ADCT-602, a Novel PBD Dimer-containing Antibody-Drug Conjugate for Treating CD22-positive Hematologic Malignancies.

Relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) and lymphomas have poor patient outcomes; novel therapies are needed. CD22 is an attractive target for antibody-drug conjugates (ADCs), being highly expressed in R/R B-ALL with rapid internalization kinetics. ADCT-602 is a novel CD22-targeting ADC, consisting of humanized mAb hLL2-C220, site specifically conjugated to the pyrrolobenzodiazepine dimer-based payload tesirine. In preclinical studies, ADCT-602 demonstrated potent, specific cytotoxicity in CD22-positive lymphomas and leukemias. ADCT-602 was specifically bound, internalized, and trafficked to lysosomes in CD22-positive tumor cells; after cytotoxin release, DNA interstrand crosslink formation persisted for 48 hours. In the presence of CD22-positive tumor cells, ADCT-602 caused bystander killing of CD22-negative tumor cells. A single ADCT-602 dose led to potent, dose-dependent, in vivo antitumor activity in subcutaneous and disseminated human lymphoma/leukemia models. Pharmacokinetic analyses (rat and cynomolgus monkey) showed excellent stability and tolerability of ADCT-602. Cynomolgus monkey B cells were efficiently depleted from circulation after one dose. Gene signature association analysis revealed IRAK1 as a potential marker for ADCT-602 resistance. Combining ADCT-602 + pacritinib was beneficial in ADCT-602-resistant cells. Chidamide increased CD22 expression on B-cell tumor surfaces, increasing ADCT-602 activity. These data support clinical testing of ADCT-602 in R/R B-ALL (NCT03698552) and CD22-positive hematologic cancers.

Preclinical Development of ADCT-601, a Novel Pyrrolobenzodiazepine Dimer-based Antibody-drug Conjugate Targeting AXL-expressing Cancers.

AXL, a tyrosine kinase receptor that is overexpressed in many solid and hematologic malignancies, facilitates cancer progression and is associated with poor clinical outcomes. Importantly, drug-induced expression of AXL results in resistance to conventional chemotherapy and targeted therapies. Together with its presence on multiple cell types in the tumor immune microenvironment, these features make it an attractive therapeutic target for AXL-expressing malignancies. ADCT-601 (mipasetamab uzoptirine) is an AXL-targeted antibody-drug conjugate (ADC) comprising a humanized anti-AXL antibody site specifically conjugated using GlycoConnect technology to PL1601, which contains HydraSpace, a Val-Ala cleavable linker and the potent pyrrolobenzodiazepine (PBD) dimer cytotoxin SG3199. This study aimed to validate the ADCT-601 mode of action and evaluate its efficacy in vitro and in vivo, as well as its tolerability and pharmacokinetics. ADCT-601 bound to both soluble and membranous AXL, and was rapidly internalized by AXL-expressing tumor cells, allowing release of PBD dimer, DNA interstrand cross-linking, and subsequent cell killing. In vivo, ADCT-601 had potent and durable antitumor activity in a wide variety of human cancer xenograft mouse models, including patient-derived xenograft models with heterogeneous AXL expression where ADCT-601 antitumor activity was markedly superior to an auristatin-based comparator ADC. Notably, ADCT-601 had antitumor activity in a monomethyl auristatin E-resistant lung-cancer model and synergized with the PARP inhibitor olaparib in a BRCA1-mutated ovarian cancer model. ADCT-601 was well tolerated at doses of up to 6 mg/kg and showed excellent stability in vivo. These preclinical results warrant further evaluation of ADCT-601 in the clinic.

CD25-targeted antibody-drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity.

BACKGROUND: Regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. They play an important role in the establishment and progression of tumors with high Tregs infiltration and present a major obstacle to tumor eradication by immunotherapies. Numerous strategies have been attempted to deplete or block Tregs, although their success has been limited. METHODS: A CD25-targeted, pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) was investigated for its ability to deplete Tregs and induce antitumor immunity. Antitumor activity of CD25-ADC either alone or in combination with an anti-programmed cell death protein 1 (PD-1) antibody was evaluated in CD25-negative syngeneic models that exhibit tumor infiltration of CD25-expressing Tregs, and its pharmacodynamics and pharmacokinetics were assessed. RESULTS: Single low doses of CD25-ADC resulted in potent and durable antitumor activity in established syngeneic solid tumor models and the combination of a suboptimal dose was synergistic with PD-1 blockade. Tumor eradication by the CD25-targeted ADC was CD8+ T cell-dependent and CD25-ADC induced protective immunity. Importantly, while CD25-ADC mediated a significant and sustained intratumoral Tregs depletion, accompanied by a concomitant increase in the number of activated and proliferating tumor-infiltrating CD8+ T effector cells, systemic Tregs depletion was transient, alleviating concerns of potential autoimmune side effects. CONCLUSIONS: This study shows that a PBD dimer-based, CD25-targeted ADC is able to deplete Tregs and eradicate established tumors via antitumor immunity. This represents a novel approach to efficiently deplete Tregs via a very potent DNA damaging toxin known to induce immunogenic cell death. Moreover, this study provides proof of concept for a completely new application of ADCs as immunotherapeutic agents, as the main mode of action relies on the ADC directly targeting immune cells, rather than tumor cells. These strong preclinical data warrant the clinical evaluation of camidanlumab tesirine (ADCT-301), a PBD-based ADC targeting human CD25, either alone or in combination with checkpoint inhibitors in solid tumors with known Tregs infiltration. A phase I trial (NCT03621982) of camidanlumab tesirine in patients with selected advanced solid tumors is ongoing.

Antitumor Activity of MEDI3726 (ADCT-401), a Pyrrolobenzodiazepine Antibody-Drug Conjugate Targeting PSMA, in Preclinical Models of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody-drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient-derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176-86. ©2018 AACR.

Development and automation of a 384-well cell fusion assay to identify inhibitors of CCR5/CD4-mediated HIV virus entry.

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/beta-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of beta-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturization to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen approximately 650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z' consistently above 0.6, which compares favourably with less complex biochemical assays.

Trypanosoma brucei glycogen synthase kinase-3, a target for anti-trypanosomal drug development: a public-private partnership to identify novel leads.

BACKGROUND: Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3ß (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT. METHODOLOGY/PRINCIPAL FINDINGS: A subset of over 16,000 inhibitors of HsGSK-3 ß from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3ß and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed. CONCLUSIONS/SIGNIFICANCE: We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.