GoKawiilClinical trial design, patients and conduct
The Algonquin study is an ongoing, multicentre, open-label, single-arm, two-part study (ClinicalTrials.gov identifier: NCT03715478)) of BPd in patients with relapsed/refractory MM. Part 1 of the trial consisted of a dose-exploration phase22. Part 2 is evaluating the safety, tolerability and clinical activity of the dose and schedule identified in part 1. Patients included in this interim analysis were enrolled in cohorts 2a and 2b, with the last patient being enrolled on 14 September 2022. The clinical data cut-off date for the presented analysis was 28 October 2025.
At screening, patients with relapsed/refractory MM were eligible if they met the following criteria: aged ≥18 years; had an Eastern Cooperative Oncology Group performance status of 0–2; had undergone autologous stem cell transplantation or were considered transplant ineligible; had experienced disease progression after ≥1 previous lines of antimyeloma treatment and must have been lenalidomide refractory and proteosome inhibitor exposed (in separate regimens or in combination); and had adequate BM, renal and cardiac function. Patients with previous pomalidomide or BCMA-target therapy exposure, concurrent corneal epithelial disease (except mild punctate keratopathy), any serious and/or unstable preexisting medical condition, a psychiatric disorder or any other condition (including laboratory abnormalities) that could interfere with the patient’s safety, obtaining informed consent or compliance with the study procedures were excluded. Sex was not considered in the study design or patient selection and was determined on the basis of self-reporting.
The Algonquin study was conducted at nine Canadian sites in accordance with the Declaration of Helsinki, International Council Harmonisation Good Clinical Practices Guidelines, local regulation governing the conduct of clinical studies, and institutional guidelines. All patients provided written, informed consent. The study protocol, amendments and informed consent were approved by the University Health Network Institutional Review Board (REB numbers 16-5260, 18-5911 and 17-5357). Eligibility criteria, trial procedures and end points, and participating sites conducting this trial, are as previously described22. In summary, belamaf was administered using the following dosages: a 30-min intravenous infusion every 4 weeks at a dose of 1.92 mg kg−1 (cohort 1); or every 4, 8 or 12 weeks at a dose of 2.5 mg kg−1 (cohorts 1a, 3a and 3b); or at total doses of 2.5 or 3.4 mg kg−1 once every 4 weeks but split evenly with 50% of the dose administered on days 1 and 8 of every cycle (cohorts 1b and 2, respectively); or as a loading dose of 2.5 mg kg−1 on cycle 1 day 1, followed by a dose reduction to 1.92 mg kg−1 from cycle 2 onwards once every 4 weeks (cohort 1c). Pomalidomide was administered at 4 mg on day 21 of 28 and dexamethasone 40 mg (20 mg if aged >75 years) weekly. Patients remained on treatment until disease progression, unacceptable toxicity or consent withdrawal. Ophthalmology examinations before each dose of belamaf and preservative-free lubricant eye drops were required throughout study treatment. Dose modifications were made independently for each drug according to predefined criteria based on the nature and toxicity grade of the event.
Acquisition of patient samples
For the Algonquin patient cohort, BM and peripheral blood samples were collected from enrolled patients at the time points indicated in Fig. 1a. Patient samples included in this investigation were selected and sequenced based on availability on an on-going basis. BM samples were included for patients with available matched baseline and C2D1 samples, except for patient ALQ-19-019, in whom poor viability prohibited baseline BM sequencing. No patient peripheral blood samples were excluded except in cases when poor sample quality prohibited sequencing or in specific analyses, which is indicated in figure legends, where applicable. For the distinct cohorts of patients used to train PreGame (discovery cohort and validation cohorts 1 and 2, n = 22), BM samples were obtained from patients with MM receiving standard-of-care BM aspirates at the Princess Margaret Cancer Centre or purchased from commercial sources (iSpecimen and Discovery Life Sciences). Samples were obtained from a mixture of untreated patients and patients having received at least one or more previous lines of therapy. BM mononuclear cells were isolated using Ficoll according to the manufacturer’s recommended protocol (Sigma) and cryopreserved in fetal calf serum (FCS) + 10% DMSO. Melanoma and lung cancer samples were obtained from surgical resections of patients receiving standard-of-care therapy at the Princess Margaret Cancer Centre. Tumour samples were digested with GentleMACS (Miltenyi) and single-cell suspensions were cryopreserved. All BM biopsy samples from patients were taken from the pelvic bone. See Supplementary Table 1 for additional details on patients in our sequencing cohorts, where available. All samples were collected with informed patient consent using protocols approved by the appropriate Institutional Review Boards.
Cell lines
The MM cell lines RPMI-8226 and MM1R were purchased from the American Type Culture Collection (ATCC), ALMC1 was purchased from Sigma, and INA6, JJN3, EJM and AMO1 were purchased from DSMZ. All MM cell lines were cultured in complete IMDM, which is IMDM (Gibco) supplemented with 10% FCS, 2 mM l-glutamine, 1% non-essential amino acids, 1% penicillin–streptomycin, 10 µg ml–1 gentamicin and 55 µM β-ME (all purchased from Gibco). Human IL-6 (BioLegend) was added to the culture media for ALMC1 (1 ng ml–1) and INA6 (10 ng ml–1) cells. To generate CD1D-overexpressing myeloma cell lines, the human CD1D full-length construct was transduced into MM cell lines using lentivirus, and CD1D+ cells were purified by FACS. Jurkat-76 cells were obtained from N.H. and cultured in the same complete IMDM as the myeloma cell lines. The melanoma cell lines A-375 (ATCC), A2058 (ATCC), 624Mel, 526Mel, 888Mel (provided by S. Rosenberg) and WM3670 (Rockland), the lung cancer cell lines A549, H1568, H1573, H1437, H1792, H1944 and H2030 (all from ATCC), and the glioblastoma cell lines LN-229 (ATCC) and A-172 (ATCC) were cultured in complete DMEM (Gibco) + 1% GlutaMAX (Gibco) and supplemented in the same manner as complete IMDM. The small-cell lung carcinoma cell line H69PR (ATCC), mesothelioma cell lines H2452 (ATCC) and MSTO-211H (ATCC), breast cancer cell lines HCC1143 (ATCC) and HCC1937 (ATCC), the pancreatic cancer cell line ASPC1 (ATCC) and the colon cancer cell lines LoVo (ATCC) and Colo205 (ATCC) were cultured in complete RPMI-1640 (Gibco) supplemented in the same manner as complete IMDM. The pancreatic cancer cell line CFPAC-1 (ATCC) was cultured in complete IMDM medium. The Merkel cell carcinoma cell lines MCC14-2, MCC26, MS-1 and MCC13 were purchased from Sigma and cultured in RPMI-1640 (Gibco) supplemented with 20% FCS, 2 mM l-glutamine, 1% penicillin–streptomycin, 25 mM HEPES and 10 µg ml–1 gentamicin. Lenti-X 293T cells were purchased from Takara and cultured in complete DMEM as described above. Healthy primary cells used for screening were obtained from healthy donors and obtained from the ATCC and Sigma and cultured according to the manufacturers’ recommended media and protocols. Tumour cell lines used were confirmed to be free of mycoplasma.
Antibodies and flow cytometry
The following antibodies were used in this study for flow cytometry as indicated: CD3 (OKT3; BioLegend, 317308 or 317322, 1:400); B2M (A17082A; BioLegend, 395716, 1:800); CD69 (FN50; BioLegend, 310910, 1:400); CD8 (RPA-T8; BioLegend, 301049, 1:400); CD4 (OKT4; BioLegend, 317420, 1:200); NGFR (ME20.4; BioLegend, 345132, 1:200); γδTCR (B1; BioLegend, 331210, 1:100); αβ TCR (IP26; BioLegend, 306720, 1:100); CD14 (61D3; ThermoFisher, 53-0149-42, 1:50); CD2 (RPA-2.10; BioLegend, 300230, 1:75); CD235 A/B (HIR2; BioLegend, 306614, 1:200); CD138 (MI15; BioLegend, 356504, 1:100); CD319 (235614; BD, 750838, 1:20); HLA-ABC (W6/32; BioLegend, 311405, 1:50); HLA-A (1082C5; BD, 568024, 1:100); HLA-C (DT-9; BD, 566372, 1:100); BTN3 (232-5; BD, 566006, 1:100); Bw4 (REA274; Miltenyi, 130-123-760, 1:100); KIR3DL1 (DX9; BioLegend, 312707, 1:100); CD94 (HP-3D9; Invitrogen, 17-5094-4210, 1:100); GPR56 (CG4; BioLegend, 358205, 1:100); 4-1BB (4B4; BioLegend, 309809, 1:200); CD1D (51.1; BioLegend, 350305, 1:100); and CD1C (L161; BioLegend, 331505, 1:100). Trustain Fc Block (BioLegend, 422302) was used to block Fc receptors, and fixable viability dye (Invitrogen, 65-0865-14), with or without Annexin V staining (BioLegend, 640920), was used to identify live and apoptotic cells. Unless used for cell sorting or annexin V staining, cells were fixed with 4% paraformaldehyde before analyses. Flow cytometry data were acquired on a BD LSR Fortessa X20 or Beckman Coulter CytoFLEX instrument and analysed using FlowJo software (v.10, BD Biosciences)
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