RESUMO
The antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs have been researched for more than 30â¯years, currently only a limited number of bispecific antibodies have achieved regulatory approval. Of the few successful examples of industrially manufacturing a bispecific antibody, the "common light chain format" is an elegant technology that simplifies the purification of a whole IgG-type bispecific antibody. Using this IgG format, the bispecific function can be introduced while maintaining the natural molecular shape of the antibody. In this article, we will first introduce the outline, prospects, and limitations of the common light chain format. Then, we will describe the identification and optimization process for ERY974, an anti-glypican-3â¯×â¯anti-CD3ε T cell-redirecting bispecific antibody with a common light chain. This format includes one of Chugai's proprietary technologies, termed ART-Ig technology, which consists of a method to identify a common light chain, isoelectric point (pI) engineering to purify the desired bispecific IgG antibody from byproducts, and Fc heterodimerization by an electrostatic steering effect. Furthermore, we describe some tips for de-risking the antibody when engineering a T cell redirecting antibody.
Assuntos
Anticorpos Biespecíficos , Imunoglobulina G , Cadeias Leves de Imunoglobulina , Engenharia de Proteínas/métodos , Animais , Complexo CD3/imunologia , Glipicanas/imunologia , Humanos , CamundongosRESUMO
Monoclonal antibodies have become a general modality in therapeutic development, and a variety of monoclonal antibodies targeting soluble antigens have been developed. However, even with infinite binding affinity to an antigen, a conventional antibody can bind to the antigen only once and results in an increase in total plasma antigen concentration in vivo. This antibody-mediated antigen accumulation generally occurs because the clearance from circulation of an antibody-antigen complex is much slower than that of a free antigen. This limitation has recently been overcome by sweeping antibodies, which are capable of actively eliminating soluble antigens from circulation. A sweeping antibody incorporates two antibody engineering technologies: one is variable region engineering to enable the antibody to bind to an antigen in plasma and dissociate from the antigen in endosome (after which the antigen undergoes lysosomal degradation), and the other is constant region engineering to increase the cellular uptake of the antibody-antigen complex into endosome. By enhancing the elimination of soluble antigens from circulation, sweeping antibodies can therapeutically target soluble antigens that conventional antibodies cannot. This review discusses the features, engineering technologies, advantages, and applications of sweeping antibodies that target soluble antigens.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Antígenos/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Formação de Anticorpos , Antígenos/sangue , Descoberta de Drogas , Engenharia Genética/métodos , Humanos , Ligação Proteica/imunologia , Receptores Fc/metabolismoRESUMO
Fc engineering can modulate the Fc-FcγR interaction and thus enhance the potency of Abs that target membrane-bound Ags, but it has not been applied to Abs that target soluble Ags. In this study, we revealed a previously unknown function of inhibitory FcγRII in vivo and, using an Ab that binds to Ag pH dependently, demonstrated that the function can be exploited to target soluble Ag. Because pH-dependent Ab dissociates Ag in acidic endosome, its Ag clearance from circulation reflects the cellular uptake rate of Ag/Ab complexes. In vivo studies showed that FcγR but not neonatal FcR contributes to Ag clearance by the pH-dependent Ab, and when Fc binding to mouse FcγRII and III was increased, Ag clearance was markedly accelerated in wild-type mice and FcR γ-chain knockout mice, but the effect was diminished in FcγRII knockout mice. This demonstrates that mouse FcγRII efficiently promotes Ab uptake into the cell and its subsequent recycling back to the cell surface. Furthermore, when a human IgG1 Fc variant with selectively increased binding to human FcγRIIb was tested in human FcγRIIb transgenic mice, Ag clearance was accelerated without compromising the Ab half-life. Taken together, inhibitory FcγRIIb was found to play a prominent role in the cellular uptake of monomeric Ag/Ab immune complexes in vivo, and when the Fc of a pH-dependent Ab was engineered to selectively enhance human FcγRIIb binding, the Ab could accelerate soluble Ag clearance from circulation. We assume such a function would enhance the therapeutic potency of Abs that target soluble Ags.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Reações Antígeno-Anticorpo/imunologia , Antígenos/sangue , Imunoglobulina G/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos/sangue , Anticorpos/imunologia , Complexo Antígeno-Anticorpo/imunologia , Antígenos/imunologia , Humanos , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/genéticaRESUMO
ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Fator IXa/imunologia , Fator X/imunologia , Hemofilia A/complicações , Hemorragia/complicações , Hemorragia/prevenção & controle , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Linhagem Celular , Modelos Animais de Doenças , Hemorragia/patologia , Humanos , Articulações/efeitos dos fármacos , Articulações/patologia , Macaca fascicularis , Masculino , CamundongosRESUMO
BACKGROUND: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. OBJECTIVES: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. METHODS: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. RESULTS: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. CONCLUSION: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.
Assuntos
Anticorpos Biespecíficos , Hemofilia A , Hemostáticos , Humanos , Hemostáticos/farmacologia , Hemostáticos/uso terapêutico , Trombina/metabolismo , Hemostasia , Coagulação Sanguínea , Fator VIIIRESUMO
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
Assuntos
Complexo CD3 , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares , Proteínas de Membrana , Carcinoma de Pequenas Células do Pulmão , Linfócitos T , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/terapia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Complexo CD3/imunologia , Animais , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Ativação Linfocitária/imunologia , Feminino , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To combat infectious diseases, vaccines are considered the best prophylactic strategy for a wide range of the population, but even when vaccines are effective, the administration of therapeutic antibodies against viruses could provide further treatment options, particularly for vulnerable groups whose immunity against the viruses is compromised. Therapeutic antibodies against dengue are ideally engineered to abrogate binding to Fcγ receptors (FcγRs), which can induce antibody-dependent enhancement (ADE). However, the Fc effector functions of neutralizing antibodies against SARS-CoV-2 have recently been reported to improve post-exposure therapy, while they are dispensable when administered as prophylaxis. Hence, in this report, we investigated the influence of Fc engineering on anti-virus efficacy using the anti-dengue/Zika human antibody SIgN-3C and found it affected the viremia clearance efficacy against dengue in a mouse model. Furthermore, we demonstrated that complement activation through antibody binding to C1q could play a role in anti-dengue efficacy. We also generated a novel Fc variant, which displayed the ability for complement activation but showed very low FcγR binding and an undetectable level of the risk of ADE in a cell-based assay. This Fc engineering approach could make effective and safe anti-virus antibodies against dengue, Zika and other viruses.
RESUMO
Efficient production of bispecific antibodies (BsAbs) in single mammalian cells is essential for basic research and industrial manufacturing. However, preventing unwanted pairing of heavy chains (HCs) and light chains (LCs) is a challenging task. To address this, we created an engineering technology for preferential cognate HC/LC and HC/HC paring called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology - Immunoglobulin), and applied it to NXT007, a BsAb for the treatment of hemophilia A. We introduced charged amino-acid substitutions at the HC/LC interface to facilitate the proper assembly for manufacturing a standard IgG-type BsAb. We generated CH1/CL interface-engineered antibody variants that achieved > 95% correct HC/LC pairing efficiency with favorable pharmacological properties and developability. Among these, we selected a design (C3) that allowed us to separate the mis-paired species with an unintended pharmacological profile using ion-exchange chromatography. Crystal structure analysis demonstrated that the C3 design did not affect the overall structure of both Fabs. To determine the final design for HCs-heterodimerization, we compared the stability of charge-based and knobs into hole-based Fc formats in acidic conditions and selected the more stable charge-based format. FAST-Ig was also applicable to stable CHO cell lines for industrial production and demonstrated robust chain pairing with different subclasses of parent BsAbs. Thus, it can be applied to a wide variety of BsAbs both preclinically and clinically.
Assuntos
Anticorpos Biespecíficos , Hemofilia A , Animais , Engenharia de Proteínas/métodos , Linhagem Celular , Dimerização , MamíferosRESUMO
In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.
Assuntos
Doença Celíaca , Glutens , Camundongos , Animais , Humanos , Coelhos , Glutens/química , Anticorpos Neutralizantes , Antígenos HLA-DQ , Peptídeos/química , Epitopos/química , Camundongos TransgênicosRESUMO
A conventional antibody targeting a soluble antigen in circulation typically requires a huge dosage and frequent intravenous administration to neutralize the antigen. This is because antigen degradation is reduced by the formation of antigen-antibody immune complexes, which escape from lysosomal degradation using neonatal Fc receptor (FcRn)-mediated recycling. To address this, we developed an antigen-sweeping antibody that combines pH-dependent antigen binding and Fc engineering to enhance Fc receptor binding. The sweeping antibody actively eliminates the plasma antigens by increasing the cellular uptake of the immune complex and dissociating the antigens in the acidic endosome for degradation. Strong antigen sweeping can reduce the dosage, potentially achieve higher efficacy, and expand the scope of antigen space available for targeting by antibodies. In this study, to further improve the sweeping efficacy, we developed a novel antibody Fc variant by enhancing Fcγ receptor IIb (FcγRIIb) binding and modulating charge characteristics for increased cellular uptake of the immune complex, together with enhancing FcRn binding for efficient salvage of the antigen-free antibodies. Our Fc variant achieved strong antigen sweeping in cynomolgus monkeys with antibody pharmacokinetics comparable to a wild-type human IgG1 antibody. The positive-charge substitutions enhanced uptake of the immune complex by FcγRIIb-expressing cells in vitro, which was completely inhibited by an anti-FcγRIIb antibody. This suggests that the strong in vivo sweeping efficacy improved by the charge engineering is more likely achieved by FcγRIIb-dependent uptake of the immune complex rather than nonspecific uptake. We expect this novel Fc engineering can maximize the antigen sweeping efficacy even in humans and create novel therapeutic antibodies that meet unmet medical needs for patients.
Assuntos
Complexo Antígeno-Anticorpo , Antígenos , Animais , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas , Macaca fascicularisRESUMO
The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/terapia , Humanos , Glicoproteínas de Membrana , Testes de Neutralização , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope ViralRESUMO
Myostatin, a member of the transforming growth factor-ß superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via "sweeping antibody technology", GYM329 reduces or "sweeps" myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.
Assuntos
Anticorpos Monoclonais/farmacologia , Força Muscular/efeitos dos fármacos , Doenças Musculares/fisiopatologia , Miostatina/imunologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Diferenciação de Crescimento/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Tamanho do Órgão , Transdução de SinaisRESUMO
Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Trifosfato de Adenosina , Neoplasias , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Glypican 3 (GPC3), a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, is expressed in a majority of hepatocellular carcinoma tissues. The murine monoclonal antibody GC33 that specifically binds to the COOH-terminal part of GPC3 causes strong antibody-dependent cellular cytotoxicity against hepatocellular carcinoma cells and exhibits strong antitumor activity in the xenograft models. To apply GC33 for clinical use, we generated a humanized GC33 from complementarity-determining region grafting with the aid of both the hybrid variable region and two-step design methods. The humanized antibody bound to GPC3 specifically and induced antibody-dependent cellular cytotoxicity as effectively as a chimeric GC33 antibody. To improve stability of the humanized GC33, we further optimized humanized GC33 by replacing the amino acid residues that may affect the structure of the variable region of a heavy chain. Substitution of Glu6 with Gln in the heavy chain significantly improved the stability under high temperatures. GC33 also has the risk of deamidation of the -Asn-Gly- sequence in the complementarity-determining region 1 of the light chain. As substitution of Asn diminished the antigen binding, we changed the neighboring Gly to Arg to avoid deamidation. The resulting humanized anti-GPC3 antibody was as efficacious as chimeric GC33 against the HepG2 xenograft and is now being evaluated in clinical trials.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Glipicanas/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Carcinoma Hepatocelular/patologia , Regiões Determinantes de Complementaridade/imunologia , Desenho de Fármacos , Humanos , Região Variável de Imunoglobulina/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Estabilidade Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.
Assuntos
Trifosfato de Adenosina/metabolismo , Anticorpos/metabolismo , Espaço Extracelular/metabolismo , Animais , Humanos , Camundongos , Microambiente TumoralRESUMO
Monoclonal antibodies (mAbs) have become an important therapeutic option for several diseases. Since several mAbs have shown promising efficacy in clinic, the competition to develop mAbs has become severe. In efforts to gain a competitive advantage over other mAbs and provide significant benefits to patients, innovations in antibody engineering have aimed at improving the pharmacokinetic properties of mAbs. Because engineering can provide therapeutics that are more convenient, safer, and more efficacious for patients in several disease areas, it is an attractive approach to provide significant benefits to patients. Further advances in engineering mAbs to modulate their pharmacokinetics were driven by the increase of total soluble target antigen concentration that is often observed after injecting a mAb, which then requires a high dosage to antagonize. To decrease the required dosage, several antibody engineering techniques have been invented that reduce the total concentration of soluble target antigen. Here, we review the various ways that antibody engineering can improve the pharmacokinetic properties of mAbs.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Engenharia Biomédica/métodos , Receptores Fc/metabolismo , Animais , Anticorpos Monoclonais/genética , Engenharia Biomédica/tendências , Vias de Administração de Medicamentos , Esquema de Medicação , Humanos , Taxa de Depuração Metabólica/efeitos dos fármacos , Taxa de Depuração Metabólica/fisiologia , Ligação Proteica/fisiologia , Receptores Fc/genéticaRESUMO
Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physicochemical stability, expression in host cells and pharmacokinetics, and the basic methodologies of antibody engineering involved. By simultaneously optimizing the antibody molecule in light of these properties, it should prove possible to shorten the research and development period necessary to identify a highly qualified clinical candidate and consequently accelerate the start of the clinical trial.
Assuntos
Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Expressão Gênica , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Mutação , Estabilidade Proteica , Proteínas RecombinantesRESUMO
The complex molecular formats of recent therapeutic antibodies, including bispecific antibodies, antibody fragments, and other fusion proteins, makes the task of purifying the desired molecules in a limited number of purification steps more and more challenging. Manufacturing these complicated biologics can be substantially improved in the affinity capture stage if the simple bind-and-elute mode is accompanied by targeted removal of the impurities, such as mis-paired antibodies and oligomers or aggregates. Here, we report a method, based on the binding valency to Protein L resin, of separating proteins during the elution step by simply controlling the conductivity at low pH. We show that the method efficiently separated targeted antibodies from mis-paired and aggregated species. Notably, the number of Protein L binding sites can be built into the molecule by design to facilitate the purification. This method may be useful for purifying various antibody formats at laboratory and manufacturing scales.
Assuntos
Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromatografia de Afinidade/métodos , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/metabolismo , Complexo CD3/imunologia , Condutividade Elétrica , Antígeno HLA-A2/imunologia , Humanos , Concentração de Íons de Hidrogênio , Resinas de Troca Iônica , Ligação Proteica , Engenharia de Proteínas , Receptor ErbB-2/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
PURPOSE: L612, a human IgM monoclonal antibody produced by an EBV-transformed human B-cell line, binds to ganglioside GM3 and kills GM3-positive human melanoma cells in the presence of complement. It has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (about 20%), pentameric IgM (about 74%), and other minor IgM molecules. Because hexameric IgM activates complement more effectively than pentameric IgM, we developed and evaluated a hexamer-dominant recombinant IgM for clinical applications. EXPERIMENTAL DESIGN: Chinese hamster ovary (CHO) cells were transfected with heavy- and light-chain genes of L612, with or without the joining-chain gene. Antitumor effects of the recombinant IgM secreted from CHO cells were evaluated in vitro and in vivo. RESULTS: Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was approximately 80% hexameric, whereas recombinant IgM from CHO cells transfected with heavy-, light-, and joining-chain genes (designated CJ45) was about 90% pentameric. Both CA19 and CJ45 recombinant IgMs caused complement-dependent cytotoxicity against human and mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller. I.v. injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing a GM3-positive mouse melanoma xenograft. CONCLUSIONS: A hexamer-dominant human IgM against GM3 may provide a more potent treatment option for patients with GM3-positive melanoma.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Gangliosídeo G(M3)/imunologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , Melanoma/química , Melanoma/patologia , Proteínas Recombinantes/uso terapêutico , Neoplasias Cutâneas/química , Neoplasias Cutâneas/patologiaRESUMO
Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. In order to neutralize the increased C5, an even higher dosage of the antibody would be required. This antigen accumulation can be suppressed by giving the antibody a pH-dependent C5-binding property so that C5 is released from the antibody in the acidic endosome and then trafficked to the lysosome for degradation, while the C5-free antibody returns back to plasma. We recently demonstrated that a pH-dependent C5-binding antibody, SKY59, exhibited long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration. Here we report the details of the antibody engineering involved in generating SKY59, from humanizing a rabbit antibody to improving the C5-binding property. Moreover, because the pH-dependent C5-binding antibodies that we first generated still accumulated C5, we hypothesized that the surface charges of the ICs partially contributed to a slow uptake rate of the C5-antibody ICs. This idea motivated us to engineer the surface charges of the antibody. Our surface-charge engineered antibody consequently exhibited a high capacity to sweep C5 and suppressed the C5 accumulation in vivo by accelerating the cycle of sweeping: uptake of ICs into cells, release of C5 from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, our engineered anti-C5 antibody, SKY59, is expected to provide significant benefits for patients with complement-mediated disorders.