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Introduction: Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities. Methods: We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15. This "PD1/IL15" selectively delivers IL-15 signaling to lymphocytes expressing PD-1. We then investigated the pharmacokinetic (PK) and pharmacodynamic (PD) effects of PD1/IL15 TaCk on immune cell subsets in cynomolgus monkeys after single and repeat intravenous dose administrations. We used these results to determine the first-in-human (FIH) dose and dosing frequency for early clinical trials. Results: The PD1/IL15 TaCk exhibited a nonlinear multiphasic PK profile, while the untargeted isotype control TaCk, containing an anti-respiratory syncytial virus antibody (RSV/IL15), showed linear and dose proportional PK. The PD1/IL15 TaCk also displayed a considerably prolonged PK (half-life range â¼1.0-4.1 days) compared to wild-type IL-15 (half-life â¼1.1 h), which led to an enhanced cell expansion PD response. The PD was dose-dependent, durable, and selective for PD-1+ lymphocytes. Notably, the dose- and time-dependent PK was attributed to dynamic TMDD resulting from test article-induced lymphocyte expansion upon repeat administration. The recommended first-in-human (FIH) dose of PD1/IL15 TaCk is 0.003 mg/kg, determined based on a minimum anticipated biological effect level (MABEL) approach utilizing a combination of in vitro and preclinical in vivo data. Conclusion: This work provides insight into the complex PK/PD relationship of PD1/IL15 TaCk in monkeys and informs the recommended starting dose and dosing frequency selection to support clinical evaluation of this novel targeted cytokine.
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Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. Obexelimab is a non-depleting anti-human CD19 mAb with an Fc region engineered to have high affinity for human FcγRIIb, thereby co-engaging BCR and FcγRIIb. To assess its ability to suppress B cell activation in vivo, we generated non-autoimmune-prone C57BL/6 (B6) and SLE-prone NZM 2328 (NZM) mice in which the human FcγRIIb extracellular domain was knocked into the mouse Fcgr2b locus (B6.hRIIb and NZM.hRIIb mice, respectively, the latter retaining features of SLE). XENP8206, a mAb which bears the same FcγRIIb-enhanced human Fc domain as does obexelimab but which recognizes murine CD19 rather than human CD19, inhibited in vitro BCR-triggered activation of B cells from both B6.hRIIb and NZM.hRIIb mice. Following administration of XENP8206 to B6.hRIIb or NZM.hRIIb mice, B cell numbers in the spleen and lymph nodes remained stable but became hyporesponsive to BCR-triggered activation for at least 14 days. These findings demonstrate proof-of-principle that pharmacologic co-engagement of BCR and human FcγRIIb inhibits B cell activation in non-autoimmune and SLE-prone hosts while preserving B cell numbers. These observations lay a strong foundation for clinical trials in human SLE with agents that co-engage BCR and FcγRIIb. Moreover, B6.hRIIb and NZM.hRIIb should serve as powerful in vivo models in the elucidation of the cellular and molecular underpinnings of the changes induced by BCR/FcγRIIb co-engagement.
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OBJECTIVE: Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. The aim of this study was to characterize B cell immunosuppression mediated by the Fc-engineered antibody, XmAb5871, which coengages FcγRIIb with the B cell antigen receptor (BCR) complex and that is currently in clinical development for the treatment of rheumatoid arthritis (RA). Because rheumatoid factor (RF) might interfere with the binding of XmAb5871 to FcγRIIb, we correlated RF titers with the potency of XmAb5871. METHODS: We analyzed the expression of CD19, FcγRIIb, and CD86 on naive and memory B cells from 50 patients with RA and 66 healthy donors, quantified XmAb5871-induced promotion of FcγRIIb phosphorylation and suppression of calcium flux in activated B cells, measured CD86 inhibition in whole blood, and correlated RF and anti-citrullinated protein antibody (ACPA) levels with drug potency. We engrafted RA peripheral blood mononuclear cells (PBMCs) into SCID mice, treated them with XmAb5871, and quantified human total IgG, total IgM, and anti-tetanus IgG antibody levels in vivo. RESULTS: B cells from all donors expressed CD19 and FcγRIIb, and the expression of FcγRIIb was higher on naive, but not memory, B cells from donors with RA compared with healthy donors. BCR-mediated calcium flux was suppressed by XmAb5871 and was associated with FcγRIIb phosphorylation. XmAb5871 inhibited CD86 induction, and the levels of RF and ACPAs did not affect efficacy. XmAb5871 suppressed B cell activation regardless of disease severity. In SCID mice engrafted with PBMCs from a patient with RA, XmAb5871 suppressed humoral responses. CONCLUSION: Coengagement of the BCR complex and FcγRIIb by XmAb5871 inhibits B cell activation and function. The similar potency in patients with RA and healthy donors and the absence of autoantibody interference suggest that XmAb5871 may represent a new therapeutic strategy to suppress autoreactive B cells in RA.
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Anticuerpos Antiidiotipos/farmacología , Antígenos CD19/inmunología , Artritis Reumatoide/patología , Linfocitos B/efectos de los fármacos , Receptores de Antígenos de Linfocitos B/efectos de los fármacos , Receptores de IgG/efectos de los fármacos , Animales , Anticuerpos Antiidiotipos/metabolismo , Antígenos CD19/metabolismo , Artritis Reumatoide/metabolismo , Linfocitos B/metabolismo , Linfocitos B/patología , Antígeno B7-2/metabolismo , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Femenino , Xenoinjertos , Humanos , Leucocitos Mononucleares/patología , Ratones , Ratones SCID , Péptidos Cíclicos/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/metabolismoRESUMEN
The mechanisms through which Caspase-2 leads to cell death are controversial. Here we show, using a combination of cell-free and cell culture-based approaches, that cleavage of the Bcl-2-family protein Bid is required for the induction of apoptosis by Caspase-2. Caspase-2 promoted cytochrome c release from mitochondria in the presence of cytosol from wild-type, but not Bid-deficient, mouse embryonic fibroblasts (MEFs). Recombinant wild-type Bid, but not a noncleavable mutant (D59E), restored cytochrome c release. Similarly, Bid-null MEFs were relatively resistant to apoptosis triggered by active Caspase-2, and apoptosis was restored in Bid-null cells by the expression of wild-type, but not D59E, Bid. Finally, Bid-null MEFs were substantially more resistant to apoptosis induced by heat shock, which has been shown to be dependent on apical activation of Caspase-2. The data are consistent with a model in which Caspase-2 induces apoptosis via cleavage of Bid at D59 and the subsequent engagement of the mitochondrial (intrinsic) pathway.
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Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasas/metabolismo , Calor , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Caspasa 2 , Células Cultivadas , Citocromos c/metabolismo , Citosol/metabolismo , Embrión de Mamíferos/citología , Activación Enzimática , Fibroblastos/metabolismo , Ratones , Mitocondrias/metabolismo , MutaciónRESUMEN
During apoptosis, engagement of the mitochondrial pathway involves a decisive event characterized by the release of mitochondrial intermembrane space proteins, such as cytochrome c. This permeabilization of the mitochondrial outer membrane depends on activation and oligomerization of multidomain Bcl-2-family proteins Bax or Bak. Although specific members of the Bcl-2 family can activate these proapoptotic proteins, we found that heat directly activated Bax or Bak to induce cytochrome c release. A preparation of mitochondria heated at 43 degrees C released cytochrome c in association with Bak oligomerization, and Bcl-xL prevented these events. Similarly, heat induced the oligomerization of recombinant Bax, conferring an ability to permeabilize mitochondria. Compared with wild-type cells, bax(-/-)bak(-/-) mouse embryonic fibroblasts and mitochondria isolated from these cells were resistant to heat-induced cytochrome c release. Cytosol from untreated cells inhibited heat-activated Bax or Bak; however, depletion of cytosolic Bcl-xL ablated this protection. Although mitochondria heated in the presence of cytosol did not release cytochrome c, they displayed a dramatic increase in sensitivity to permeabilization by the BH3-only protein Bid. Additionally, a peptide corresponding to the BH3 domain of Puma counteracted the inhibitory effect of cytosol and permitted heat-activated Bak to permeabilize the mitochondria. Therefore, heat represents a condition under which multidomain proapoptotic proteins are activated, and this activation is regulated by both antiapoptotic and BH3-only members of the Bcl-2 family. Our results support an emerging paradigm, wherein the activation of Bax or Bak and the blockade of antiapoptotic Bcl-2 proteins are pivotal steps in the mitochondrial pathway of apoptosis.
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Apoptosis/fisiología , Calor , Mitocondrias Hepáticas/fisiología , Membranas Mitocondriales/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Células Cultivadas , Citocromos c/metabolismo , Citosol/metabolismo , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/metabolismo , Permeabilidad , Proteína bcl-X/metabolismoRESUMEN
Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.
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Apoptosis , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteína 11 Similar a Bcl2 , Proteínas Portadoras/metabolismo , Citocromos c/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Permeabilidad , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/química , Homología de Secuencia de Aminoácido , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mm KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes.