Humanized pigs
Transgenic mini-pigs have been created for the study of monoclonal antibodies
Elena Kleshchenko, PCR.news
A scientific group from Germany and Switzerland, with the participation of the Research and Development department of Roche, created humanized minipigs for preclinical testing of monoclonal antibodies (MCAT). The COVID-19 pandemic introduced everyone to antiviral MCATs that recognize important parts of viral proteins. MCAT is widely used in the treatment of oncological, autoimmune and neurological diseases. However, the patient's immune system can produce antibodies against the therapeutic antibody, and this leads to complications, sometimes life-threatening. Theoretically, it is very difficult to predict these side effects and the effect of anti-drug antibodies on the effectiveness of the drug, and in vivo trials are complicated by the fact that any human protein causes an immune reaction in a wild-type animal. To avoid this, they make an antibody analogue specific to the experimental animal, or create transgenic animals that express human immunoglobulins and recognize them as "their own". In such animals, the immune response to various MCAT should be similar to that of humans.
Several research groups have already obtained transgenic mice that express sets of human immunoglobulin genes. This work was done by the authors of a new article in Nature Biomedical Engineering. Their mice have their own immunoglobulins (and are therefore fully immunocompetent), but they also express a mini-repertoire of human immunoglobulins of subclass 1 G (IgG1) — only the secreted form of human heavy chains Ig-γ1, as well as light chains Ig-k and Ig-λ. These chains are most often used in the production of therapeutic antibodies, and MCAT preparations have already been studied on such mice.
However, the data obtained on small rodents cannot always be transferred to humans, in addition, the small size of the mouse makes many manipulations difficult, for example, the introduction of the drug into the vitreous body of the eye. Preclinical tests of pharmaceuticals must necessarily be carried out on one species that does not belong to the rodent order. Until now, primates were the only option. Laboratory pigs are a great alternative: a short pregnancy, numerous offspring, relatively low requirements for the conditions of detention and at the same time a large size and similarity of anatomy and physiology with human ones. In preclinical studies, different lines of minipigs are used — Hanford, Yucatan, Yucatan Micro, Sinclair, Goettingen; in the Russian Federation there are Novosibirsk mini-sibs.
The authors of the study took the Goettingen minipigs as a basis. They created two expression vectors, one with segments of the IGH gene, which, when re-arranged, give sequences encoding a variety of secretory heavy chains of IgG1 immunoglobulins, the other with segments of the IGK gene, respectively, giving light chains of Ig-k. These gene elements should generate soluble human IgG without affecting the repertoire of porcine antibodies (this can only happen with the expression of membrane-bound immunoglobulins).
Kidney fibroblasts obtained from male minipigs were transfected with vectors; after PCR screening for transgenicity, the selected nuclei were transplanted into eggs. Eight transgenic piglets were obtained, four reached puberty, three of them expressed both heavy and light human IgG chains. All descendants of the founding animals demonstrated Mendelian inheritance of transgenics, which indicates integration into one genomic locus, and stable levels of human immunoglobulin in the blood serum. Analysis of the matrix RNA confirmed that the rearrangement of gene segments is adequate, and the variable sequence contains amino acid substitutions due to somatic mutations.
The authors tested how transgenes affected the immune system. The minipigs did not suffer from increased infectious load, the morphology of the spleen, lymph nodes and bone marrow did not change. The reaction to the model antigen — snail lymph hemocyanin — turned out to be normal.
Finally, the reaction of humanized minipigs to prototype preparations of human therapeutic antibodies — bevacizumab and daratumumab - was investigated. The animals were given seven injections and regularly detected antibodies against antibodies in the blood. Antibodies against bevacizumab were found in wild-type pigs, but not in humanized ones. Antibodies against daratumumab did not appear in either of them. Apparently, both of these drugs are non-immunogenic for humans. Then the immunogenicity of therapeutic antibodies atezolizumab or cergutuzumab, which induce antibody responses in 39% and 70% of patients, respectively, was evaluated; a similar difference in immunogenicity was observed in pigs.
The authors consider transgenic minipigs to be an ideal model for evaluating the safety of therapeutic antibodies and predicting possible side effects.
Article by Flisikowska et al. A humanized minipig model for the toxicological testing of therapeutic recombinant antibodies is published in the journal Nature Biomedical Engineering.
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