RESUMEN
PF-07209960 is a novel bispecific fusion protein composed of an anti-PD-1 antibody and engineered IL-15 cytokine mutein with reduced binding affinity to its receptors. The pharmacokinetics (PK), pharmacodynamics (PD), and toxicity of PF-07209960 were evaluated following once every other week subcutaneous (SC) or intravenous (IV) administration to cynomolgus monkeys in a repeat-dose PKPD (0.01-0.3 mg/kg/dose) and GLP toxicity study (0.1-3 mg/kg/dose). PF-07209960 showed dose dependent pharmacokinetics with a terminal T1/2 of 8 and 13 hours following IV administration at 0.03 and 0.1 mg/kg, respectively. The clearance is faster than a typical IgG1 antibody. Slightly faster clearance was also observed following the second dose, likely due to increased target pool and formation of anti-drug antibodies (ADA). Despite a high incidence rate of ADA (92%) observed in GLP toxicity study, PD-1 receptor occupancy, IL-15 signaling (STAT5 phosphorylation) and T cell expansion were comparable following the first and second doses. Activation and proliferation of T cells were observed with largest increase in cell numbers found in gamma delta T cells, followed by CD4+ and CD8+ T cells, and then NK cells. Release of cytokines IL-6, IFNγ, and IL-10 were detected, which peaked at 72 hours postdose. There was PF-07209960-related mortality at ≥1 mg/kg. At scheduled necropsy, microscopic findings were generalized mononuclear infiltration in various tissues. Both the no observed adverse effect level (NOAEL) and the highest non severely toxic dose (HNSTD) were determined to be 0.3 mg/kg/dose, which corresponded to mean Cmax and AUC48 values of 1.15 µg/mL and 37.9 µg*h/mL, respectively.
Asunto(s)
Anticuerpos Monoclonales , Receptor de Muerte Celular Programada 1 , Animales , Macaca fascicularis , Interleucina-15 , Administración Intravenosa , Citocinas , Inhibidores de Puntos de Control InmunológicoRESUMEN
Although cytokine support can enhance CAR T-cell function, coadministering cytokines or engineering CAR T cells to secrete cytokines can result in toxicities. To mitigate these safety risks, we engineered iTurboCAR T cells that coexpress a novel inducible Turbo (iTurbo) cytokine signaling domain. iTurbo domains consist of modular components that are customizable to a variety of activating inputs, as well as cytokine signaling outputs multiplexable for combinatorial signaling outcomes. Unlike most canonical cytokine receptors that are heterodimeric, iTurbo domains leverage a compact, homodimeric design that minimizes viral vector cargo. Using an iTurbo domain activated by the clinically validated dimerizer, AP1903, homodimeric iTurbo domains instigated signaling that mimicked the endogenous heterodimeric cytokine receptor. Different iTurbo domains programmed iTurboCAR T cells toward divergent phenotypes and resulted in improved antitumor efficacy. iTurbo domains, therefore, offer the flexibility for user-programmable signaling outputs, permitting control over cellular phenotype and function while minimizing viral cargo footprint.
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Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Citocinas , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal , Linfocitos TRESUMEN
INTRODUCTION: Following the approval of the T cell engaging bispecific antibody blinatumomab, immune cell retargeting with bispecific or multispecific antibodies has emerged as a promising cancer immunotherapy strategy, offering alternative mechanisms compared to immune checkpoint blockade. As we gain more understanding of the complex tumor microenvironment, rules and design principles have started to take shape on how to best harness the immune system to achieve optimal anti-tumor activities. AREAS COVERED: In the present review, we aim to summarize the most recent advances and challenges in using bispecific antibodies for immune cell retargeting and to provide insights into various aspects of antibody engineering. Discussed herein are studies that highlight the importance of considering antibody engineering parameters, such as binding epitope, affinity, valency, and geometry to maximize the potency and mitigate the toxicity of T cell engagers. Beyond T cell engaging bispecifics, other bispecifics designed to recruit the innate immune system are also covered. EXPERT OPINION: Diverse and innovative molecular designs of bispecific/multispecific antibodies have the potential to enhance the efficacy and safety of immune cell retargeting for the treatment of cancer. Whether or not clinical data support these different hypotheses, especially in solid tumor settings, remains to be seen.
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Anticuerpos Biespecíficos , Neoplasias , Anticuerpos Biespecíficos/metabolismo , Anticuerpos Biespecíficos/uso terapéutico , Humanos , Inmunoterapia , Linfocitos T , Microambiente TumoralRESUMEN
CD70 is highly expressed in renal cell carcinoma (RCC), with limited expression in normal tissue, making it an attractive CAR T target for an immunogenic solid tumor indication. Here we generated and characterized a panel of anti-CD70 single-chain fragment variable (scFv)-based CAR T cells. Despite the expression of CD70 on T cells, production of CAR T cells from a subset of scFvs with potent in vitro activity was achieved. Expression of CD70 CARs masked CD70 detection in cis and provided protection from CD70 CAR T cell-mediated fratricide. Two distinct classes of CAR T cells were identified with differing memory phenotype, activation status, and cytotoxic activity. Epitope mapping revealed that the two classes of CARs bind unique regions of CD70. CD70 CAR T cells displayed robust antitumor activity against RCC cell lines and patient-derived xenograft mouse models. Tissue cross-reactivity studies identified membrane staining in lymphocytes, thus matching the known expression pattern of CD70. In a cynomolgus monkey CD3-CD70 bispecific toxicity study, expected findings related to T-cell activation and elimination of CD70-expressing cells were observed, including cytokine release and loss of cellularity in lymphoid tissues. Finally, highly functional CD70 allogeneic CAR T cells were produced at large scale through elimination of the T-cell receptor by TALEN-based gene editing. Taken together, these efficacy and safety data support the evaluation of CD70 CAR T cells for the treatment of RCC and has led to the advancement of an allogeneic CD70 CAR T-cell candidate into phase I clinical trials. SIGNIFICANCE: These findings demonstrate the efficacy and safety of fratricide-resistant, allogeneic anti-CD70 CAR T cells targeting renal cell carcinoma and the impact of CAR epitope on functional activity. See related commentary by Adotévi and Galaine, p. 2517.
Asunto(s)
Carcinoma de Células Renales , Trasplante de Células Madre Hematopoyéticas , Neoplasias Renales , Animales , Ligando CD27 , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva , Neoplasias Renales/patología , Macaca fascicularis , Ratones , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: T cell checkpoint immunotherapies have shown promising results in the clinic, but most patients remain non-responsive. CD47-signal regulatory protein alpha (SIRPα) myeloid checkpoint blockade has shown early clinical activity in hematologic malignancies. However, CD47 expression on peripheral blood limits αCD47 antibody selectivity and thus efficacy in solid tumors. METHODS: To improve the antibody selectivity and therapeutic window, we developed a novel affinity-tuned bispecific antibody targeting CD47 and programmed death-ligand 1 (PD-L1) to antagonize both innate and adaptive immune checkpoint pathways. This PD-L1-targeted CD47 bispecific antibody was designed with potent affinity for PD-L1 and moderate affinity for CD47 to achieve preferential binding on tumor and myeloid cells expressing PD-L1 in the tumor microenvironment (TME). RESULTS: The antibody design reduced binding on red blood cells and enhanced selectivity to the TME, improving the therapeutic window compared with αCD47 and its combination with αPD-L1 in syngeneic tumor models. Mechanistically, both myeloid and T cells were activated and contributed to antitumor activity of αCD47/PD-L1 bispecific antibody. Distinct from αCD47 and αPD-L1 monotherapies or combination therapies, single-cell RNA sequencing (scRNA-seq) and gene expression analysis revealed that the bispecific treatment resulted in unique innate activation, including pattern recognition receptor-mediated induction of type I interferon pathways and antigen presentation in dendritic cells and macrophage populations. Furthermore, treatment increased the Tcf7+ stem-like progenitor CD8 T cell population in the TME and promoted its differentiation to an effector-like state. Consistent with mouse data, the compounds were well tolerated and demonstrated robust myeloid and T cell activation in non-human primates (NHPs). Notably, RNA-seq analysis in NHPs provided evidence that the innate activation was mainly contributed by CD47-SIRPα but not PD-L1-PD-1 blockade from the bispecific antibody. CONCLUSION: These findings provide novel mechanistic insights into how myeloid and T cells can be uniquely modulated by the dual innate and adaptive checkpoint antibody and demonstrate its potential in clinical development (NCT04881045) to improve patient outcomes over current PD-(L)1 and CD47-targeted therapies.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antígeno CD47/metabolismo , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Biespecíficos/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunidad Innata , Inmunoterapia/métodos , Macaca fascicularis , Ratones , Microambiente TumoralRESUMEN
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
The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Neoplasias del Colon/terapia , Inmunoterapia , Interleucina-15/uso terapéutico , Melanoma Experimental/terapia , Animales , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Modelos Animales de Enfermedad , Humanos , Interleucina-15/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
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
BACKGROUND: CD40 is a compelling target for cancer immunotherapy, however, attempts to successfully target this pathway have consistently been hampered by dose-limiting toxicity issues in the clinic that prevents the administration of efficacious doses. METHODS: Here, using cytokine and cytokine receptor depletion strategies in conjunction with a potent CD40 agonist, we investigated mechanisms underlying the two primary sources of CD40 agonist-associated toxicity, hepatotoxicity and cytokine release syndrome (CRS). RESULTS: We demonstrate that CD40 agonist -induced hepatotoxicity and CRS are mechanistically independent. Historical data have supported a role for interleukin-6 (IL-6) in CRS-associated wasting, however, our findings instead show that an inflammatory cytokine network involving TNF, IL-12p40, and IFNγ underlie this process. Deficiency of TNF or IFNγ did not influence CD40-induced hepatitis however loss of IL-12p40 significantly decreased circulating concentrations of liver enzymes and reduced the frequency of activated CD14+MHCII+ myeloid cells in the liver, indicating a role for IL-12p40 in liver pathology. CONCLUSIONS: As clinical research programs aim to circumnavigate toxicity concerns while maintaining antitumor efficacy it will be essential to understand which features of CD40 biology mediate antitumor function to develop both safe and efficacious agonists.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Inmunoterapia/métodos , Subunidad p40 de la Interleucina-12/efectos adversos , Animales , Femenino , Humanos , RatonesRESUMEN
Patients with relapsed or refractory acute myeloid leukemia (AML) have a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell leukemias and lymphomas and could prove highly efficacious in AML. However, a significant number of patients with AML may not receive treatment with an autologous product due to manufacturing failures associated with low lymphocyte counts or rapid disease progression while the therapeutic is being produced. We report the preclinical evaluation of an off-the-shelf CAR T cell therapy targeting Fms-related tyrosine kinase 3 (FLT3) for the treatment of AML. Single-chain variable fragments (scFvs) targeting various epitopes in the extracellular region of FLT3 were inserted into CAR constructs and tested for their ability to redirect T cell specificity and effector function to FLT3+ AML cells. A lead CAR, exhibiting minimal tonic signaling and robust activity in vitro and in vivo, was selected and then modified to incorporate a rituximab-responsive off-switch in cis. We found that allogeneic FLT3 CAR T cells, generated from healthy-donor T cells, eliminate primary AML blasts but are also active against mouse and human hematopoietic stem and progenitor cells, indicating risk of myelotoxicity. By employing a surrogate CAR with affinity to murine FLT3, we show that rituximab-mediated depletion of FLT3 CAR T cells after AML eradication enables bone marrow recovery without compromising leukemia remission. These results support clinical investigation of allogeneic FLT3 CAR T cells in AML and other FLT3+ hematologic malignancies.
Asunto(s)
Inmunoterapia Adoptiva , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Tirosina Quinasa 3 Similar a fms/inmunología , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/diagnóstico , Ratones , Receptores Quiméricos de Antígenos/genética , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
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
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
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
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
Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Complejo CD3/inmunología , Claudinas/inmunología , Inmunoconjugados/uso terapéutico , Neoplasias Pancreáticas/terapia , Neoplasias Gástricas/terapia , Adenocarcinoma/terapia , Animales , Carcinoma Ductal Pancreático/terapia , Línea Celular Tumoral , Humanos , Inmunoconjugados/sangre , Ratones , Neoplasias Pancreáticas/sangre , Ratas , Neoplasias Gástricas/sangreRESUMEN
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
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
Monoclonal antibodies are the largest class of therapeutic proteins due in part to their ability to bind an antigen with a high degree of affinity and specificity. A precise determination of their epitope is important for gaining insights into their therapeutic mechanism of action and to help differentiate antibodies that bind the same antigen. Here, we describe a method to precisely and efficiently map the epitopes of multiple antibodies in parallel over the course of just several weeks. This approach is based on a combination of rational library design, yeast surface display, and next generation DNA sequencing and provides quantitative insights into the epitope residues most critical for the antibody-antigen interaction. As an example, we will use this method to map the epitopes of several antibodies that neutralize alpha toxin from Staphylococcus aureus.