RESUMEN
CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.
Asunto(s)
Ligando CD27/química , Complejos Multiproteicos/química , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/química , Ligando CD27/genética , Ligando CD27/inmunología , Cristalografía por Rayos X , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/inmunología , Estructura Cuaternaria de Proteína , Linfocitos T/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
T-cell engaging biologics is a class of novel and promising immune-oncology compounds that leverage the immune system to eradicate cancer. Here, we compared and contrasted a bispecific diabody-Fc format, which displays a relatively short antigen-binding arm distance, with our bispecific IgG platform. By generating diverse panels of antigen-expressing cells where B cell maturation antigen is either tethered to the cell membrane or located to the juxtamembrane region and masked by elongated structural spacer units, we presented a systematic approach to investigate the role of antigen epitope location and molecular formats in immunological synapse formation and cytotoxicity. We demonstrated that diabody-Fc is more potent for antigen epitopes located in the membrane distal region, while bispecific IgG is more efficient for membrane-proximal epitopes. Additionally, we explored other parameters, including receptor density, antigen-binding affinity, and kinetics. Our results show that molecular format and antigen epitope location, which jointly determine the intermembrane distance between target cells and T cells, allow decoupling of cytotoxicity and cytokine release, while antigen-binding affinities appear to be positively correlated with both readouts. Our work offers new insight that could potentially lead to a wider therapeutic window for T-cell engaging biologics in general.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígeno de Maduración de Linfocitos B/metabolismo , Productos Biológicos/farmacología , Epítopos , Inmunoglobulina G/farmacología , Ingeniería de Proteínas , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígeno de Maduración de Linfocitos B/inmunología , Sitios de Unión de Anticuerpos , Productos Biológicos/inmunología , Productos Biológicos/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Línea Celular Tumoral , Citocinas/metabolismo , Mapeo Epitopo , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/inmunología , Sinapsis Inmunológicas/metabolismo , Cinética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tirosina Quinasa 3 Similar a fms/inmunología , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Antibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.
Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoterapia Adoptiva/métodos , Metotrexato/administración & dosificación , Neoplasias/terapia , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/efectos de los fármacos , Animales , Línea Celular Tumoral , Terapia Combinada/métodos , Femenino , Células HEK293 , Humanos , Inmunoterapia Adoptiva/efectos adversos , Ratones , Neoplasias/inmunología , Cultivo Primario de Células , Receptores Quiméricos de Antígenos/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/efectos de los fármacos , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The restricted expression pattern of B-cell maturation antigen (BCMA) makes it an ideal tumor-associated antigen (TAA) for the treatment of myeloma. BCMA has been targeted by both CD3 bispecific antibody and antibody-drug conjugate (ADC) modalities, but a true comparison of modalities has yet to be performed. Here we utilized a single BCMA antibody to develop and characterize both a CD3 bispecific and 2 ADC formats (cleavable and noncleavable) and compared activity both in vitro and in vivo with the aim of generating an optimal therapeutic. Antibody affinity, but not epitope was influential in drug activity and hence a high-affinity BCMA antibody was selected. Both the bispecific and ADCs were potent in vitro and in vivo, causing dose-dependent cell killing of myeloma cell lines and tumor regression in orthotopic myeloma xenograft models. Primary patient cells were effectively lysed by both CD3 bispecific and ADCs, with the bispecific demonstrating improved potency, maximal cell killing, and consistency across patients. Safety was evaluated in cynomolgus monkey toxicity studies and both modalities were active based on on-target elimination of B lineage cells. Distinct nonclinical toxicity profiles were seen for the bispecific and ADC modalities. When taken together, results from this comparison of BCMA CD3 bispecific and ADC modalities suggest better efficacy and an improved toxicity profile might be achieved with the bispecific modality. This led to the advancement of a bispecific candidate into phase I clinical trials.
Asunto(s)
Anticuerpos Biespecíficos/administración & dosificación , Antígeno de Maduración de Linfocitos B/metabolismo , Complejo CD3/inmunología , Inmunoconjugados/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , Animales , Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/farmacología , Afinidad de Anticuerpos , Antígeno de Maduración de Linfocitos B/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoconjugados/efectos adversos , Inmunoconjugados/farmacología , Ratones , Mieloma Múltiple/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Determine the differential effect of a FcγR-binding, mIgG2a anti-GITR antibody in mouse tumor models, and characterize the tumor microenvironment for the frequency of GITR expression in T-cell subsets from seven different human solid tumors.Experimental Design: For mouse experiments, wild-type C57BL/6 mice were subcutaneously injected with MC38 cells or B16 cells, and BALB/c mice were injected with CT26 cells. Mice were treated with the anti-mouse GITR agonist antibody 21B6, and tumor burden and survival were monitored. GITR expression was evaluated at the single-cell level using flow cytometry (FC). A total of 213 samples were evaluated for GITR expression by IHC, 63 by FC, and 170 by both in seven human solid tumors: advanced hepatocellular carcinoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, pancreatic carcinoma, head and neck carcinoma, melanoma, and ovarian carcinoma. RESULTS: The therapeutic benefit of 21B6 was greatest in CT26 followed by MC38, and was least in the B16 tumor model. The frequency of CD8 T cells and effector CD4 T cells within the immune infiltrate correlated with response to treatment with GITR antibody. Analysis of clinical tumor samples showed that NSCLC, renal cell carcinoma, and melanoma had the highest proportions of GITR-expressing cells and highest per-cell density of GITR expression on CD4+ Foxp3+ T regulatory cells. IHC and FC data showed similar trends with a good correlation between both techniques. CONCLUSIONS: Human tumor data suggest that NSCLC, renal cell carcinoma, and melanoma should be the tumor subtypes prioritized for anti-GITR therapy development.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Proteína Relacionada con TNFR Inducida por Glucocorticoide/genética , Melanoma Experimental/genética , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Proteína Relacionada con TNFR Inducida por Glucocorticoide/antagonistas & inhibidores , Humanos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Receptores de IgG/inmunología , Subgrupos de Linfocitos T/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.
Asunto(s)
Antígeno de Maduración de Linfocitos B/inmunología , Trasplante de Células/métodos , Inmunoterapia Adoptiva/métodos , Mieloma Múltiple/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno de Maduración de Linfocitos B/genética , Donantes de Sangre , Línea Celular Tumoral , Trasplante de Células/efectos adversos , Citotoxicidad Inmunológica/genética , Edición Génica , Vectores Genéticos , Enfermedad Injerto contra Huésped/terapia , Humanos , Inmunoterapia Adoptiva/efectos adversos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/patología , Supervivencia sin Progresión , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Rituximab/uso terapéutico , Linfocitos T/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Transducción Genética , Trasplante Homólogo/métodosRESUMEN
Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.