RESUMO
Prolactin (PRL) has recently been demonstrated to elicit female-selective nociceptor sensitization and increase pain-like behaviors in female animals. Here we report the discovery and characterization of first-in-class, humanized PRL neutralizing monoclonal antibodies (PRL mAbs). We obtained two potent and selective PRL mAbs, PL 200,031 and PL 200,039. PL 200,031 was engineered as human IgG1 whereas PL 200,039 was reformatted as human IgG4. Both mAbs have sub-nanomolar affinity for human PRL (hPRL) and produce concentration-dependent and complete inhibition of hPRL signaling at the hPRL receptor (hPRLR). These two PRL mAbs are selective for hPRL as they do not inhibit other hPRLR agonists such as human growth hormone or placental lactogen. They also cross-react with non-human primate PRL but not with rodent PRL. Further, both mAbs show long clearance half-lives after intravenous administration in FcRn-humanized mice. Consistent with their isotypes, these mAbs only differ in binding affinities to Fcγ receptors, as expected by design. Finally, PL 200,019, the murine parental mAb of PL 200,031 and PL 200,039, fully blocked stress-induced and PRL-dependent pain behaviors in female PRL-humanized mice, thereby providing in vivo preclinical proof-of-efficacy for PRL mAbs in mechanisms relevant to pain in females.
Assuntos
Prolactina , Receptores da Prolactina , Feminino , Camundongos , Animais , Gravidez , Prolactina/metabolismo , Prolactina/farmacologia , Receptores da Prolactina/metabolismo , Anticorpos Monoclonais , Placenta/metabolismo , Ligação ProteicaRESUMO
We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/JH (H3J) fragments. One IGHV gene provided a universal VH scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal VH scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated KD values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies.
Assuntos
Regiões Determinantes de Complementaridade , Biblioteca Gênica , Imunoglobulina G , Anticorpos de Cadeia Única , Regiões Determinantes de Complementaridade/biossíntese , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Leucócitos Mononucleares/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genéticaRESUMO
The construction of Fab phage libraries requires the cloning of domains from both the light and the heavy chain of antibodies. Despite the advent of powerful strategies such as splicing-by-overlap extension PCR, obtaining high quality libraries with excellent coverage remains challenging. Here, we explored the use of type IIS restriction enzymes for the seamless cloning of Fab libraries. We analyzed human, murine and rabbit germline antibody repertoires and identified combinations of restriction enzymes that exhibit very few or no recognition sites in the antibody sequences. We describe three phagemid vectors, pUP-22Hb, pUP-22Mc and pUP-22Rc, which were employed for cloning the Fab repertoire of these hosts using BsmBI and SapI (human) or SapI alone (mouse and rabbit). Using human serum albumin as a model immunization, we built a mouse/human chimeric Fab library and a mouse Fab library in a single step ligation and successfully panned multiple cognate antibodies. The overall process is highly scalable and faster than PCR-based techniques, with a Fab insertion success rate of around 80%. By using carefully chosen overhangs on each end of the antibody domains, this approach paves the way to the universal, sequence- and vector-independent cloning and reformatting of antibody libraries.
Assuntos
Técnicas de Visualização da Superfície Celular , Clonagem Molecular , Fragmentos Fab das Imunoglobulinas/genética , Biblioteca de Peptídeos , Albumina Sérica/imunologia , Animais , Bacteriófagos/genética , Bacteriófagos/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Epitopos , Feminino , Vetores Genéticos , Humanos , Imunização , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos Endogâmicos BALB C , Coelhos , Albumina Sérica/administração & dosagem , Albumina Sérica HumanaRESUMO
Gene delivery vectors based on adenovirus, particularly human adenovirus serotype 5 (hAd5) have great potential for the treatment of variety of diseases. However, the tropism of hAd5 needs to be modified to achieve tissue- or cell- specific therapies for the successful application of this vector system to clinic. Here, we modified hAd5 tropism by replacing the fiber knob which contains the coxsackievirus B and adenovirus receptor (CAR)-binding sites with a biotin acceptor peptide, a truncated form of Propionibacterium shermanii 1.3 S transcarboxylase domain (PSTCD), to enable metabolically biotinylation of the virus. We demonstrate here that the new adenovirus no longer shows CAR-dependent cell uptake and transduction. When metabolically biotinylated and avidin-coated, it forms a nano-complex that can be retargeted to distinct cells using biotinylated antibodies. This vector may prove useful in the path towards achieving targeted gene delivery.
Assuntos
Adenovírus Humanos/fisiologia , Biotina/metabolismo , Proteínas do Capsídeo/genética , Deleção de Genes , Técnicas de Transferência de Genes , Vetores Genéticos , Tropismo Viral , Adenovírus Humanos/genética , Animais , Carboxil e Carbamoil Transferases/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Terapia Genética/métodos , Humanos , Receptores Virais/genética , Proteínas Recombinantes/genética , Coloração e Rotulagem/métodosRESUMO
Molecular medicine can benefit greatly from antibodies that deliver therapeutic and imaging agents to select organs and diseased tissues. Yet the development of complex and defined composite nanostructures remains a challenge that requires both designed stoichiometric assembly and superior in vivo testing ability. Here, we generate nanostructures called nanostreptabodies by controlled sequential assembly of biotin-engineered antibody fragments on a streptavidin scaffold with a defined capacity for additional biotinylated payloads such as other antibodies to create bispecific antibodies as well as organic and non-organic moieties. When injected intravenously, these novel and stable nanostructures exhibit exquisite targeting with tissue-specific imaging and delivery, including rapid transendothelial transport that enhances tissue penetration. This "tinkertoy construction" strategy provides a very flexible and efficient way to link targeting vectors with reporter and/or effector agents, thereby providing virtually endless combinations potentially useful for multipurpose molecular and functional imaging in vivo as well as therapies.
Assuntos
Anticorpos/metabolismo , Técnicas de Transferência de Genes , Nanoestruturas/química , Especificidade de Órgãos , Estreptavidina/metabolismo , Animais , Anticorpos Biespecíficos/metabolismo , Formação de Anticorpos , Sequência de Bases , Biotinilação , Cromatografia Líquida de Alta Pressão , Expressão Gênica , Vetores Genéticos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos , Modelos Biológicos , Dados de Sequência MolecularRESUMO
The molecular diversity of the luminal endothelial cell surface arising in vivo from local variations in genetic expression and tissue microenvironment may create opportunities for achieving targeted molecular imaging and therapies. Here, we describe a strategy to identify probes and their cognate antigens for targeting vascular endothelia of specific organs in vivo. We differentially screen phage libraries to select organ-targeting antibodies by using luminal endothelial cell plasma membranes isolated directly from tissue and highly enriched in natively expressed proteins exposed to the bloodstream. To obviate liver uptake of intravenously injected phage, we convert the phage-displayed antibodies into scFv-Fc fusion proteins, which then are able to rapidly target select organ(s) in vivo as visualized directly by gamma-scintigraphic whole-body imaging. Mass spectrometry helps identify the antigen targets. This comprehensive strategy provides new promise for harnessing the power of phage display for mapping vascular endothelia natively in tissue and for achieving vascular targeting of specific tissues in vivo.
Assuntos
Anticorpos/análise , Anticorpos/imunologia , Células Endoteliais/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Aminopeptidases/imunologia , Animais , Antígenos/imunologia , Células Endoteliais/metabolismo , Pulmão/irrigação sanguínea , Pulmão/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Especificidade de Órgãos , Ratos , Reprodutibilidade dos TestesRESUMO
Cryptococcus neoformans causes a life-threatening meningoencephalitis in a significant percentage of AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid (TT) produce Abs that, based on the epitope recognized, can be either protective or nonprotective. Since nonprotective Abs block the efficacy of protective Abs, we are interested in developing a vaccine that would focus the immune response specifically to protective epitopes. Previously, we screened a phage display library with 2H1, a protective anti-GXM mAb, and isolated PA1, a representative peptide that had a K(d) of 295 nM for 2H1. Mice immunized with PA1 conjugated to keyhole limpet hemocyanin developed high anti-peptide (1/13,000), but low anti-GXM (maximum, 1/200) titers. We now report our efforts to improve this vaccine by screening a sublibrary with six random amino acids added to either end of the PA1 motif to identify higher affinity peptides. P206.1, a peptide isolated from this sublibrary, had 80-fold higher affinity for 2H1 (K(d) = 3.7 nM) than PA1. P206.1 bound protective, but not nonprotective, anti-GXM Abs. Mice immunized with P206.1 conjugated to various carriers did not mount an Ab response to GXM despite developing high anti-peptide titers. However, mice primed with GXM-TT and boosted with P206.1-TT developed significant anti-GXM titers (maximum, 1/180,000). This latter immunization scheme focused the immune response on protective epitopes, since only 2-5% of these titers were directed against nonprotective de-O-acetylated GXM epitopes compared with 20-60% in animals primed and boosted with GXM-TT.