Phase 1, first-in-human study of TYRP1-TCB (RO7293583), a novel TYRP1-targeting CD3 T-cell engager, in metastatic melanoma: active drug monitoring to assess the impact of immune response on drug exposure

Introduction: Although checkpoint inhibitors (CPIs) have improved outcomes for patients with metastatic melanoma, those progressing on CPIs have limited therapeutic options. To address this unmet need and overcome CPI resistance mechanisms, novel immunotherapies, such as T-cell engaging agents, are being developed. The use of these agents has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs), which is challenging to predict preclinically and can lead to neutralization of the drug and loss of efficacy. Methods: TYRP1-TCB (RO7293583; RG6232) is a T-cell engaging bispecific (TCB) antibody that targets tyrosinase-related protein 1 (TYRP1), which is expressed in many melanomas, thereby directing T cells to kill TYRP1-expressing tumor cells. Preclinical studies show TYRP1-TCB to have potent anti-tumor activity. This first-in-human (FIH) phase 1 dose-escalation study characterized the safety, tolerability, maximum tolerated dose/optimal biological dose, and pharmacokinetics (PK) of TYRP1-TCB in patients with metastatic melanoma (NCT04551352). Results: Twenty participants with cutaneous, uveal, or mucosal TYRP1-positive melanoma received TYRP1-TCB in escalating doses (0.045 to 0.4 mg). All participants experienced ≥1 treatment-related adverse event (TRAE); two participants experienced grade 3 TRAEs. The most common toxicities were grade 1–2 cytokine release syndrome (CRS) and rash. Fractionated dosing mitigated CRS and was associated with lower levels of interleukin-6 and tumor necrosis factor-alpha. Measurement of active drug (dual TYPR1- and CD3-binding) PK rapidly identified loss of active drug exposure in all participants treated with 0.4 mg in a flat dosing schedule for ≥3 cycles. Loss of exposure was associated with development of ADAs towards both the TYRP1 and CD3 domains. A total drug PK assay, measuring free and ADA-bound forms, demonstrated that TYRP1-TCB-ADA immune complexes were present in participant samples, but showed no drug activity in vitro. Discussion: This study provides important insights into how the use of active drug PK assays, coupled with mechanistic follow-up, can inform and enable ongoing benefit/risk assessment for individuals participating in FIH dose-escalation trials. Translational studies that lead to a better understanding of the underlying biology of cognate T- and B-cell interactions, ultimately resulting in ADA development to novel biotherapeutics, are needed

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine

[Background]:Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.[Methodology/Principal Findings]:Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.[Conclusions/Significance]:The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis.This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (SAF2014-59716-R and BIO2014-56930-P), Instituto de Salud Carlos III (RD12/0036/0065 from Red TemaÂtica de Investigación Cooperativa en Cáncer, cofunded by the EU's European Regional Development Fund ± FEDER),European Community's Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS), and Spain-UK International Joint Project grant from The Royal Society-CSIC (2004GB0032). CG was supported by the RamoÂn y Cajal Program from the Spanish Ministerio de Ciencia e Innovación.AÂCM was recipient of Formación de Profesorado Universitario predoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación.Peer reviewe

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine.

Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis

Schematic model of mitochondria involvement in the killing activity of edelfosine against <i>Leishmania</i> parasites and tumor cells.

<p>This is a schematic diagram to portray one currently plausible mechanism of how edelfosine induces cell death in <i>Leishmania</i> parasites and tumor cells through its main mitochondrial localization in both biological systems. Protection of mitochondria by Bcl-X_L ectopic expression restrains cell death. See text for details.</p

Edelfosine resistance of <i>atp7</i>Δ mutant in <i>Saccharomyces cerevisiae</i> yeast.

<p>Growth curves of wild-type (BY4741) (<b>A</b>), <i>ATP7</i> knock-out mutant (<i>atp7</i>Δ) (<b>B</b>) and the mutant strain harboring the corresponding cognate gene (<i>atp7</i>Δ+pRS416-<i>ATP7</i>) (<b>C</b>) in SDC medium containing different concentrations of edelfosine. The cultures were carried out in duplicate and in at least three independent experiments. Data shown are mean values of three independent experiments. SD values were less than 10% of the mean values.</p

Inhibition of apoptosis-like cell death by ectopic expression of Bcl-X_L in <i>L</i>. <i>infantum</i> promastigotes and HeLa tumor cells.

<p>Inhibition of apoptosis-like cell death by ectopic expression of Bcl-X_L in <i>L</i>. <i>infantum</i> promastigotes and HeLa tumor cells.</p

<i>In vitro</i> and <i>in vivo</i> antileishmanial activity of the antitumor ether phospholipid edelfosine.

<p>Apoptosis-like cell death was quantitated by flow cytometry as percentage of hypodiploid cells (sub-G₀/G₁) in different <i>Leishmania</i> spp. promastigotes (<b>A</b>) and human cancer cell lines (myeloid leukemia HL-60, multiple myeloma MM144, and cervical carcinoma HeLa) (<b>B</b>), following 24-h incubation with distinct APLs (10 μM). Untreated control cells were run in parallel. ErPC, erucylphosphocholine. (<b>C</b>) Time-course of 10 μM edelfosine-induced apoptosis-like cell death (% hypodiploid cells) in <i>L</i>. <i>panamensis</i> promastigotes. Untreated control parasites were run in parallel. (<b>D</b>) Induction of apoptosis-like cell death in <i>L</i>. <i>panamensis</i> axenic amastigotes treated with the indicated concentrations of edelfosine for 16 h. (<b>E</b>) GFP-<i>L</i>. <i>panamensis</i> (<i>GFP-Lp</i>)-infected J774 macrophages were incubated for 1 h with 10 μM PTE-ET and analyzed by fluorescence microscopy. Incubation of GFP-<i>Lp</i>-infected J774 macrophages with 10 μM edelfosine for 24 h decreased parasite infection (<b>F</b>) and the number of parasites per macrophage (<b>G</b>), as compared to untreated infected cells (Control). (<b>H</b>) Murine BMM were infected with <i>L</i>. <i>panamensis</i> and subsequently treated with distinct APLs (10 μM) or vehicle (Control). Parasite load was measured after 3-day incubation. (<b>I-K</b>) Hamsters were inoculated in the nose with <i>L</i>. <i>panamensis</i> promastigotes, and then treated orally with edelfosine (20 mg/kg, <i>n</i> = 8) or with water vehicle (Control) for 28 days. Evolution index during treatment (<b>I</b>) and parasite load in the nose at the end of the 4-week treatment (<b>J</b>) were determined. Edelfosine treatment dramatically reduced nose inflammation and damage at the end of treatment (<b>K</b>). Data shown are means ± SD or representative of three independent experiments performed. Asterisks indicate that the differences between control and edelfosine-treated groups are statistically significant. (*) <i>P</i><0.05. (**) <i>P</i><0.01.</p

Edelfosine induces breakage of kinetoplast DNA prior to nuclear DNA breakdown, and accumulates in mitochondria in <i>Leishmania</i> parasites and cancer cells.

<p>(<b>A</b>) <i>L</i>. <i>panamensis</i> promastigotes were untreated (Control) or treated with 10 μM edelfosine (EDLF) for 6 and 9 h, and then analyzed by confocal microscopy for propidium iodide (PI) staining and TUNEL assay. The positions of the nucleus (N) and kinetoplast (K) are indicated by arrows. Merging of PI and TUNEL panels (Merge) shows the DNA-containing organelles with DNA disruption in yellow. The corresponding differential interference contrast (DIC) images were included in the Merge panels to highlight parasite morphology and facilitate kinetoplast identification. (<b>B</b>) <i>L</i>. <i>panamensis</i> promastigotes and (<b>C</b>) HeLa cancer cells were incubated with 10 μM PTE-ET (blue fluorescence) for 1 h, 100 nM MitoTracker (red fluorescence) for 20 min to localize mitochondria, and then analyzed by fluorescence microscopy. Areas of colocalization between mitochondria and PTE-ET in merge panels are purple. The corresponding differential interference contrast (DIC) images are also shown. Images are representative of three independent experiments. Bar, 20 μm.</p

F_OF₁-ATPase recruitment into rafts in the antileishmanial activity of edelfosine and oligomycin inhibitory effect on cytotoxicity.

<p>(<b>A</b>) <i>L</i>. <i>panamensis</i> promastigotes untreated (Control) and treated with 10 μM edelfosine for 9 h were lysed in 1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. Individual fractions were electrophoresed, and location of GM1 was determined. (<b>B</b>) Proteins from lipid rafts of untreated control and edelfosine-treated <i>L</i>. <i>panamensis</i> promastigotes were subjected to two-dimensional gel electrophoresis followed by MALDI-TOF analysis. Mitochondrial F_OF₁-ATP synthase β subunit is indicated by an arrow. (<b>C</b>) Mass spectrum of the tryptic peptides of the F_OF₁-ATP synthase β subunit spot. Mass values (m/z) and putative amino acid position assignments are indicated above peaks. (<i>Inset</i>) Peptide coverage map of <i>Leishmania</i> F_OF₁-ATP synthase β subunit; the peptides used for identification are highlighted in bold characters and underlined. (<b>D</b>) <i>L</i>. <i>panamensis</i> were untreated (Control) or preincubated with 1 μM oligomycin for 1 h and then incubated in the absence or presence of 10 μM edelfosine for 9 h, and ΔΨ_m disruption (Low ΔΨ_m) and DNA breakdown (hypodiploids) were evaluated. Data shown are means ± SD or representative of three independent experiments. (*) <i>P</i><0.05. (**) <i>P</i><0.01.</p