"It's a small personal step, but it's a big step for humanity" — said Armstrong, the first astronaut to land on the moon, at the moment he stepped on the moon.
Today, scientists like Daniel-Adriano Silva, K. Christopher Garcia and David Baker of the University of Washington in Seattle and Stanford University have taken such a small step together. For the first time, they achieved the de novo design synthesis of a protein anti-cancer drug, an analogue of cytokine IL-2 Neo-2/15. This protein design strategy can be widely used in signaling proteins, which will create more and better drug candidates.
Neo-2/15 has only 14% of the amino acid sequence of human IL-2, but retains most of the function of IL-2, except for removing the ability to bind CD25 that may cause toxic side effects. In mouse models of colon cancer and melanoma, Neo-2/15 strongly inhibited tumor growth and even completely eliminated tumors in some mice, resulting in better treatment than natural IL-2. The paper was published in Nature[1].
Neo-2/15 binds to β subunits and γ subunits of IL-2 receptors
IL-2 is arguably very important in the history of immunology, occupying several firsts: it is the first type I cytokine to be cloned, the first short-chain type I cytokine to be deciphered from the receptor structure, or the first cytokine to be found to act in a growth factor-like manner through a specific high-affinity receptor [2].
In addition, IL-2 is the first effective cancer immunotherapy. In November 1984, a patient with metastatic melanoma began treatment with high-dose IL-2, and after several months, her entire body was gone,[3] and there was no recurrence for 29 years [4]. This is also the first time that it has been proved that simply by stimulating T cells, it is possible to completely cure tumors.
However, in this trial, IL-2 also showed strong toxic side effects. IL-2 induces extensive capillary leakage syndrome in vivo, resulting in interstitial infiltration of the lungs and significant weight gain in patients. In addition, half of patients had elevated serum creatinine, and two-thirds had elevated bilirubin.
The role of IL-2
These toxic side effects greatly limit the use of IL-2. It has been shown that the receptor of IL-2 consists of three subunits of α, β, and γ, in which the binding of β subunit and γ subunit to IL-2 activates T cells, producing the therapeutic effect of IL-2, while the α subunit (also known as CD25) mediates the toxic side effects of IL-2 [5].
On the other hand, the presence of α subunits also increases the binding capacity of IL-2 and β, γ subunits by about 100 times. This may seem good at first glance, but α subunits are mainly expressed on immunosuppressive Treg cells, and there are almost no α subunits on CD8+ effector T cells and NK cells. With the use of IL-2 in small doses, almost all of the Treg cells are activated, but the immune system is suppressed [6].
In clinical trials, the IL-2 long-acting formulation NKTR-214 was used in combination with nivolumab, and the response rate of nivolumab was also reduced [7].
To this end, many scientists have attempted to modify the structure of IL-2 so that it can only bind to β subunits, γ subunits, and not to α subunits.[6]
To engineer a protein, it is common practice to start with the natural protein and let it mutate randomly until a mutant protein with the right properties is found. But this approach hits a wall in IL-2. After all, this method is to let the blind cat kill the rat, and IL-2 itself is not very stable, and the mutant is usually more unstable. It's time for the cat to open its eyes.
The researchers used the method of X-ray crystal diffraction to obtain the structure of the complex formed by the binding of IL-2 and its receptors, and carefully analyzed the interaction between IL-2 and IL-2 receptor α, β and γ subunits.
Using the data, the researchers used rosetta, a protein design software they developed themselves, to design 40 IL-2 analogues that act only with β and γ subunits, but not with α subunits. From them, 22 were selected for synthetic testing and their structure fine-tuned to further enhance their stability and effectiveness.
The core of IL-2 is four α-helixes, and the four α-helixes of natural IL-2 (middle) and Neo-2/15 have different connections
Eventually, the researchers obtained neo-2/15, an IL-2 analogue with the strongest affinity for the IL-2 β receptor and the γ subunit affinity. It is only 14% identical to IL-2 in humans and only 24% in mice.
In terms of stability, Neo-2/15 can still bind to the β, γ subunits of human IL-2 receptors after 2 hours of incubation at 80 °C. Even after 1 h incubation at 95 °C, Neo-2/15 is effective in promoting T cell survival. Under these conditions, natural IL-2 has long been denatured and inactivated.
Next, the researchers tested neo-2/15 treatment effects with mouse models of colon cancer and melanoma. Compared with IL-2, Neo-2/15 was more effective in inhibiting tumor growth, and even allowed 4 (a total of 10) melanoma model mice to not grow tumors.
In colon cancer model mice, Neo-2/15 (red) inhibited tumor growth better than IL-2 (gray) and no treatment (black),
As a protein drug, immunogenicity is also a problem that cannot be ignored. The researchers gave healthy mice (4 weeks) and tumor-bearing mice (2 weeks) daily continuous injections of Neo-2/15, respectively, and no antibodies against Neo-2/15 were detected. Only by adding complete Freund's adjuvants to enhance immunogenicity can the corresponding polyclonal antibodies be produced, and these antibodies do not cross-react with endogenous IL-2, and the safety is still good.
"People have been trying to engineer IL-2 for 30 years to make it safer and more effective, but it's hard to do that because natural proteins tend to be less stable," said Silva, the paper's first author. Because we designed it from scratch, we know all the parts of it, and we can continue to improve it to make it more stable and more active. ”
Corresponding author David Baker also believes that "this is the first completely new design with very obvious anti-cancer potential protein. This may be a new drug with better performance than the previous drug. ”
At present, the University of Washington has authorized Neolurukin Therapeutics to conduct further research on Neo-2/15 to promote it to the clinic.
Along the way from the Human Genome Project, human beings have deepened their understanding of themselves step by step. Today, we have finally realized a new functional protein that is completely artificially designed according to its own needs. It also brings us one step closer to "God."
Editor God nagging
The protein is designed from scratch, and the function is similar to that of the natural protein, but the sequence is only 14% the same. It seems that DNA→ RNA→ protein → function have been at least partially opened. Good luck to David Baker soon!
bibliography:
1. SILVA D-A, YU S, ULGE U Y, et al. De novo design of potent and selective mimics of IL-2 and IL-15[J]. Nature, 2019, 565(7738): 186-191.
2. PAUL W E. Fundamental Immunology[M]. Wolters Kluwer/Lippincott Williams & Wilkins, 2008.
3. Rosenberg S A, Lotze M T, Muul L M, et al. Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer[J]. New England journal of medicine, 1985, 313(23): 1485-1492.
4. Rosenberg S A. IL-2: the first effective immunotherapy for human cancer[J]. The Journal of Immunology, 2014, 192(12): 5451-5458.
5. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system[J]. Nature Reviews Immunology, 2012, 12(3): 180.
6. Kureshi R, Bahri M, Spangler J B. Reprogramming immune proteins as therapeutics using molecular engineering[J]. Current opinion in chemical engineering, 2018, 19: 27-34.
7. Diab A, Hurwitz M E, Cho D C, et al. NKTR-214 (CD122-biased agonist) plus nivolumab in patients with advanced solid tumors: Preliminary phase 1/2 results of PIVOT[J]. J Clin Oncol, 2018, 36(suppl; abstr 3006).
The author of this article | Kong Shaofan
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