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1.
Blood ; 138(25): 2655-2669, 2021 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-34280257

RESUMEN

Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121).


Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Péptidos/uso terapéutico , Proteínas WT1/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Antígeno HLA-A2/inmunología , Humanos , Leucemia Mieloide Aguda/inmunología , Ratones , Péptidos/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas
2.
Proc Natl Acad Sci U S A ; 108(27): 11187-92, 2011 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-21690412

RESUMEN

We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.


Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Biespecíficos/química , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Angiopoyetina 2/inmunología , Animales , Anticuerpos Biespecíficos/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Modelos Moleculares , Neovascularización Fisiológica , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Factor A de Crecimiento Endotelial Vascular/inmunología
3.
Arch Biochem Biophys ; 526(2): 206-18, 2012 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-22464987

RESUMEN

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Receptores ErbB/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Neoplasias/terapia , Receptor IGF Tipo 1/inmunología , Animales , Anticuerpos Biespecíficos/genética , Afinidad de Anticuerpos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Clonación Molecular , Receptores ErbB/metabolismo , Femenino , Humanos , Inmunoglobulina G/genética , Inmunoterapia , Ratones , Ratones SCID , Modelos Moleculares , Neoplasias/inmunología , Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Ingeniería de Proteínas , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéutico
4.
MAbs ; 11(8): 1402-1414, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31526159

RESUMEN

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the 'Fc-load' on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Anticuerpos Biespecíficos/química , Anticuerpos Monoclonales/química , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química
5.
MAbs ; 9(7): 1076-1087, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28805498

RESUMEN

The determination of the binding strength of immunoglobulins (IgGs) to targets can be influenced by avidity when the targets are soluble di- or multimeric proteins, or associated to cell surfaces, including surfaces introduced from heterogeneous assays. However, for the understanding of the contribution of a second drug-to-target binding site in molecular design, or for ranking of monovalent binders during lead identification, affinity-based assessment of the binding strength is required. Typically, monovalent binders like antigen-binding fragments (Fabs) are generated by proteolytic cleavage with papain, which often results in a combination of under- and over-digestion, and requires specific optimization and chromatographic purification of the desired Fabs. Alternatively, the Fabs are produced by recombinant approaches. Here, we report a lean approach for the functional assessment of human IgG1s during lead identification based on an in-solution digestion with the GingisKHAN™ protease, generating a homogenous pool of intact Fabs and Fcs and enabling direct assaying of the Fab in the digestion mixture. The digest with GingisKHAN™ is highly specific and quantitative, does not require much optimization, and the protease does not interfere with methods typically applied for lead identification, such as surface plasmon resonance or cell-based assays. GingisKHAN™ is highly suited to differentiate between affinity and avidity driven binding of human IgG1 monoclonal and bispecific antibodies during lead identification.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Péptido Hidrolasas
6.
Leuk Lymphoma ; 54(11): 2500-5, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23452151

RESUMEN

Both complement and antibody-dependent cellular cytotoxicity (ADCC) contribute to the clinical efficacy of anti-CD20 monoclonal antibody (mAb) therapy. Paradoxically, the C3b component of complement can block interaction between mAb and natural killer (NK) cells. The present study compared the effect of complement on the ability of two anti-CD20 mAbs, rituximab and GA101, to activate NK cells and mediate ADCC. Complement blocked adherence of NK cells to rituximab, but had little effect on NK binding to GA101. Target cells coated with rituximab or GA101 were able to activate NK cells in the absence of serum. Complement in serum blocked NK activation induced by rituximab, but not GA101. Complement blocked rituximab-induced NK-cell mediated ADCC, but not GA101-induced ADCC. These results demonstrate that the decreased ability of GA101 to fix complement relative to rituximab results in an enhanced ability of GA101 to bind to NK cells, activate NK cells and induce ADCC when serum is present.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales de Origen Murino/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Proteínas del Sistema Complemento/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales de Origen Murino/metabolismo , Línea Celular , Complemento C1q/inmunología , Complemento C1q/metabolismo , Complemento C5/inmunología , Complemento C5/metabolismo , Proteínas del Sistema Complemento/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Unión Proteica/inmunología , Rituximab
7.
PLoS One ; 8(4): e61953, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23613981

RESUMEN

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.


Asunto(s)
Angiopoyetina 2/inmunología , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estabilidad Proteica , Estructura Terciaria de Proteína , Electricidad Estática , Relación Estructura-Actividad , Temperatura
8.
Clin Cancer Res ; 19(24): 6730-40, 2013 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-24097868

RESUMEN

PURPOSE: VEGF-A blockade has been clinically validated as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of tumor angiogenesis and metastasis. EXPERIMENTAL DESIGN: We have applied the recently developed CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab (Avastin), and the other arm recognizing Ang-2 based on LC06, an Ang-2 selective human IgG1 antibody. The potency of Ang-2-VEGF CrossMab was evaluated alone and in combination with chemotherapy using orthotopic and subcutaneous xenotransplantations, along with metastasis analysis by quantitative real-time Alu-PCR and ex vivo evaluation of vessels, hypoxia, proliferation, and apoptosis. The mechanism of action was further elucidated using Western blotting and ELISA assays. RESULTS: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient or cell line-derived human tumor xenografts, especially at later stages of tumor development. Ang-2-VEGF-A CrossMab treatment led to a strong inhibition of angiogenesis and an enhanced vessel maturation phenotype. Neoadjuvant combination with chemotherapy resulted in complete tumor regression in primary tumor-bearing Ang-2-VEGF-A CrossMab-treated mice. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiologic vessels. Moreover, treatment with Ang-2-VEGF-A CrossMab resulted in inhibition of hematogenous spread of tumor cells to other organs and reduced micrometastatic growth in the adjuvant setting. CONCLUSION: These data establish Ang-2-VEGF-A CrossMab as a promising antitumor, antiangiogenic, and antimetastatic agent for the treatment of cancer.


Asunto(s)
Angiopoyetina 2/inmunología , Anticuerpos Biespecíficos/administración & dosificación , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/inmunología , Angiopoyetina 2/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Bevacizumab , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/inmunología , Ratones , Metástasis de la Neoplasia , Neoplasias/inmunología , Neovascularización Patológica/inmunología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores
9.
PLoS One ; 8(2): e54923, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23405099

RESUMEN

There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.


Asunto(s)
Angiopoyetina 1/antagonistas & inhibidores , Angiopoyetina 2/antagonistas & inhibidores , Angiopoyetina 2/inmunología , Anticuerpos Neutralizantes/farmacología , Antineoplásicos/farmacología , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/tratamiento farmacológico , Angiopoyetina 1/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Antineoplásicos/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Córnea/efectos de los fármacos , Córnea/inmunología , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Microvasos/efectos de los fármacos , Microvasos/inmunología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/inmunología , Fosforilación , Distribución Aleatoria , Receptor TIE-2/antagonistas & inhibidores , Receptor TIE-2/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Protein Eng Des Sel ; 25(10): 571-80, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22976197

RESUMEN

We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.


Asunto(s)
Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/metabolismo , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismo , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Sitios de Unión de Anticuerpos , Línea Celular , Disulfuros/química , Expresión Génica , Humanos , Metaloproteinasa 2 de la Matriz/inmunología , Metaloproteinasa 9 de la Matriz/inmunología , Péptido Hidrolasas/metabolismo , Ingeniería de Proteínas , Proteolisis , Receptor ErbB-3/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/inmunología
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