RESUMO
T cells recognize small fragments of microorganisms (antigens) on the surface of other cells using T cell antigen receptors. The mechanism by which these receptors signal into T cells is controversial, but two recent studies provide important new clues.
Assuntos
Antígenos/imunologia , Imunidade Celular/imunologia , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Antígenos/metabolismo , Humanos , Complexo Principal de Histocompatibilidade/imunologiaRESUMO
BACKGROUND: The VEGF/VEGFR and the HGF/cMET pathways are key mediators of the interplay of tumor cells and their microenvironment. However, inhibition of VEGF has been shown to produce only limited clinical benefit and inhibition of the activation of cMET by HGF has not translated into clinical benefit in pivotal trials. MP0250, a DARPin® molecule that specifically inhibits both VEGF and HGF has been developed to explore the clinical potential of dual inhibition of these pathways. RESULTS: MP0250 binding to VEGF and HGF inhibited downstream signalling through VEGFR2 and cMET resulting in inhibition of proliferation of VEGF- and HGF-dependent cells. Antitumor activity was demonstrated in VEGF- and HGF-dependent xenograft and syngeneic models with activity superior to that of individual VEGF- and HGF-blocking DARPin® molecules. Combination therapy studies showed potentiation of the antitumor activity of chemotherapy and immunotherapy agents, including an anti-PD1 antibody. MATERIALS AND METHODS: Potency of MP0250 was assessed in cellular models and in a variety of xenograft models as monotherapy or in combination with standard-of-care drugs. CONCLUSIONS: Dual inhibition of VEGF and HGF by MP0250 produced powerful single agent and combination antitumor activity. This, together with increasing understanding of the role of the HGF/cMET pathway in resistance to VEGF (and other agents), supports testing of MP0250 in the clinic.
RESUMO
MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of â¼ 30 hours in mouse and â¼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antineoplásicos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Administração Intravenosa , Animais , Repetição de Anquirina/genética , Repetição de Anquirina/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Desenho de Fármacos , Feminino , Meia-Vida , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/imunologia , Humanos , Infusões Intravenosas , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica Humana/genética , Albumina Sérica Humana/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
The B cell surface molecule CD22 is a member of the Siglec family. Siglecs possess a conserved membrane-distal immunoglobulin domain that mediates binding to sialylated glycoproteins or glycolipids. Although the structural basis of sialic acid recognition by Siglecs is quite well understood, the binding properties of the interaction between Siglecs and their native ligands have not been investigated. CD22 binding requires alpha2-6-linked sialic acid, which is mostly carried on N-glycans. One protein that carries such N-glycans is CD45. In this study we used surface plasmon resonance to perform thermodynamic and kinetic analysis of CD22 binding to native CD45. CD22 bound with a low affinity (K(d) 130 microM at 25 degrees C) and very fast kinetics (k(off) >or=18 s(-1), calculated k(on) >or=1.5 x 10(5) M(-1)s(-1)). Van't Hoff analysis revealed that binding was enthalpically driven at physiological temperatures, as is typical of most lectin-carbohydrate interactions. Since there is evidence that CD22 binds preferably to CD45, even though many cell surface proteins carry alpha2-6-linked sialic acid, we compared the affinities of CD22 binding to CD45, to CD4 carrying alpha2-6-linked sialic acid, and to a synthetic alpha2-6-sialoglycoconjugate. The affinities did not differ substantially, suggesting that CD22 binds preferentially to CD45 not because the latter presents higher affinity ligands but because it carries multiple copies of thereof.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Moléculas de Adesão Celular/metabolismo , Lactose/metabolismo , Lectinas , Antígenos Comuns de Leucócito/metabolismo , Oligossacarídeos/metabolismo , Resinas Acrílicas/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos CD/isolamento & purificação , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Diferenciação de Linfócitos B/isolamento & purificação , Células CHO , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/isolamento & purificação , Cricetinae , Cinética , Lactose/análogos & derivados , Ligantes , Camundongos , Polímeros/metabolismo , Ratos , Proteínas Recombinantes/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , SolubilidadeRESUMO
The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR zeta-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.
Assuntos
Antígenos CD , Glicoesfingolipídeos/metabolismo , Antígenos Comuns de Leucócito/química , Antígenos Comuns de Leucócito/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Lipídeos de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Antígenos CD2/química , Antígenos CD2/genética , Antígenos CD2/metabolismo , Linhagem Celular , Dimerização , Humanos , Antígenos Comuns de Leucócito/genética , Leucossialina , Camundongos , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígenos Thy-1/química , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , TransfecçãoRESUMO
Considerable progress has been made in characterizing four key sets of interactions controlling antigen responsiveness in T cells, involving the following: the T cell antigen receptor, its coreceptors CD4 and CD8, the costimulatory receptors CD28 and CTLA-4, and the accessory molecule CD2. Complementary work has defined the general biophysical properties of interactions between cell surface molecules. Among the major conclusions are that these interactions are structurally heterogeneous, often reflecting clear-cut functional constraints, and that, although they all interact relatively weakly, hierarchical differences in the stabilities of the signaling complexes formed by these molecules may influence the sequence of steps leading to T cell activation. Here we review these developments and highlight the major challenges remaining as the field moves toward formulating quantitative models of T cell recognition.