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Immunotherapy has become very important in medicine thanks to the Nobel Medicine prize awarded to James P. Allison and Tasuku Honjo in 2018, for their discoveries in key molecules to the regulation of the immune response (CTLA4 and PD1) (Ishida et al., 1992; Leach et al., 1996). Immunotherapy consists on the modification of any element of the immune response to promote the prognosis of a pathology. There is extensive evidence of its benefits and importance in autoimmune diseases and cancer (Nishimura et al., 1999; Yang 2015). From the second half of the past century, studies have reported the use of human dialyzable leukocyte extracts (hDLE) as immunotherapy with a possible immunomodulatory effect. Several studies have proven that hDLE can modify the immune response. In the 1970s, Dr. Sergio Estrada Parra started the production of hDLE at laboratory scale in Mexico. In recent years, his efforts were completed with the patent registration of an industrial scale manufacturing process, which allows for its protection in USA, Mexico, Colombia and, recently, the European Union.
Transferon® is a blood-derived product with immunomodulatory properties composed of a mixture of low-molecular weight peptides (<10 kDa) originated from human leukocyte rupture. It is considered a complex drug and has batch-to-batch reproducibility.
Transferon® production is conducted under strict quality standards that result in a robust manufacturing process. The release of Transferon® batches is done complying with quality specifications, which are based on the physicochemical and functional characterization of the product.
Over 15,000 patients are currently benefited with the use of Transferon® every year, which has been proven to be an effective coadjuvant in the treatment of diseases such as allergy, autoimmunity, and infections. These pathologies involve a failure or deregulation of the immune system, hence the immunomodulatory ability of Transferon® is of great importance, which has been proven through in vitro assays and animal models.
It must also be considered that Transferon® has been proven to be safe and has gone through a pharmacovigilance process for the past eight years, which has reported no serious adverse effects. In addition, UDIMEB1, through USEIC2, provides free medical consultation of specialists in different medical areas, allowing for the correct patient diagnosis and follow-up. The specialist determines whether the patient is a candidate to use Transferon®, the appropriate dose, and considers the greater benefit for the patient in all cases.
This drug has been largely used in our country; however, there is an information gap regarding its function and effects. Recently, UDIMEB has committed to a rigorous molecular and clinical analysis of the product. Therefore, we consider that spreading this knowledge is essential to a better use of the product by the general population. Additionally, the implemented strategy can be useful for the development of other products or extracts with similar or different therapeutic properties to Transferon®. In this document, we present the current scientific information of the characteristics and effects of Transferon® after years of research by several scientific groups of renowned institutions in our country.
Physicochemical characterization
The physicochemical characterization of a complex drug is not only useful to determine its properties, but also is fundamental to establish its attributes based on batch-to-batch reproducibility (Nicholas, 2012). Transferon® has been analyzed by different chromatographic, electrophoretic, and spectroscopic techniques, which allowed to define and control its content (total peptides), assess its identity and peptide origin, and identify its most relevant batch-to-batch consistent physicochemical characteristics. This is especially important since the characteristics of a complex drug entirely depend on the manufacturing process and slight alterations to the process will generate drugs with different safety, quality, and efficacy (De Stefano et al., 2009). Furthermore, all the physicochemical and biological properties of Transferon® depend on a standardized manufacturing process that is intellectually protected in USA, EU, and Mexico (Estrada-Parra et al., 2013, 2016); and they cannot be directly extrapolated to other dialyzable extracts since each product might have different components when obtained through different manufacturing processes, thus they must be independently evaluated.
Total peptide content
Historically, the concentration of DLE of peripheral blood, spleen, or other sources and species have been defined based on their biological activity. This process uses arbitrary units defined as the total mass of peptides obtained from the lysis process of a defined number of leukocytes (Jose et al., 1976). However, the magnitude of these units varies among products since the lysis efficiency is also different among processes. In order to control the variability of the biological activity in each batch, and the expected therapeutic effect, the concentration of Transferon® was defined based on the total peptide content (0.4 mg/mL) (Medina-Rivero et al., 2014).
Mass distribution profile
All drugs must have an identity test that differentiate them from other drugs. Complex drugs, in specific, must account for analytical tests that demonstrate the dispersion of all its components. In this sense, the identity test of Transferon® is based on the analysis of its components’ mass distribution. The identity profile of Transferon®, obtained through size exclusion chromatography, is composed of eight main populations with molecular weight under 10 kDa (Medina-Rivero et al., 2014). This profile has been fundamental to the quality control of Transferon® and to develop comparability studies against other dialyzable extracts.
Peptide characterization
The characterization of Transferon® has focused on its most abundant fraction, peptides. Therefore, the used techniques analyze the characteristics of these biopolymers: size, the proportion of amino acids, and consistency of peptide polarity. These studies have proven that Transferon® peptides have a molecular weight lower than 10 kDa, are mostly hydrophilic, and have glycine as the most abundant amino acid. These properties are batch-to-batch reproducible (Medina-Rivero et al., 2016). Recently, the polydispersity index was determined at 1.11 orthogonally using mass spectrometry and nuclear magnetic resonance. This proves the applicability of high-technology techniques for a characterization that will allows identifying the sequence of the active peptide components in this complex drug (Vázquez-Leyva et al., 2019).
Effects of Transferon® in different in vitro models
Given the high molecular complexity of Transferon®, it is expected to find diverse in vitro effects related to is immunomodulatory properties, which also depend on the model employed, the use of concomitant stimuli, and the concentration of Transferon®.
One of the used models to evaluate the effects of Transferon® consisted on the use of THP-1 cell line which is derived from human monocytes. Cells were differentiated into macrophages (CD11b+ and CD14+) stimulated with LPS and low and high doses of Transferon®. No changes in IL-1β, TNF-a, and IL-6 levels were observed under the experimental conditions tested when stimulated only with the drug (10 pg/mL-10µ/mL). Conversely, significant differences were observed in the expression of costimulatory CD80 and CD86 molecules and in the production of IL-6 when THP-1 macrophages were treated with LPS (1 µg/mL) and high doses of Transferon® (0.1-10 µg/mL). Although the only detected correlation was the expression of CD80 (obtained while stimulating with 10µg/mL Transferon® and LPS) and IL-6. This result suggests a positive feedback between IL-6 production and the expression of CD80, as reported in other cell populations (Jiménez-Uribe et al., 2019).
Another system used in the evaluation of the immunomodulatory effects of Transferon® is the Jurkat cell line which is derived from human T lymphocytes. In which the levels of IFN-γ produced when treated with Transferon® were analyzed. A discrete production of the cytokine was observed, which allowed the development of a limit test for batch release (Medina-Rivero et al., 2014). Later, research continued until obtaining a test that not only considered the concentration limits of an inflammatory molecule but had a dose-response behavior, and complied with the pharmaceutical industry standards. The test had to meet all the validation parameters of any bioassay used for batch release, such as concentration interval, precision, accuracy, specificity, and system suitability. This was achieved by adding different concentrations of the drug to “rescue” cells from proliferation inhibition induced by azathioprine, a purine analog that interferes with DNA replication. This methodology allows for the replacement of less sensitive and costly methodologies and can be routinely applied for batch release purposes (Carballo-Uicab et al., 2019).
Another study uses an elegant model to induce a specific type of NK CD56+CD16+CD11c+ cells produced by treatment with Transferon® from progenitor cells (CD34+) obtained from human umbilical cord. It was observed that the resulting cell population showed distinctive characteristics of NK cells, such as IFN-γ production and cytotoxicity to tumor cells, as well as to induce the proliferation of Tγδ lymphocytes. It is suggested that Transferon® exerts a direct effect in vitro on progenitor cells (CD34+) as well as the activation and remodeling of the support hematopoietic microenvironment (Ramírez-Ramírez et al., 2016). The models described contribute with knowledge of the pharmacodynamic of the product by proving immunomodulatory and stimulation effects of early hematopoiesis.
Safety and efficacy tests of Transferon® in animal models
There are four relevant studies in animals that comprise the establishment of a test for batch release to the production of support information on safety and clinical use of Transferon®. The first study was conducted by Salinas-Jazmín and colleagues (Salinas-Jazmín et al., 2015), who developed a murine model of infection with Herpes simplex virus type 1 (HSV-1, strain KOS). The virus can induce a neuropathic effect in mice, clinically expressed as reduced motility, paralysis, weight loss, and death. Mice were administered with different doses of Transferon® orally at an interval from 12.5 ng to 1.50 µg every other day for 10 days and were continuously followed-up for 20 days to observe the development of clinical signs. During the study, serum levels of proinflammatory cytokines were evaluated. The results of the study demonstrated that the treatment with Transferon® increased the survival of infected mice with respect to controls. In addition, Transferon® was observed to increase IFN-γ concentration, which suggests that it indirectly stimulates the activation of HSV-1-specific T lymphocytes. This proves that the protective effect of this drug is mediated by its activity on the immune system.
Another study conducted by Hernández-Esquivel and colleagues (Hernández-Esquivel et al., 2018) developed a xenotransplantation model of transformed murine prostate epithelial cells (PEC-Src), representing the transcriptional profiles of human prostate cancer. These cells can be tracked through bioluminescence in an in vivo imaging system Lumina XR (Caliper Life Sciences). Cells were injected in mice to induce metastasis and tumors. Animals were then administered with different doses of Transferon® (1-25 µg/kg). Results showed a dose-dependent effect of Transferon® to reduce tumoral growth and metastasis in the brain. The study concluded that, despite Transferon® does not have direct cytotoxic or antitumoral effects, it can regulate the production of proinflammatory cytokines and several growth factors that inhibit tumor growth. These results make evident the potential uses of Transferon® in this type of human cancer.
Immunogenicity is another factor to be considered when protein or protein-derived drugs are used. At this regard, there are two important works on Transferon®. The first was conducted by Ávila and colleagues (Avila et al., 2017) to evaluate the immunogenicity of Transferon® combined with adjuvants. Mice were immunized at days 1, 7, and 14; ovalbumin was used as positive control and hydrolyzed collagen as negative control. The results proved that, although Transferon® and the controls increased total IgGs serum levels, only ovalbumin was able to induce the production of specific antibodies. Therefore, the study concluded that Transferon® is not immunogenic. In a study conducted by Mellado-Sánchez and colleagues (Mellado-Sánchez et al., 2019), Transferon® was covalently conjugated to carrier proteins, as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), to increase its immunogenic potential. Mice and rabbits immunized with the conjugates showed a significant production of antibodies able to recognize Transferon® components. Additionally, rabbits exhibited higher titration with respect to mice and their antibodies blocked IL-2 production in Jurkat cells after costimulation with Transferon®.
Collectively, these studies demonstrated that Transferon® is capable to induce a biological response in animal models not by a direct effect against the etiological agent, but through the modulation of the immune response. Besides, it was found that Transferon® has a low immunogenic potential on its own.
Clinical research
Although scarce, clinical research have revealed significant data, among which are the adverse effects induced by Transferon® consumption (Homberg et al., 2015). The effects were evaluated in 3844 patients with different conditions, such as arthritis, allergies, and infections, all of them treated canonically and using Transferon® as a therapeutic adjuvant. The adverse effects commonly reported were headache, increase in symptomatology of the main clinical condition, rhinorrhea, cough, fatigue, and rash. All of them were exhibited by 0.01 % of the participants and, interestingly, were more frequent in women, thyroid-hormone or estrogen consumers, and subjects in glucocorticoid therapy.
This finding is relevant considering the data published on major depressive disorder patients treated with serotonin reuptake inhibitors (SSRIs) and Transferon® (Hernández et al., 2013). This work proved that the biggest difference between depressive patients treated with SSRI antidepressants and those treated with SSRIs + Transferon® is a 50 % reduction of cortisol levels from week 20 of a 52-week treatment. Contrastingly, the canonical treatment with SSRIs only reached a 30 % decrease in cortisol levels at week 52. It must be noted that the decrease in circulating cortisol levels is considered a marker of clinical improvement since it indicates a decrease in hyperactivity of the hypothalamic-pituitary-adrenal axis characteristic of this disease.
Furthermore, patients treated with SSRIs + Transferon® versus SSRIs show higher circulatory IFN-γ levels than those induced by SSRIs consumption from week 5 until the end of the study. Circulatory IFN-γ levels are directly related to the number of neuronal connections of the brain regions involved with social behaviors (Filiano et al., 2016).
Finally, a recently published study consisted of a follow-up from January 2010 to December 2016 and evaluated 123 pediatric patients with sepsis; 15 of them were treated with standard medication and Transferon® while 108 received canonical treatment (Castrejón et al., 2019). When compared against the control group, patients treated with Transferon® showed decreased levels of C reactive protein, increased leukocyte count, and reduced levels of neutrophils in the first 72 h after hospitalization. In addition, those patients treated with Transferon® showed a higher rate of survival. This supports the relevance of safety and the immunomodulatory ability of the inflammatory response. Indeed, the drug has positive effects as a therapeutic adjuvant in pathologies with a considerable inflammatory component.
Conclusions
Transferon® is a safe and effective product of high quality. Its quality is supported by an extensive physicochemical characterization which confirms that the manufacturing process is robust and yields consistent batches. On the other hand, the safety of this drug has been proven through immunogenicity studies and an active pharmacovigilance program. In addition, the published evidence suggests that Transferon® is beneficial as an adjuvant treatment of conditions in which the inflammatory response is deregulated since it promotes the patient’s recovery. Therefore, Transferon® can be considered as another therapeutic tool. Clearly, more studies should be conducted in upcoming years to improve the conditions of use of Transferon®.
