Development and characterization of bispecific antibodies
Antibodies to CD27 and PD-L1 were generated by immunization of H2L2 human Ig transgenic mice (Harbour Antibodies BV) with recombinant human CD27 or PD-L1. Splenocytes were used for hybridoma preparation by standard polyethylene glycol fusion techniques. The variable heavy and light chain regions of selected antibodies were cloned into a human IgG1κ expression vector, expressed in ExpiCHO cells (Invitrogen), and further characterized. The PD-L1 antagonist mAb avelumab (AVE) was similarly prepared using the published sequence (WHO Drug Information, Vol. 29, No. 2, p. 203, 2015).
For the BsAb, an expression vector encoded the full-length anti-PD-L1 mAb 9H9 IgG1κ heavy and light chains and the scFv of the anti-CD27 2B3 mAb genetically linked in VL–VH orientation to the C-terminus of the 9H9 mAb heavy chain. Cysteine residues were introduced, one in 2B3 VL and one in 2B3 VH, to stabilize the scFv domains. An analogous vector was prepared in which the 9H9 VH and VL were replaced by the avelumab VH and VL. These vectors were transfected into HD-BIOP3 cells (Horizon Discovery) and proteins were purified by protein A and size-exclusion chromatography. All purified mAbs and BsAbs contained < 0.5 endotoxin units/mg.
Binding and blocking assays
Affinity determination using bio-layer interferometry
The mAbs or BsAbs were captured on anti-human Fc capture biosensors (ForteBio). Binding was determined by exposing the loaded biosensor to human PD-L1-HIS (R&D Systems) or human CD27 (generated in-house). Affinity measurements were determined using twofold serial dilutions of analyte ranging from 50 to 0.195 nM. The association and dissociation curves were fitted to a 1:1 binding model using the data analysis software according to the manufacturer’s guidelines.
Extracellular domains of human and cynomolgus CD27 were generated and purified from transient transfections using Protein L. The fusion protein of human PD-L1 and mouse Ig Fc (msFc) domain was purified from transient transfections using Protein A. The following fusion proteins were purchased: mouse PD-L1 with mouse Ig Fc domain (ACROBioSystems), and mouse, cynomolgus macaque and rat PD-L1 with human Ig Fc (R&D Systems). For ELISA, plates coated with recombinant protein were exposed to samples and binding was detected using an HRP-labeled goat-anti-human IgG (Fc-specific) antibody and developed with 3,3′,5,5′-tetramethylbenzidine substrate. For BsAb binding, wells were coated with CD27 protein. BsAb dilutions were allowed to bind before adding human or mouse PD-L1-msFc which was detected with an HRP-labeled goat anti-mouse IgG (Fc specific) antibody.
HEK293 cells transfected with human CD27 or PD-L1 (Crown Bioscience) were incubated with mAbs for 20 min, and the bound antibodies were detected with a phycoerythrin (PE)-labeled goat anti-human IgG Fc-specific probe (Jackson ImmunoResearch). Cell-associated fluorescence was determined by analysis using a FACSCanto II™ instrument (BD Biosciences). To assess the effect of mAbs or BsAbs on ligand binding, CD27 expressing Ramos cells (ATCC) or HEK293-PD-L1 cells were briefly pre-incubated with the mAbs, BsAbs or controls, followed by the addition of 0.5 µg/ml human CD70-biotin (US Biological) or 0.5 µg/ml PD-1-biotin (R&D Systems), respectively. Binding of biotinylated ligands was detected with streptavidin PE (SA–PE) and analyzed on a FACSCanto II™ instrument.
CD27 agonist assays
NFκB reporter assay
A stable cell line was developed from HEK293 NFκB-luciferase reporter cell line (Signosis, Inc.) transfected with human CD27. Cells were incubated with mAbs or BsAb for 6 h at 37 °C, 6% CO2. Luciferase was detected with the Luciferase Assay System (Promega).
Human T cell costimulation
Ninety-six well tissue culture plates were prepared by adding 1 µg/ml anti-CD3 mAb (OKT3- eBioscience), and/or 2 µg/ml recombinant human PD-L1 and coated overnight at 4 °C. After washing the wells with PBS, 100,000 CD3+ cells isolated by magnetic bead separation from peripheral blood mononuclear cells (PBMC) were added to each well in media. The plates were incubated for 3 days at 37 °C and 5% CO2, and supernatants were harvested and analyzed for IL-2 or IFN-γ production by ELISA (R&D Systems).
Cell-based PD-1 signaling assay
The effect of the BsAb on blockade of PD-1 signaling was performed per manufacturer’s instructions with a cell-based method in which blocking PD-1 signaling allows T cell receptor (TCR) activation and induces luminescence via the NFAT pathway (Promega). Luminescence was detected by the addition of Bio-Glo reagent and quantitated on a PerkinElmer Victor X luminometer.
Mixed lymphocyte reaction (MLR)
Human PBMCs were isolated from buffy coats using Ficoll separation, and CD4+ cells were further isolated using magnetic bead separation (Miltenyi). Monocyte-derived dendritic cells (DC) were generated from PMBCs by adhering to plastic and then cultured for 7 days in RPMI medium containing 10% FBS, 10 ng/ml IL-4 plus 100 ng/ml GM-CSF (R&D Systems). Cells were harvested and confirmed to be 80% DCs by expression of CD11c. The CD4+ cells and DCs from allogeneic donors were co-incubated at a 10:1 ratio in the presence of mAb or BsAb for 3 days. Supernatants were harvested and analyzed for IL-2 or IFN-γ production by ELISA (R&D Systems).
Antibody-dependent cellular cytotoxicity (ADCC)
ADCC activity was evaluated using a commercially available ADCC Reporter Bioassay Kit (Promega). This assay indirectly measures ADCC through quantitation of the FcγRIIIa receptor activity. Target cells included tumor cell lines Ramos (CD27+) and MDA-MB-231 (PD-L1+) (ATCC) or HEK293 cell lines expressing either CD27 or PD-L1.
Enhancement of vaccine-specific T cell responses
The huCD27-Tg mice  received intraperitoneal (i.p.) administrations of 0.05 mg of BsAb or mAbs and 5 mg of ovalbumin (OVA) (Sigma-Aldrich). After 1 week, spleen cells were harvested and ELISPOT analysis was performed with and without 2 µg/ml SIINFEKL peptide (GenScript) incubated overnight in IFN-γ Ab-coated 96-well filtration plates (Sigma-Aldrich). Spots were developed by using an IFN-γ antibody set and a 3-amino-9-ethylcarbazole substrate (BD Biosciences) and counted by ZellNet Consulting, Inc.
Mouse tumor models
For the syngeneic lymphoma model, huCD27-Tg Balb/C mice were injected intravenously (i.v.) with BCL1 cells (1 × 106) on day 0, followed by i.p. injection of mAbs or BsAbs (0.2 mg) on days 5 or 7. Mice were observed daily for survival. Xenograft tumor studies were performed using the human B cell lymphoma cell line Raji (ATCC) with 0.5 × 106 cells implanted subcutaneously (s.c.) on day 0 into the flanks of SCID mice cells followed by i.p. administration of mAbs or BsAbs (0.1 mg) on days 5, 8, 12, 15, 19, and 22. Tumors were measured by caliper twice per week, and mice were euthanized according to pre-defined endpoint criteria.
Pilot non-human primate study
Three male cynomolgus monkeys (Citoxlab, Stilwell KS) received a single 7 mg/kg slow bolus i.v. injection (2–3 min) of CDX-527, via a cephalic catheter. Animals were followed for 21 days. Evaluations included clinical signs, body temperature, clinical pathology parameters (hematology, coagulation, clinical chemistry and urinalysis), and toxicokinetic parameters. Body weights were recorded once prior to BsAb administration and weekly thereafter. This was designed as a survival study with no planned necropsy.
Quantitation of CDX-527 concentration and anti-drug antibodies (ADA) was performed using a Mesoscale Discovery platform (MSD). For pharmacokinetics (PK), the plates were coated with a human CD27-Fc. The bound CDX-527 was detected by adding human PD-L1-msFc and a ruthenium-labeled SULFO-Tag F(ab’)2 (Mesoscale diagnostics) goat anti-mouse IgG (Fc-specific) followed by tripropylamine. The ADA assay used streptavidin-coated plates to capture biotinylated CDX-527. Serum samples were then added, and reactive antibodies were detected with ruthenium-conjugated CDX-527 and tripropylamine.
Statistical significance was evaluated using two-way ANOVA or paired Student’s t-test as appropriate. For tumor survival studies, the Mantel-Cox test was used.