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Small Proteins: Into the Gap

We medicinal chemists are used to thinking about small molecule drugs – it’s what we do. And we’re also comfortable with having a category in our worldview that we assign to “biologics” – proteins, mostly, many of them antibodies, which can also be extremely therapeutically effective under the right conditions. But we really need to expand our thinking a bit, because there’s a whole world in between these two.

Consider the odd sorts of antibodies found in the camelids (camels, llamas, alpacas, and so on). They have only the “heavy chain” part, and stripping it down to just that domain (patented under the name “Nanobody”) gives you a much smaller protein piece that’s still capable of selective antigen recognition. You can get something similar from sharks as well. These smaller proteins can have rather different properties from traditional antibodies, and being smaller, you have more opportunity to modulate those properties in a defined way. Similarly, there have been many proposals over the years for “antibody-ish” protein platforms, and you can imagine that there are a lot of possibilities.

Here’s another example from the recent literature. The authors are, as they say, taking advantage of

. . .advances in both DNA manufacturing and protein design that have led to a fortunate convergence between the upper limit of the size of oligonucleotides (230 bp) that can be synthesized as pools of 10,000 or larger, and the lower limit of the size of genetically encodable computationally designed proteins (roughly 40 amino acids).

As a test case, they’re looking for a protein binder to influenza A H1 haemagglutinin (HA) and botulinum toxin, which was discussed here just the other day. The work starts out with computational filtering of possible proteins (using Rosetta software), trying different combinations of alpha-helix and beta-strands, and for the actual binding surfaces they built on past work to find such species and on the geometry of natural binding partners. (This definitely gives you a leg up – doing this from scratch, without known structures, would be a steep climb, you’d have to think). They picked several thousand likely variants for each target, including some random changes as well as more defined ones, and expressed the lot (about 17,000 proteins) in yeast.

Fluorescent binding assays on these libraries, followed by cell sorting on said fluorescent readouts, gave dose-responsive readouts of hits, which is reassuring. With botulin, for example, at 100, 10, and 1 nanomolar, they pulled out 2685, 987, and 355 proteins (from a starting pool of 5306 that actually expressed, out of the 5311 designed). Exposing the last set to a protease treatment beforehand, to leave only the hardy ones, took that down to 57 sequences. For both this and the HA pool, the computationally designed sequences were enriched in the final winners, which is a good sign. Interestingly, in both sets, the only ones that remained after protease exposure were the ones that incorporated disulfides, which clearly had an impact on structural stability, as they should.

The authors claim that this may well be the largest scale attempt at confirming computational ability to design protein-protein interactions, and I certainly can’t refute them. Some crystal structures were solved with some of the best binders, and they fit the calculations quite well.They proved highly active in cell protection assays, and here comes the small-protein property advantage: incubating these at 80C for an hour before running the assay did not change their activity at all, in marked contrast to what happens with conventional antibodies. Similarly, multiple rounds of dosing elicited little or no immune response in rodents.  Moreover, intranasal administration of an HA candidate to mice, followed by a lethal challenge with influenza virus, led to 100% survival at doses down to 0.03 mg/kg, which is 100-fold lower on a mass basis than the known broadly neutralizing antibody. A 3 mg/kg dose was 100% effective 72 hours after exposure as well. Notably, iv administration was useless.

This is all pretty impressive, I’d say, and what I like even more is that we’re just in the beginning phases of this sort of work. We’re going to get better at producing these proteins, designing them, and selecting them, and there could be some major opportunities out there. Stay tuned!

19 comments on “Small Proteins: Into the Gap”

  1. luysii says:

    These things might be called antibodies with antibodies. Another approach to the influenza virus hemagglutinin (HA) used the solved structure of broadly neutralizing antibodies and the site that they hit [ Science vol. 358 pp. 450 – 451, 496 – 502 ’17 ] which is crucial for viral function. Binding of the antibody here prevents the conformational change required for the virus to escape the endosome, a fact interesting in itself in that it implies that it only works after the virus enters the cell, although the authors do not explicitly state this.

    Study of one broadly neutralizing antibody showed that binding to the site was mediated by a single hypervariable loop. So the authors worked with a cyclic peptide mimicking the loop. This has several advantages, in particular the fact that the entropic work of forcing a floppy protein chain into the binding conformation is already done before the peptide meets its target.

    The final cyclic peptide contained 11 amino acids, of which 5 weren’t natural. It neutralized pandemic H1 and avaian H5 influenza A strains at nanoMolar concentration.

    It’s important that crystal structures of the broadly neutralizing antibody binding to HA were available — this requires atomic level resolution.

  2. Barry says:

    Genentech worked long and hard to carve away 2/3 of the molecular weight of Avastin to produce a single-chain Lucentis. Disappointingly, the transport properties of the two are the same. But this new paper demonstrates other virtues to reducing molecular weight.
    And it’s implicit that the useful hit rate might be boosted further by imposing some disulfides (maybe not as many as in conotoxins) in the library design.

    1. Imaging guy says:

      Did they really have to work long and hard just to make the Fab fragment of a whole antibody? Moreover, I don’t think transport properties would be the same if given parenterally.

      1. Barry says:

        Yes, it was a lot of engineering to build the binding site–that had spanned light- and heavy-chain–into a correctly-folded smaller single chain. And they looked very hard for any hint that their transport properties were different to justify the work.

    2. P says:

      I am quite curious what transport type are you referning to.

  3. Ted says:

    I am working with some of these people, and some of the things they have been able to pull off lately is just ridiculous – check out this variant:

    They designed a protein binder to fentanyl with nM potency, tied it to a luciferase reporter system, then stuck it into a plant. Now you’ve got a plant that lights up when it gets lit up… so to speak.

    The ability to design parallel features into these minimally sized proteins will eventually make mAbs obsolete. It’s the difference between painting and paint-by-numbers…


  4. anon electrochemist says:

    Shelf-stable antivenoms, anyone?

    1. David says:

      Yes please.

  5. Biotechchap says:

    Intranasal worked well but IV uselless…..can somebody elaborate the possible reasons…i would expect better performance in IV….given that way more antibodies are commercially administered thru IV than Intra-nasal route!!!

    Did they tried oncology animal models instead of just fly??

    1. Barry says:

      Mucosal immunity is the first-line against a pathogen like flu which may never circulate in the plasma compartment

      1. Mol Biologist says:

        Barry, you are always great with your questions. In order to understand what is an analog of Juvenile Hormone in human physiology you have to refer to pleiotropy of immune system.
        Here is a good example that hormonal signaling is important for regulating immune system activity and, more generally, for governing trade-offs between physiological processes. You can not attach wings to the caterpillar and make it fly, change must occurs inside.

    2. Druid says:

      These mini-proteins are direct blocking antagonists, not immunogenic. I estimate that the concentration of mini-protein in nasal mucosa is about 1000x higher when applied topically than when given IV, at least for a while, so if that is where the infection gets started, it is in the right place. I think that is how the flu virus is administered to the mouse.

      1. Barry says:

        yes, this is effectively “passive immunity” like taking gamma-globulin i.v. or i.m., except it’s on the mucosal surface (and presumably doesn’t persist as long)

        1. Druid says:

          I reckon the plasma half-life will be about 1 hr. The intra-nasal topical half-life is probably faster, so it’s time to call the drug delivery department for a bioadhesive polymer formulation!

  6. Biotechchap says:

    Intranasal worked well but IV uselless…..can somebody elaborate the possible reasons…i would expect better performance in IV….given that way more antibodies are commercially administered thru IV than Intra-nasal route!!!

    Did they tried oncology animal models instead of just flu (correction).

  7. dfdsg says:


  8. anon says:

    Antibodies have other immunological functions other than antigen binding ( e.g. complement mediated lysis, antibody dependent cytotoxicity, antibody dependent phagocytosis, FcR n mediated recycling (critical for long half life)). Most of these functions are mediated through the FC region which accounts for a large portion of their molecular weight . Nanobodies without FC region may be small, it also loses a lot of functions associated with the antibody modality.

    1. luysii says:

      Point well taken

  9. Old says:

    Check out Complix’alphabody technology:

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