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1.
MAbs ; 12(1): 1792130, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32684124

RESUMO

Conditionally active COBRA™ (COnditional Bispecific Redirected Activation) T cell engagers are engineered to overcome the limitations of inherently active first-generation T cell engagers, which are unable to discern between tumor and healthy tissues. Designed to be administered as prodrugs, COBRAs target cell surface antigens upon administration, but engage T cells only after they are activated within the tumor microenvironment (TME). This allows COBRAs to be preferentially turned on in tumors while safely remaining inactive in healthy tissue. Here, we describe the development of the COBRA design and the characterization of these conditionally active T cell engagers. Upon administration COBRAs are engineered to bind to tumor-associated antigens (TAAs) and serum albumin (to extend their half-life in circulation), but are inhibited from interacting with the T cell receptor complex signaling molecule CD3. In the TME, a matrix metalloproteinase (MMP)-mediated linker cleavage event occurs within the COBRA construct, which rearranges the molecule, allowing it to co-engage TAAs and CD3, thereby activating T cells against the tumor. COBRAs are conditionally activated through cleavage with MMP9, and once active are highly potent, displaying sub-pM EC50s in T cell killing assays. Studies in tumor-bearing mice demonstrate COBRA administration completely regresses established solid tumor xenografts. These results strongly support the further characterization of the novel COBRA design in preclinical development studies.


Assuntos
Anticorpos Biespecíficos , Antígenos de Neoplasias , Antineoplásicos Imunológicos , Imunoterapia , Ativação Linfocitária , Neoplasias Experimentais/terapia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Células HT29 , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Experimentais/imunologia , Engenharia de Proteínas , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Protein Expr Purif ; 149: 75-83, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29655788

RESUMO

Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.


Assuntos
Anticorpos Monoclonais Humanizados/biossíntese , Engenharia de Proteínas/métodos , Animais , Anticorpos Monoclonais Humanizados/genética , Células CHO , Cricetulus , Expressão Gênica , Vetores Genéticos , Células HEK293 , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Conformação Proteica , Multimerização Proteica , Transfecção
3.
Obesity (Silver Spring) ; 24(8): 1687-94, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27330016

RESUMO

OBJECTIVE: Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half-life and low immunogenicity could be useful in the treatment of this condition. METHODS: Phage display technology coupled with flow cytometry and cell-based in vitro assays were employed to identify an allosteric agonist of the mouse LEPR. LEP-deficient Lep(ob) /Lep(ob) mice were used to compare in vivo effects of LEP to antibody administration. To evaluate hypothalamic effects of treatment, changes in mRNA levels of neuropeptide Y and proopiomelanocortin were measured. RESULTS: XPA.80.037 is a monoclonal antibody that demonstrates allosteric agonism of the mouse LEPR. Treatment of Lep(ob) /Lep(ob) mice with XPA.80.037 markedly reduced hyperphagia and body weight, normalized blood glucose and plasma insulin levels, and corrected dyslipidemia. These metabolic alterations correlated with changes in mRNA levels of neuropeptide Y and proopiomelanocortin, suggesting that XPA.80.037 had hypothalamic effects. CONCLUSIONS: Agonist allosteric monoclonal antibodies to the LEPR can correct metabolic effects associated with LEP deficiency in vivo and thereby have the potential to treat conditions of LEP deficiency.


Assuntos
Glicemia/metabolismo , Leptina/metabolismo , Leptina/fisiologia , Obesidade/metabolismo , Pró-Opiomelanocortina/metabolismo , Receptores para Leptina/metabolismo , Regulação Alostérica , Animais , Peso Corporal , Diabetes Mellitus/metabolismo , Meia-Vida , Hipotálamo/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo
4.
MAbs ; 3(1): 49-60, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21048425

RESUMO

Interleukin-1ß (IL-1ß) is a potent mediator of inflammatory responses and plays a role in the differentiation of a number of lymphoid cells. In several inflammatory and autoimmune diseases, serum levels of IL-1ß are elevated and correlate with disease development and severity. The central role of the IL-1 pathway in several diseases has been validated by inhibitors currently in clinical development or approved by the FDA. However, the need to effectively modulate IL-1ß-mediated local inflammation with the systemic delivery of an efficacious, safe and convenient drug still exists. To meet these challenges, we developed XOMA 052 (gevokizumab), a potent anti-IL-1ß neutralizing antibody that was designed in silico and humanized using Human Engineering™ technology. XOMA 052 has a 300 femtomolar binding affinity for human IL-1ß and an in vitro potency in the low picomolar range. XOMA 052 binds to a unique IL-1ß epitope where residues critical for binding have been identified. We have previously reported that XOMA 052 is efficacious in vivo in a diet-induced obesity mouse model thought to be driven by low levels of chronic inflammation. We report here that XOMA 052 also reduces acute inflammation in vivo, neutralizing the effect of exogenously administered human IL-1ß and blocking peritonitis in a mouse model of acute gout. Based on its high potency, novel mechanism of action, long half-life, and high affinity, XOMA 052 provides a new strategy for the treatment of a number of inflammatory, autoimmune and metabolic diseases in which the role of IL-1ß is central to pathogenesis.


Assuntos
Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos/imunologia , Inflamação/prevenção & controle , Interleucina-1beta/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Linhagem Celular , Reações Cruzadas/imunologia , Relação Dose-Resposta a Droga , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Inflamação/sangue , Inflamação/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Interleucina-6/metabolismo , Cinética , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos
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