RESUMO
Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.
Assuntos
Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/antagonistas & inibidores , Mutação , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Antineoplásicos Imunológicos/química , Complexo CD3/química , Desenho de Fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , Relação Estrutura-AtividadeRESUMO
Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF.
Assuntos
Proteínas Mutantes/química , Mutação , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Animais , Anti-Inflamatórios/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Sítios de Ligação , Varredura Diferencial de Calorimetria , Linhagem Celular Tumoral , Citocinas/metabolismo , Desenho de Fármacos , Feminino , Fibroblastos/metabolismo , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/química , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica , Receptores Tipo II do Fator de Necrose Tumoral/antagonistas & inibidores , Proteínas Recombinantes de Fusão/químicaRESUMO
Tumor necrosis factor-α (TNF), which is an immuno-modulatory cytokine, has been suggested to cause inflammatory responses as well as protection against tissue dysfunction by binding two types of TNF receptor (TNFR1/TNFR2). However, the physiological effects of TNFR2-specific activation remain unclear. We therefore aimed to generate a TNF mutant with full TNFR2-selective agonist activity as a functional analytical tool. In this study, we utilized a phage display technique to create mouse TNFR2 (mTNFR2)-selective TNF mutants that bind specifically to mTNFR2 and show full bioactivity compared with wild-type TNF. A new phage library displaying TNF mutants was created, in which nine amino acid residues at the predicted receptor-binding site were randomized. From this library, an agonistic TNF mutant exhibiting high binding selectivity and bioactivity to mTNFR2 was isolated. We propose that this TNF mutant would be a powerful tool with which to elucidate the functional roles of mTNFR2.
RESUMO
Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10(-1) µg/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors.
Assuntos
Anticorpos Biespecíficos/imunologia , Neoplasias da Mama/tratamento farmacológico , Complexo CD3/imunologia , Receptores da Família Eph/imunologia , Animais , Anticorpos Biespecíficos/uso terapêutico , Especificidade de Anticorpos/imunologia , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologia , Células Th1/metabolismoRESUMO
The EPH receptor A10 (EphA10) is up-regulated in breast cancer but is not normally expressed in healthy tissue, thus it has been suggested that EphA10 may be a useful target for cancer therapy. This study reports a diabody, an antibody derivative binding two different target molecules, EphA10 expressed in tumor cells and CD3 expressed in T cells, which showed T cell dependent-cytotoxicity. The diabody, which has His-tagged and FLAG-tagged chains, was expressed in Escherichia coli and purified in both heterodimer (Db-1) and homodimer (Db-2) formulations by liquid chromatography. Flow cytometry analysis using EphA10-expressing cells showed that binding activity of heterodimers was stronger than that of homodimers. Addition of diabodies to PBMC cultures resulted in T-cell mediated redirected lysis, and the bioactivity was consistent with the stronger binding activity of heterodimeric diabody formulations. Our results indicate that diabodies recognizing both EphA10 and CD3 could have a range of potential applications in cancer therapy, such as breast cancers that express the EPH receptor A10, especially triple negative breast cancer.
Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Receptores da Família Eph/imunologia , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , Ligação Proteica , TransfecçãoRESUMO
One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.
Assuntos
Anticorpos Biespecíficos/imunologia , Antineoplásicos/imunologia , Imunoglobulina G/imunologia , Neoplasias/imunologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacocinética , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Área Sob a Curva , Sítios de Ligação/imunologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Relação Dose-Resposta a Droga , Receptores ErbB/imunologia , Feminino , Humanos , Imunoglobulina G/genética , Imunoglobulina G/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Células MCF-7 , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ligação Proteica/imunologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The domains of bispecific diabodies (BsDbs) can be ordered in four different ways; however, the influence of domain order on the cytotoxicity of BsDbs that retarget immune cells against tumor cells had not been addressed. We previously reported the marked antitumor effects of a humanized BsDb that targets epidermal growth factor receptor and CD3 (hEx3-Db). Here, we rearranged the domains of hEx3-Db to examine the influence of domain order on the function of BsDbs. We successfully prepared homogenous dimers of hEx3-Db in all four domain configurations. Interestingly, all three rearranged hEx3s inhibited cancer growth more effectively than did the original hEx3-Db, in which both components were in variable heavy domain (VH)-variable light domain (VL) order (redesignated as hEx3-HL), and the highest effects were observed with hEx3-LH (hEx3-Db with both components in VL-VH order). In addition, hEx3-LH had comparable in vitro growth inhibitory effects to those of the tandem single-chain variable fragment (scFv) format of hEx3-Db (hEx3-tandem scFv (taFv)), which we previously showed to have greater cytotoxicity than does hEx3-HL. Flow cytometry suggested that the enhanced cytotoxicity of hEx3-LH is attributable to structural superiority for cross-linking, similar to that of hEx3-taFv. Furthermore, hEx3-LH inhibited cancer growth in mice more effectively than did hEx3-taFv; this difference may be due to differences in antibody stability. Our results show that merely rearranging the domain order of BsDbs can enhance their effects beyond those with structural format conversion.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Complexo CD3/imunologia , Receptores ErbB/imunologia , Neoplasias/terapia , Animais , Anticorpos Biespecíficos/imunologia , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/imunologia , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.
Assuntos
Anticorpos Biespecíficos/imunologia , Antineoplásicos/metabolismo , Complexo CD3/imunologia , Receptores ErbB/imunologia , Anticorpos de Cadeia Única/imunologia , Linfócitos T/imunologia , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Citotoxinas/química , Citotoxinas/genética , Citotoxinas/imunologia , Citotoxinas/farmacologia , Humanos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacologiaRESUMO
We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy.