RESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Humanos , SARS-CoV-2/genética , Proteínas de Repetição de Anquirina Projetadas , Microscopia Crioeletrônica , Anticorpos Monoclonais/uso terapêutico , Terapia Combinada de Anticorpos , Anticorpos NeutralizantesRESUMO
Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Autoanticorpos/imunologia , Interleucina-17/imunologia , Timoma/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Afinidade de Anticorpos/imunologia , Autoanticorpos/genética , Autoanticorpos/isolamento & purificação , Linhagem Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interleucina-17/genética , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Timoma/sangueRESUMO
BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety. METHODS: We recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation. RESULTS: ING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells. CONCLUSION: A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.
RESUMO
Epidermal growth factor receptor (EGFR)-specific monoclonal antibodies predominantly inhibit colorectal cancer (CRC) growth by interfering with receptor signaling. Recent analyses have shown that patients with CRC with mutated KRAS and BRAF oncogenes do not profit from treatment with such antibodies. Here we have used the binding domains of cetuximab and pantitumumab for constructing T cell-engaging BiTE antibodies. Both EGFR-specific BiTE antibodies mediated potent redirected lysis of KRAS- and BRAF-mutated CRC lines by human T cells at subpicomolar concentrations. The cetuximab-based BiTE antibody also prevented at very low doses growth of tumors from KRAS- and BRAF-mutated human CRC xenografts, whereas cetuximab was not effective. In nonhuman primates, no significant adverse events were observed during treatment for 3 wk at BiTE serum concentrations inducing, within 1 d, complete lysis of EGFR-overexpressing cancer cells. EGFR-specific BiTE antibodies may have potential to treat CRC that does not respond to conventional antibodies.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Receptores ErbB/antagonistas & inibidores , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados , Cetuximab , Neoplasias Colorretais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes ras/efeitos dos fármacos , Humanos , Mutação/efeitos dos fármacos , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf , Fatores de RiscoRESUMO
muS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. MT110, its human-specific analog, is in a clinical phase 1 trial for treatment of patients with adenocarcinoma of the lung or gastrointestinal tract. Recent studies have shown a therapeutic window for muS110, have explored single-dose toxicity of muS110, and have found that a 1-week low-dose treatment dramatically increased the tolerability of mice to very high doses of muS110 (Cancer Immunol. Immunother. 2009;58:95-109). Here we analyzed the impact of long-term, high-dose treatment of mice with muS110 on antitumor activity and functionality of T cells. After an initial self-limiting cytokine release, the 1-week adaptation period effectively blunted further cytokine production in response to a subsequent high-dose treatment with muS110. The much-increased tolerability of mice adapted to muS110 was not because of anergy of T cells. T cells isolated from chronically muS110-treated mice fully retained their cytotoxic potential, proliferative capacity, and responsiveness to stimulation by either muS110 or anti-CD3/anti-CD28/interleukin-2 when compared with T cells from control mice. Unimpaired T-cell performance was also evident from the effective prevention of orthotopic 4T1 breast tumor outgrowth in mice treated long term with escalating doses of muS110. Finally, we show that muS110 and MT110 recognize orthologous epitopes on mouse and human EpCAM proteins, suggesting that the target-related safety profile of muS110 in mice may be predictive for MT110 in humans.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Antígenos de Neoplasias/imunologia , Complexo CD3/imunologia , Moléculas de Adesão Celular/imunologia , Imunoterapia , Neoplasias Mamárias Experimentais/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/efeitos adversos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Complexo CD3/genética , Complexo CD3/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Anergia Clonal/efeitos dos fármacos , Citocinas/metabolismo , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/efeitos adversos , Ativação Linfocitária/efeitos dos fármacos , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Engenharia de Proteínas , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologia , Fatores de TempoRESUMO
Carcinoembryonic antigen (CEA, CD66e) is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/terapia , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Anticorpos Biespecíficos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células CHO , Antígeno Carcinoembrionário/sangue , Cricetinae , Cricetulus , Humanos , Imunoterapia , Camundongos , Camundongos SCID , Proteínas Recombinantes de Fusão/imunologia , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/imunologiaRESUMO
NKG2D is a surface receptor expressed on NK cells but also on CD8(+) T cells, gammadelta T cells, and auto-reactive CD4(+)/CD28(-) T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (KD) of 2.7 +/- 1.4 x 10(-11) M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFalpha, IFNgamma and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after seven days in human serum at 37 degrees C, and resisted thermal inactivation up to 70 degrees C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Afinidade de Anticorpos , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunoglobulinas/metabolismo , Células Matadoras Naturais/metabolismo , Macaca fascicularis , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Biblioteca de Peptídeos , Linfócitos T/imunologiaRESUMO
GM-CSF (granulocyte-macrophage colony stimulating factor) plays a central role in inflammatory processes. Treatment with antibodies neutralizing murine GM-CSF showed significant therapeutic effects in mouse models of inflammatory diseases. We constructed by phage display technology a human scFv, which could potently neutralize human GM-CSF. At first, a human V(L) repertoire was combined with the V(H) domain of a parental GM-CSF-neutralizing rat antibody. One dominant rat/human scFv clone was selected, neutralizing human GM-CSF with an IC50 of 7.3 nM. The human V(L) of this clone was then combined with a human V(H) repertoire. The latter preserved the CDR 3 of the parental rat V(H) domain to retain binding specificity. Several human scFvs were selected, which neutralized human GM-CSF at low nanomolar concentrations (IC50 > or = 2.6 nM). To increase serum half-life, a branched 40 kDa PEG-polymer was coupled to the most potent GM-CSF-neutralizing scFv (3077) via an additional C-terminal cysteine. PEG conjugation had a negligible effect on the in vitro neutralizing potential of the scFv, although it caused a significant drop in binding affinity owing to a reduced on-rate. It also significantly increased the stability of the scFv at elevated temperatures. In mouse experiments, the PEGylated scFv 3077 showed a significantly prolonged elimination half-life of 59 h as compared with 2 h for the unconjugated scFv version. PEGylated scFv 3077 is a potential candidate for development of a novel antibody therapy to treat pro-inflammatory human diseases.