RESUMEN
IL2 signals pleiotropically on diverse cell types, some of which contribute to therapeutic activity against tumors, whereas others drive undesired activity, such as immunosuppression or toxicity. We explored the theory that targeting of IL2 to CD8+ T cells, which are key antitumor effectors, could enhance its therapeutic index. To this aim, we developed AB248, a CD8 cis-targeted IL2 that demonstrates over 500-fold preference for CD8+ T cells over natural killer and regulatory T cells (Tregs), which may contribute to toxicity and immunosuppression, respectively. AB248 recapitulated IL2's effects on CD8+ T cells in vitro and induced selective expansion of CD8+T cells in primates. In mice, an AB248 surrogate demonstrated superior antitumor activity and enhanced tolerability as compared with an untargeted IL2Rßγ agonist. Efficacy was associated with the expansion and phenotypic enhancement of tumor-infiltrating CD8+ T cells, including the emergence of a "better effector" population. These data support the potential utility of AB248 in clinical settings. Significance: The full potential of IL2 therapy remains to be unlocked. We demonstrate that toxicity can be decoupled from antitumor activity in preclinical models by limiting IL2 signaling to CD8+ T cells, supporting the development of CD8+ T cell-selective IL2 for the treatment of cancer. See related article by Kaptein et al. p. 1226.
Asunto(s)
Linfocitos T CD8-positivos , Interleucina-2 , Animales , Linfocitos T CD8-positivos/inmunología , Interleucina-2/farmacología , Ratones , Humanos , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Neoplasias/inmunología , Neoplasias/tratamiento farmacológicoRESUMEN
SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC50) values as low as 9 × 10-14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/química , SARS-CoV-2/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Apoferritinas/química , Disponibilidad Biológica , Mapeo Epitopo , Humanos , Inmunoglobulina G/inmunología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ingeniería de Proteínas/métodos , Subunidades de Proteína/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Distribución TisularRESUMEN
FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines in vitro and in vivo. 7370 was also capable of activating T cells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3+ dendritic cells (DCs) and bone marrow FLT3+ stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/farmacología , Complejo CD3/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/uso terapéutico , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Complejo CD3/química , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Inmunoglobulina G/farmacología , Inmunofenotipificación , Leucemia Mieloide Aguda , Depleción Linfocítica , Macaca fascicularis , Ratones , Modelos Moleculares , Dominios Proteicos/efectos de los fármacos , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/químicaRESUMEN
Agonistic antibodies targeting the tumor necrosis factor (TNF) superfamily of co-stimulatory receptors (TNFRSF) are progressing through various stages of clinical development for cancer treatment, but the desired and defining features of these agents for optimal biological activity remain controversial. One idea, based on recent studies with CD40, is that non-ligand-blocking antibodies targeting membrane-distal cysteine-rich domain 1 (CRD1) have superior agonistic activities compared with ligand-blocking antibodies targeting more membrane-proximal CRDs. Here, we determined the binding and functional characteristics of a panel of antibodies targeting CRDs 1-4 of OX40 (also known as TNFRSF4 or CD134). In striking contrast to CD40, we found that ligand-blocking CRD2-binding and membrane-proximal CRD4-binding anti-OX40 antibodies have the strongest agonistic and anti-tumor activities. These findings have important translational implications and further highlight that the relationship between epitope specificity and agonistic activity will be an important issue to resolve on a case-by-case basis when optimizing antibodies targeting different co-stimulatory tumor necrosis factor receptors (TNFRs).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Inmunoterapia/métodos , Neoplasias Experimentales/terapia , Ligando OX40/inmunología , Receptores OX40/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Epítopos/química , Epítopos/inmunología , Humanos , Células Jurkat , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ligando OX40/química , Ratas , Ratas Endogámicas Lew , Receptores OX40/químicaRESUMEN
4-1BB (CD137, TNFRSF9) is an inducible costimulatory receptor expressed on activated T cells. Clinical trials of two agonist antibodies, utomilumab (PF-05082566) and urelumab (BMS-663513), are ongoing in multiple cancer indications, and both antibodies demonstrate distinct activities in the clinic. To understand these differences, we solved structures of the human 4-1BB/4-1BBL complex, the 4-1BBL trimer alone, and 4-1BB bound to utomilumab or urelumab. The 4-1BB/4-1BBL complex displays a unique interaction between receptor and ligand when compared with other TNF family members. Furthermore, our ligand-only structure differs from previously published data. Utomilumab, a ligand-blocking antibody, binds 4-1BB between CRDs 3 and 4. In contrast, urelumab binds 4-1BB CRD-1, away from the ligand binding site. Finally, cell-based assays demonstrate utomilumab is a milder agonist than urelumab. Collectively, our data provide a deeper understanding of the 4-1BB signaling complex, providing a template for future development of next generation 4-1BB targeted biologics.
Asunto(s)
Ligando 4-1BB/química , Ligando 4-1BB/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/química , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Anticuerpos Monoclonales Humanizados , Sitios de Unión , Células HEK293 , Humanos , Células Jurkat , Modelos Moleculares , Dominios ProteicosRESUMEN
Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Cisteína/química , Inmunoconjugados/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Trastuzumab/farmacología , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteína-1 Reguladora de Fusión/inmunología , Humanos , Inmunoconjugados/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratas , Ratas Sprague-Dawley , Receptor ErbB-2/antagonistas & inhibidores , Trastuzumab/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Soft tissue sarcoma (STS) is a heterogenous tumor arising from the embryonic mesoderm represented by approximately 50 histological subtypes. Effective therapeutic intervention is lacking for recurrent, late stage and metastatic disease. CD39, a cell-surface ectonucleotidase, has previously been shown to be upregulated in hematological malignancies and various epithelial tumors, but not in STS. Here, we show by mass spectrometry and immunohistochemistry that CD39 is highly expressed in primary patient sarcoma samples. Moreover, CD39 nucleotidase activity is enhanced in fibrosarcoma compared with normal control cells. We demonstrate that an inhibitory monoclonal anti-CD39 antibody, abrogates CD39 enzymatic activity significantly and prolongs survival in a lethal metastatic patient-derived sarcoma model. Taken together, the data suggest CD39 is a novel therapeutic target for the treatment of STS.
RESUMEN
Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Citometría de Flujo , Colorantes Fluorescentes/química , Maleimidas/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Antígenos de Superficie/inmunología , Línea Celular Tumoral , Humanos , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Receptor EphA2/inmunología , Espectrometría de FluorescenciaRESUMEN
A simulation of tensile strength of various alginate-based hollow microfibers using FEA analysis has been conducted with the hypothesis of macroscopic isotropy and linear elastic-plastic behavior. Results of student t-tests indicated that there was no significant difference between the experimental and simulated tensile strengths (p = 0.37, α = 0.05), while there was a significant reduction in elasticity as a result of chitosan coating (p = 0.024, α = 0.05). The hypothesis of macroscopic isotropy was verified by highly correlated (R(2) ≥ 0.92) theoretical and experimental elongation at break measurements, findings that could be extended to the failure analysis of alginate microfibers used in regenerative medicine.
Asunto(s)
Alginatos/química , Materiales Biocompatibles/química , Quitosano/química , Fibras Minerales/análisis , Animales , Elasticidad , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Ensayo de Materiales , Medicina Regenerativa/instrumentación , Resistencia a la TracciónRESUMEN
The diversity of a panel of antibodies that target a specific antigen can be established in various assay formats. In conventional epitope binning assays purified antibodies are tested in a pairwise manner: two antibodies that compete with each other for binding to an antigen are grouped into the same cluster or bin, while they are assigned to two different clusters when they do not compete. Here we present a high through put assay that enables grouping of crude hybridoma supernatants without a need for antibody purification. In addition, the assay does not require recombinant protein, because it is conducted on cells that express the antigen of interest. Hence, one can use the antibody-clustering assay for cell surface proteins that are not amenable to purification. Heavy chain variable region (VH) sequencing shows that VH composition within clusters is conserved. Finally, the assay is in good agreement with a conventional epitope binning assay with purified antigen.