducible ON-switches and heat-inducible ON-switches, we chose the best mutant, TEVts#6 and TCI42 to build our double-status switch. But actually, users can choose other thermo-sensitive protease or transcription factors to build other more suitable double-status switches based on their needs.
AS shown in Figure 6, our cold-inducible ON-switch works when the temperature is low and then express green fluorescence. When the temperature rises, the activity of cold-inducible ON-switch is inhibited and the heat-inducible ON-switch works. Therefore, we can turn on different genes’ expression at different temperatures. Similar with our single thermo-sensitive parts, this double-status switch also shows high performance and versatility.
Figure 6. The induction curve of the double-status switches (TOP10)
Incubated in the anaerobic environment in vitro, our engineered bacteria successfully expressed CD47 nanobody, indicating its potential for precisely controlled expression near the cancer tissues.
Figure 7. The SDS-PAGE analysis result of CD47 nanobody purification product. An around 14kDa protein can be observed, which is corresponding to the theoretical molecular weight of CD47 nanobody (17.42kDa) our bacteria are supposed to express. The bacteria were incubated in a 500mL conical flask in the anaerobic environment.
To control the unintended proliferation of our engineered bacteria into healthy tissues, also prevent the possible side effect to healthy tissues caused by CD47nb, our design of combining TCI42 with a lysis gene, ΦX174, precisely restricted the bacteria lysis and the drug release in the cancer tissues specifically heated by our hardware. We tested the efficiency of bacteria lysis under 42℃. As we expected, we could observe a prominent decrease of OD600 when our engineered bacteria were incubated under a circumstance of 42 ℃, where the controlled group bacteria were observed to grow in a steady increasing trend. What’s more, in the environment of 37 ℃, the growth of our bacteria was similar to the controlled group bacteria, indicating rather low leakage or burden of the whole system.
Figure 8. The curve of OD600 change of experiment/ controlled group under 37/42℃ condition.
These results further confirmed that our ARK.micro could function as a unprecedentedly promising platform to promote the development of microbial therapy, meanwhile further exploiting its unique advantages on cancer treatment.
WHO(World Health Organization) (2018). World health statistics.
Zu C , Wang J . Tumor-colonizing bacteria: A potential tumor targeting therapy[J]. Critical Reviews in Microbiology, 2014, 40(3):225-235.
Chowdhury, S., Castro, S., Coker, C., Hinchliffe, T. E., Arpaia, N., & Danino, T. (2019). Programmable bacteria induce durable tumor regression and systemic antitumor immunity. Nature Medicine, 25(7), 1057-1063.
Willingham S B , Volkmer J P , Gentles A J , et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(17):6662-6667.