Abstract
Transferon® is an immunomodulator made of a complex mixture of peptides from human dialyzable leucocyte extracts (hDLEs). Development of surrogate antibodies directed to hDLE is an indispensable tool for studies during process control and preclinical trials. These antibodies are fundamental for different analytical approaches, such as identity test and drug quantitation, as well as to characterize its pharmacokinetic and mechanisms of action. A previous murine study showed the inability of the peptides of Transferon® to induce antibody production by themselves; therefore, in this work, two approaches were tested to increase its immunogenicity: chemical conjugation of the peptides of Transferon® to carrier proteins and the use of a rabbit model. Bioconjugates were generated with Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA) through maleimide-activated carrier proteins. BALB/c mice and New Zealand rabbits were immunized with Transferon® conjugated to KLH or nonconjugated Transferon®. Animals that were immunized with conjugated Transferon® showed significant production of antibodies as evinced by the recognition of Transferon®-BSA conjugate in ELISA assays. Moreover, rabbits showed higher antibody titers when compared with mice. Neither mouse nor rabbits developed antibodies when immunized with nonconjugated Transferon®. Interestingly, rabbit antibodies were able to partially block IL-2 production in Jurkat cells after costimulation with Transferon®. In conclusion, it is feasible to elicit specific and functional antibodies anti-hDLE with different potential uses during the life cycle of the product.
Highlights
Transferon® is a hemoderivative used as biotherapeutic with immunomodulatory properties; its components are a complex mixture of peptides from human dialyzable leucocyte extracts; these peptides are constituted by different proportions of amino acids among them Gly, Glu, and Ala [1]
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 Journal of Immunology Research of antidrug antibodies (ADAs) that could diminish the effect of the drug or the formation of immunocomplexes that would lead to an inflammatory response
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 human dialyzable leucocyte extracts (hDLEs)
Summary
Transferon® is a hemoderivative used as biotherapeutic with immunomodulatory properties; its components are a complex mixture of peptides from human dialyzable leucocyte extracts (hDLEs); these peptides are constituted by different proportions of amino acids among them Gly, Glu, and Ala (almost 44% of the total content of proteinogenic amino acids) [1]. 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. In spite of poor immunogenicity and enzymatic degradation sensibility, there are several advantages using peptides as immunogens, such as they can be produced, purified, and characterized ; the immunogenicity can be directed to known, relevant targets instead of undesirable virulence factors from pathogens in the antigen preparation, rendering in safer preparations with lower adverse effects in the host, no biosafety concerns for the researchers because they do not need to manage the whole virulent pathogen, there is no reversion leading to virulence and no risk of genetic integration or recombination, they can be produced on a large scale at a reasonable cost, and they are easy to store and transport [4]. Several strategies to enhance peptides immunogenicity have been tested, including the design of multiepitope vaccines using different platforms for production and delivery (DNA vaccines, recombinant or synthetic peptides, dendrimers, nanoparticles, etc.) [5,6,7,8,9]; even for more than one pathogen [10], mixture with classical and novel adjuvants [11, 12], built-in vaccine adjuvants such as interleukins, TLR-agonists, or dendritic cell targeting [13,14,15,16], chemical conjugation to a carrier protein [4, 17, 18], or the use of different animal models instead of mice, like rabbits and chickens [19, 20] have been tested
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