DNA Logic Meets Affibodies: DNA Circuits Enable Smart Drug Delivery!

Antibody-drug conjugates are the latest fashion in biology. It turns out that targeting cytotoxic drugs only where they are needed is a pretty good strategy!

Can DNA nanotechnology make them even better?

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Antibodies are the workhorses of biology.

They are everywhere and do everything. Of course, they’re at the center of your body’s immune response. But they’re also the basis of your Western blot, the reason diagnostic tests work, and even power pregnancy tests!

And they’re huge in medicine too!

Especially monoclonal antibodies (mAbs). These proteins recognize a single target with high affinity. And with modern bioengineering, you can make a mAb that binds to pretty much any extracellular protein!

This binding can have a few outcomes:

And the applications are many. Cancer therapy is the most common, but autoimmune diseases, infections, or blood clot prevention are also up there!

But scientists, of course, wanted to make them even better.

The problem with antibodies?

They’re mostly passive. They bind, they block something, or they mark a cell for destruction, and that’s it. Not exactly an active contribution! So, how do we make them more dynamic?

Well, you attach a drug to them!

This is the idea behind antibody-drug conjugates (ADCs). They have 3 parts:

Now, the antibody is a guided missile for cancer cells. ADCs work, with over 20 FDA-approved therapies across solid and non-solid tumors. And over 400 more are in development!

But they still have a major problem: tissue penetration.

Antibodies are big, around 150 kDa. They can’t easily enter the tumor site. In some cases, less than 1% of the administered dose reaches the tumor! Not good.

One solution? Smaller binders.

Affibodies are one of the most common alternatives to antibodies, and they have a few nice advantages:

Affibodies have already shown their power in clinical trials! Can we make them even better?

This is where today’s paper comes in!

The authors combined affibodies (and sometimes aptamers!) with DNA circuits to turn cell surface recognition into amplified payload delivery. This new drug delivery system combines the strengths of ADCs with DNA nanotech: amazing!

The DNA circuit they use is called a hybridization chain reaction (HCR).

HCR is an enzyme-free DNA amplification method. It has two parts:

The initiator opens H1. H1 can now open H2, which will bind to another copy of H1, and so on. The result of this cycle is a long, double-stranded DNA polymer.

HCR acts as an amplification system: for example, if you have a fluorophore on the hairpins, you will get multiple copies and a stronger signal. Like a limited, enzyme-free version of PCR!

To turn this system into a DNA circuit, the team used a split initiator. Instead of one DNA strand, the trigger is split into two pieces, S1 and S2, each attached to a different biomarker binder.

When the two biomarkers are close enough, S1 and S2 bind to H1, form a three-way junction, and start the HCR! This creates a logic AND gate: the reaction starts only when both biomarker 1 and biomarker 2 are present.

The core idea is:

The team used their DNA-binder system for 3 applications.

They first tested the gate using different biomarker combinations:

The team tested the system’s specificity using fluorescently labeled hairpins.

The HCR signal is strong only when the right pair is present. For EGFR+PD-L1, A-431 cells show the strongest response. If the biomarker is not present, there is almost no signal! The same for EGFR+PTK7. And the most responsive cell lines show a 59x amplification!

The authors also showed a small variant of the system. By using two copies of the same biomarker binder, the HCR can report the local density of that receptor! In A-431 cells, EGFR is highly expressed, and the HCR gave a 217x increase in signal!

The coolest part of the paper!

Building on the fluorescence data, they developed a targeted drug delivery system. The main payload? MMAE, an anti-cancer drug conjugated to the HCR hairpins. They also tested Dxd, another clinically relevant payload.

The most interesting result?

The dose-response is not linear. They compared 3 hairpins in various combinations:

Why does it happen? The authors think that when 3 MMAEs are close, they better interact with the membrane. This results in better internalization and better toxicity!

All of this while keeping incredible selectivity! In mixed cell populations, only the target cells are eliminated, while the untargeted HeLa cells continue to grow, unbothered.

Finally, the team showed that the HCR product can recruit antibodies. How? Simple: add a target, and they’ll come. They used a fluorescein-labelled HCR and tested binding for an anti-fluorescein antibody.

The HCR created multivalent spacing that improved antibody affinity compared to monovalent DNA, reaching up to 3.4 nM for the HCR product! Much stronger than monovalent binding.

And it even worked on cells! Adding output to their system: payloads and antibody recruitment! This could enable pre-targeting strategies or recruit immune effectors from inside the body!

A cool paper!

DNA nanotech and antibodies are a great match:

But antibodies are complicated to make!

Using affibodies (or aptamers) and a relatively simple HCR system keeps binding and logic without the overhead. And it makes it modular! You swap binders, and you get a new system! Amazing.

Now, it still has limitations:

But still, a very cool idea! Go and read it here!

If you made it this far, thank you! What do you think of antibody-drug conjugates? Do you think they are the future of biologics? Reply and let me know!