RESUMO
The large molecular size of antibody drugs is considered one major factor preventing them from becoming more efficient therapeutics. Variable regions of heavy chain antibodies (HCAbs), or single-domain antibodies (sdAbs), are ideal building blocks for smaller antibodies due to their molecular size and enhanced stability. In the search for better antibody formats for in vivo imaging and/or therapy of cancer, three types of sdAb-based molecules directed against epidermal growth factor receptor (EGFR) were constructed, characterized and tested. Eleven sdAbs were isolated from a phage display library constructed from the sdAb repertoire of a llama immunized with a variant of EGFR. A pentameric sdAb, or pentabody, V2C-EG2 was constructed by fusing one of the sdAbs, EG2, to a pentamerization protein domain. A chimeric HCAb (cHCAb), EG2-hFc, was constructed by fusing EG2 to the fragment crystallizable (Fc) of human IgG1. Whereas EG2 and V2C-EG2 localized mainly in the kidneys after i.v. injection, EG2-hFc exhibited excellent tumor accumulation, and this was largely attributed to its long serum half life, which is comparable to that of IgGs. The moderate size (approximately 80 kDa) and intact human Fc make HCAbs a unique antibody format which may outperform whole IgGs as imaging and therapeutic reagents.
Assuntos
Sistemas de Liberação de Medicamentos , Receptores ErbB/efeitos dos fármacos , Fragmentos de Imunoglobulinas/farmacologia , Sequência de Aminoácidos , Animais , Camelídeos Americanos , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Humanos , Fragmentos de Imunoglobulinas/administração & dosagem , Fragmentos de Imunoglobulinas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Engenharia de Proteínas , Alinhamento de SequênciaRESUMO
BACKGROUND: Francisella tularensis subsp. tularensis is classified as a Category A bioweapon that is capable of establishing a lethal infection in humans upon inhalation of very few organisms. However, the virulence mechanisms of this organism are not well characterized. Francisella tularensis subsp. novicida, which is an equally virulent subspecies in mice, was used in concert with a microPET scanner to better understand its temporal dissemination in vivo upon intranasal infection and how such dissemination compares with other routes of infection. Adult mice were inoculated intranasally with F. tularensis subsp. novicida radiolabeled with 64Cu and imaged by microPET at 0.25, 2 and 20 hours post-infection. RESULTS: 64Cu labeled F. tularensis subsp. novicida administered intranasally or intratracheally were visualized in the respiratory tract and stomach at 0.25 hours post infection. By 20 hours, there was significant tropism to the lung compared with other tissues. In contrast, the images of radiolabeled F. tularensis subsp. novicida when administered intragastrically, intradermally, intraperitoneally and intravenouslly were more generally limited to the gastrointestinal system, site of inoculation, liver and spleen respectively. MicroPET images correlated with the biodistribution of isotope and bacterial burdens in analyzed tissues. CONCLUSION: Our findings suggest that Francisella has a differential tissue tropism depending on the route of entry and that the virulence of Francisella by the pulmonary route is associated with a rapid bacteremia and an early preferential tropism to the lung. In addition, the use of the microPET device allowed us to identify the cecum as a novel site of colonization of Francisella tularensis subsp. novicida in mice.
