In medical practice, monoclonal and polyclonal antibodies have been increasingly used over the past decades. The remarkable specificity of antibodies opens up unique possibilities for their use in the diagnosis and treatment of various diseases. Antibodies are used in the treatment of cancer, autoimmune, infectious diseases, as well as in organ and tissue transplantation (you can read more about antibodies here and here ). A number of antibody-based drugs are being tested in various phases of clinical trials . However, the development and production costs of such drugs are very high and, in addition, serious side effects are commonly associated with their use. Teams of scientists around the world are developing a variety of strategies to overcome these limitations. One of such approaches, as suggested by several researchers, is the use of ultra-high dilutions of antibodies obtained by serial dilution. A study published by an international team of scientists in The Journal of Immunology, is devoted to this approach. They studied the possible mechanisms of action of ultrahigh dilutions of biologics using ultra-high dilutions of antibodies to human interferon gamma (“highly-diluted Abs to IFN”, hd-anti-IFN) as an example. The scientists applied a molecular, cellular and systems approach to find out whether hd-anti-IFNγ can affect the molecular structure of IFNγ changing its biological activity and its ability to interact with other molecules.

IFNγ plays an extremely important immunostimulating and immunomodulatory role in both innate and acquired immunity against viral, as well as some bacterial and protozoal infections. It is a pro-inflammatory cytokine produced primarily by activated T lymphocytes and natural killer cells (NK cells). IFNγ is a homodimeric glycoprotein and binds IFNγ receptors on the surface of various cells of the immune system.

In the study, an aqueous-alcoholic mixture of ultrahigh dilutions of affinity-purified rabbit polyclonal antibodies to human IFN was used. The mixture contained 12th, 30th and 50th centesimal dilutions of antibodies to IFNγ. The method of serial dilution with shaking was used: the antibodies were mixed with a solvent (water-alcohol solution) and subjected to vigorous vibration stirring. All subsequent dilutions were obtained by mixing one part of the previous dilution and 99 parts of the solvent, followed by stirring.

To study the effect of hd-anti-IFNγ on the structure (conformation) of IFN molecule, scientists used nuclear magnetic resonance (NMR) spectroscopy. In addition to standard proton magnetic resonance (¹H-NMR), two-dimensional NMR with isotope labeling (¹H-¹⁵N-NMR) was also used to improve the resolution of overlapping signals (due to the high number of atoms in the interferon molecule). The scientists found a number of differences in the NMR spectra of IFNγ and IFNγ, to which hd-anti-IFNγ was added. Several peaks changed their intensity, new peaks appeared, and some peaks, on the contrary, disappeared. Thus, the addition of hd-anti-IFNγ to the IFNγ sample caused both global and specific changes in the NMR spectrum. In general, the intensity and resolution of the spectrum have improved. A total of 13 amino acids were found to show a change in position or intensity upon addition of the hd-anti-IFNγ but not placebo. This indicates certain conformational changes in the IFNγ molecule. These changes in the structure of IFNγ were focused mainly at the interface between the two monomers and, thus, can probably affect the equilibrium of the formation of the dimeric structure of IFNγ. In turn, the kinetics of IFNγ oligomerization can affect the stoichiometry of the IFNγ /IFNγ receptor complex.

Analysis of the interaction between hd-anti-IFNγ and IFNγ using two-dimensional NMR spectroscopy. In the center is the IFNγ model with probable locations of conformational changes. Monomers in the dimer are highlighted in blue and pink. Amino acid residues with the greatest chemical shift (change in the local environment) are marked in red, blue and yellow, in decreasing order. Left - parts of 2D NMR spectra showing a chemical shift in positions located in the contact surface between monomers, on the right - outside this region.
Source: https://www.jimmunol.org/content/205/5/1345
Originally published in The Journal of Immunology. Tarasov S.A., Gorbunov E.A., Don E.S., Emelyanova A.G., Kovalchuk A.L., Yanamala N., Schleker A.S.S., Klein-Seetharaman J., Groenestein R., Tafani J-P., van der Meide P., Epstein O.I. 2020. Insights into the mechanism of action of highly diluted biologics. J. Immunol. Vol: 205(5), 1345-1354. Copyright © 2020. The American Association of Immunologists, Inc.

At the next step, the effect of hd-anti-IFNγ on the interaction of IFNγ with its receptor was studied. For this, a human monocyte-like cell line U-937 with high expression of IFNγ receptors was used. When these cells were treated with hd-anti-IFNγ, an increase in the specific binding of IFNγ was observed by competitive radioligand binding assay using IFNγ labeled with 125I, a radioactive isotope of iodine. In addition, hd-anti-IFNγ appeared to regulate IFNγ levels, enhancing its secretion and/or inducing the proliferation of IFNγ-producing cells. This conclusion was made based on the results of an ELISpot assay on human peripheral blood mononuclear cells (PBMC). These cells secrete IFNγ upon specific stimulation (for example, by viral peptides). After treatment with hd-anti-IFNγ, an increase in IFNγ secretion was observed.

Since IFNγ plays an important role in innate and adaptive immunity against viral infections, the next step was to test the antiviral effect of hd-anti-IFNγ in an in vivo model of viral infection. Mice were challenged with influenza A virus (subtype H1N1 or H3N8). Starting 5 days before viral challenge and up to 21 or 26 days after (for H1N1 and H3N8, respectively), hd-anti-IFNγ were administered by oral gavage twice daily. In addition, for the mice infected with the H1N1 virus hd-anti-IFNγ were added to their drinking water in a 1:3 ratio. The classic antiviral drug oseltamivir (Tamiflu) and the combination of hd-anti-IFNγ and oseltamivir (only for H1N1) were used as controls. The body weight of the animals and their mortality were assessed as indicators of the effectiveness of the treatments. The survival rate of animals receiving hd-anti-IFNγ was even slightly higher than that of those receiving oseltamivir (80% versus 70% for H1N1 and 95% versus 75% for H3N8, while in the “no treatment” control this indicator was 30% and 20% for H1N1 and H3N8, respectively). Co-administration of oseltamivir and hd-anti-IFNγ to mice infected with H1N1 also increased their survival rate (up to 75%). In addition, in mice infected with H3N8, body weight loss was significantly lower with any treatment (hd-anti-IFNγ or oseltamivir), while in groups of animals infected with H1N1, no significant differences in body weight loss were found.

In previous studies, scientists have demonstrated that the biological effects of high dilutions of antibodies are specific. The present study also showed that treatment with hd-anti-IFNγ resulted in an increase in IFNγ binding to its receptor, while highly diluted antibodies to another pro-inflammatory cytokine, TNF-α, did not have a significant effect. Based on these data, it was suggested that ultra-high dilutions of antibodies can act on their antigen (in this case, IFNγ) like allosteric modulators, causing conformational changes in its structure. This, in turn, may affect the functional activity of the antigen, for example, its interaction with receptors.

Thus, hd-anti-IFNγ have an antiviral effect by directly affecting the structure of IFNγ molecules. At this stage, both preclinical and clinical studies of the antiviral activity of hd-anti-IFNγ are underway. In the present study, an attempt was made to elucidate its possible mechanisms.

The authors suggest that unlike traditional Ab-based drugs, the highly diluted Abs act by inducing conformational modifications in their targets, which then affect interactions of the modified targets with the respective receptors and thus finally orchestrate the target-dependent biological pathway. Therefore, that may be the key point that allows achieving various therapeutic effects.