Transferon™, a peptide mixture with immunomodulatory properties is not immunogenic when administered with various adjuvants

Abstract

Transferon, a human dialyzable leukocyte extract (hDLE), is a biotherapeutic that comprises a complex mixture of low-molecular-weight peptides (< 10 kDa) and is used to treat diseases with an inflammatory component. Some biotherapeutics, including those composed of peptides, can induce anti-drug antibodies (ADA) that block or diminish their therapeutic effect. Nevertheless, few studies have evaluated peptide-derived drug immunogenicity. In this study, the immunogenicity of Transferon was examined in a murine model during an immunization scheme using the following adjuvants: Al(OH), incomplete Freund's adjuvant (IFA), or Titermax Gold. The inoculation scheme entailed three routes of administration (intraperitoneal, Day 1; subcutaneous, Day 7; and intramuscular, Day 14) using 200 μg Transferon/inoculation. Serum samples were collected on Day 21. Total IgG levels were quantitated by affinity chromatography, and specific antibodies against components of Transferon were analyzed by dot-blot and ELISA. Ovalbumin (OVA, 44 kDa) and peptides from hydrolyzed collagen (PFHC, < 17 kDa) were used as positive and negative controls, respectively, in the same inoculation scheme and analyses for Transferon. OVA, PFHC, and Transferon increased total IgG concentrations in mice. However, only IgG antibodies against OVA were detected. Based on the results, it is concluded that Transferon does not induce generation of specific antibodies against its components in this model, regardless of adjuvant and route of administration. These results support the safety of Transferon by confirming its inability to induce ADA in this animal model.

CD80 Expression Correlates with IL-6 Production in THP-1-Like Macrophages Costimulated with LPS and Dialyzable Leukocyte Extract (Transferon®)

“…In these assays, we did not observe morphological changes nor alterations of the production of IL-1 β , TNF- α , or IL-6 in macrophage-like THP1 cells (Figure 2). This result agrees with the null immunogenicity previously reported for Transferon® [24]. In contrast, stimulation with LPS (1 μ g/mL) significantly increased the fraction of positive cells, as expected [25]; treatment with LPS was used as positive control in these experiments.…”

Development of Functional Antibodies Directed to Human Dialyzable Leukocyte Extract (Transferon®)

“…These results are in agreement with those obtained in our previous study [2], where no antibodies were detected in mice immunized with Transferon® and different adjuvants. But a question arose, the influence of adjuvant over the response obtained with unconjugated peptides of Transferon®, given that in the preliminary study two extra adjuvants were analyzed (Al(OH) 3 and TiterMax® Gold®), we reanalyzed those sera using the bioconjugates as antigens, searching for a signal; nonetheless, this was negligible (Supplemental Figure) in spite of using a more sensible system through the conjugation of Transferon® peptides.…”

“…In a previous study carried out by our group [2], we demonstrated the null immunogenicity of Transferon® in a murine model even though different immunization routes and adjuvants were employed, highlighting the safeness of the product but delaying the development of a surrogate antibody directed to this hDLE. Therefore, this work was aimed at inducing immunogenicity against this hemoderivative through two strategies; one of them was chemical conjugation of the Transferon® peptides to a carrier protein as the immunogen and the second one was to use the rabbit to achieve the induction of antibodies against peptides in general [21].…”

“…Samples from a previous study were tested for reactivity against TCRP-KLH antigen [2] (Supplemental Figure).…”

“…The safeness of a biotherapeutic is a mandatory requirement for its use in humans; in this regard, one of the nondesirable characteristics for this kind of products is the induction of antidrug antibodies (ADAs) that could diminish the effect of the drug or the formation of immunocomplexes that would lead to an inflammatory response. So far, there are no reports of ADA induction in patients treated with Transferon®, nor in mice [2], giving evidence of the safeness of this biotherapeutic along with null acute toxicity in mice (unpublished data).…”