Development and Preliminary Clinical Activity of PD-1-Guided CTLA-4 Blocking Bispecific DART Molecule

Alexey Berezhnoy, Bradley J Sumrow, Kurt Stahl, Kalpana Shah, Daorong Liu, Jonathan Li, Su-Shin Hao, Anushka De Costa, Sanjeev Kaul, Johanna Bendell, Gregory M Cote, Jason J Luke, Rachel E Sanborn, Manish R Sharma, Francine Chen, Hua Li, Gundo Diedrich, Ezio Bonvini, Paul A Moore, Alexey Berezhnoy, Bradley J Sumrow, Kurt Stahl, Kalpana Shah, Daorong Liu, Jonathan Li, Su-Shin Hao, Anushka De Costa, Sanjeev Kaul, Johanna Bendell, Gregory M Cote, Jason J Luke, Rachel E Sanborn, Manish R Sharma, Francine Chen, Hua Li, Gundo Diedrich, Ezio Bonvini, Paul A Moore

Abstract

Combination immunotherapy with antibodies directed against PD-1 and CTLA-4 shows improved clinical benefit across cancer indications compared to single agents, albeit with increased toxicity. Leveraging the observation that PD-1 and CTLA-4 are co-expressed by tumor-infiltrating lymphocytes, an investigational PD-1 x CTLA-4 bispecific DART molecule, MGD019, is engineered to maximize checkpoint blockade in the tumor microenvironment via enhanced CTLA-4 blockade in a PD-1-binding-dependent manner. In vitro, MGD019 mediates the combinatorial blockade of PD-1 and CTLA-4, confirming dual inhibition via a single molecule. MGD019 is well tolerated in non-human primates, with evidence of both PD-1 and CTLA-4 blockade, including increases in Ki67+CD8 and ICOS+CD4 T cells, respectively. In the ongoing MGD019 first-in-human study enrolling patients with advanced solid tumors (NCT03761017), an analysis undertaken following the dose escalation phase revealed acceptable safety, pharmacodynamic evidence of combinatorial blockade, and objective responses in multiple tumor types typically unresponsive to checkpoint inhibitor therapy.

Keywords: CTLA-4; PD-1; bispecific; checkpoint; combinatorial; immunotherapy; pharmacodynamics.

Conflict of interest statement

A.B., B.J.S., K.S., D.L., J.L., S.-S.H., A.D., K.S., F.C., H.L., E.B., G.D., and P.A.M. are contracted or employed by MacroGenics, and received stock options as a condition of employment. A.B., B.J.S., K.S., E.B., G.D., and P.A.M. are inventors on MacroGenics patent applications based on the work described herein.

© 2020.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Cells Co-expressing PD-1 and CTLA-4 Are More Prevalent in the Tumor Microenvironment (A) In situ RNA hybridization of PD-1 and CTLA-4 probes in ovarian cancer tumor cores (N = 21) analyzed using RNAscope and quantified with HALO software. Each square represents an individual core, with red and blue circles representing the indicated frequency of PD-1 and CTLA-4 expression, respectively. The first square shows PD-1 and CTLA-4 expression in a non-malignant ovary sample. (B) In situ RNA hybridization of PD-1 (red) and CTLA-4 (blue) probes visualized by RNAscope in representative tumor microarray core or healthy tonsil samples. (C) Fraction of cells co-expressing PD-1 and CTLA-4 RNA detected by ISH in lymphoid organs from healthy donors (N = 7) or tumor samples from randomly selected patients (N = 12). Means and standard deviations (SDs) are shown. (D) Peripheral blood mononuclear cells (PBMCs) from healthy donors (N = 8) and PBMCs (N = 27) or dissociated tumor cells (DTCs) (N = 7) from patients with various cancers were stained for PD-1 and CTLA-4 expression and analyzed by flow cytometry. Box and whiskers plots depict the minimum, first quartile, median, third quartile, and maximum. Gated on viable CD45+/CD3+ cells. (E) Representative fluorescence-activated cell sorting (FACS) images from (D) gated on viable T cells. See also Figure S1.
Figure 2
Figure 2
MGD019 Molecular Structure and Bispecific Binding to PD-1 and CTLA-4 (A) MGD019 is a tetravalent bispecific (2 × 2) Fc-bearing DART molecule. (B) Binding of MGD019 (red diamonds), parental PD-1 mAb retifanlimab (blue squares), parental CTLA-4 mAb 4B6 (green triangles), or isotype control (black circles) to Jurkat/PD-1 cells and blockade of PD-L1 binding to the cells. (C) Binding to Jurkat/CTLA-4 cells and blockade of B7-1 binding to the cells. (D) Re-activation of β-galactosidase (β-gal) upon co-engagement of PD-1 and CTLA-4 by MGD019 in PathHunter PD-1+CTLA-4+ assay. Error bars depict standard errors of the mean (SEMs). (E) Binding to in vitro-stimulated, PD-1+/CTLA-4+ primary T cells and blockade of B7.1 binding to Jurkat PD-1+/CTLA-4+ cells. (F) Blockade B7.1 binding to Jurkat PD-1+/CTLA-4+ by MGD019 or CTLA-4 mAbs alone or in the presence of a 10× concentration of competing PD-1 mAbs (open red diamonds and purple crosses, respectively). (G) Interaction of MGD019 with single- and dual-expressing cells. Average (EC50) values of PD-1 (blue) and CTLA-4 (yellow) ligand binding blockade (Table S1). Representative experiments out of ≥3 independent repeats are shown in (B)–(F). See also Table S1 and Figures S2 and S3.
Figure 3
Figure 3
PD-1 x CTLA-4 Bispecific Inhibitor Enhances T Cell Activation (A) Dual reporter cell line (Jurkat-PD-1/CTLA-4 cells) and artificial APCs (Raji-PD-L1/B7 cells) were co-cultured in the presence of MGD019 (red diamonds), its parental PD-1 (blue squares) or CTLA-4 (green triangles) mAbs, their combination (purple crosses), nivolumab ( blue blue crosses), ipilimumab (yellow triangles), or their combination (tan hexagons) and isotype control (black circles). Representative experiments out of 3 independent repeats are shown. (B) Mean fold change of IL-2 concentrations in the samples treated with 10 μg/mL of MGD019 or control mAbs relative to samples treated with control IgG plus indicated concentrations of SEB. The experiments were performed individually with PBMCs from healthy donors (N = 39). Error bars depict SEMs. Inset: subset of donors (N = 9/39) with reduced effects to PD-1 blockade (IL-2 fold change [f.c.] in vitro with anti-CD3 and treated with MGD019 or replicas of ipilimumab featuring its original IgG1 Fc or replaced with IgG4. Fraction of FoxP3+ T cells was measured after 48-h incubation. Representative graphs of 18 independent replicates are shown. (D) Mean values of CD4+FoxP3+ cells measurements across multiple (N = 6) independent repeats using PBMCs of different donors (N = 2). See also Figure S4.
Figure 4
Figure 4
MGD019 Provides Dual Checkpoint Blockade In Vivo (A and B) Cynomolgus monkeys (5 females and 5 males per group) were infused with 10, 40, or 100 mg/kg MGD019 at days 1, 8, 15, and 22. Mean serum concentrations of MGD019 and mean percentages of PD-1+ cells with MGD019 detectable at cell surface by flow cytometry are shown. Error bars depict SEMs, vertical dotted lines indicate dose administration, and the horizontal dotted line marks 100% cell-surface binding. (C–E) Splenocytes obtained from cynomolgus monkeys treated with 4 weekly administrations of MGD019 or vehicle control and necropsied 3 days after the last infusion were stained for CD28/CD95 (C), ICOS (D), or CD25/FoxP3 (E) expression. Means and SDs are shown. Gated on CD45+/CD3+/CD4+ cells. (F) Cynomolgus monkeys were infused i.v. Q1W for 3 weeks with 75 mg/kg MGD019 (3 males/3 females) or, in a separate study, 100 mg/kg parental PD-1 mAb (2 males/2 females). Ki67 expression on circulating CD4+ T cells was quantified by flow cytometry. Means and SEMs are depicted. See also Figure S5.
Figure 5
Figure 5
Clinical Benefits of Dual PD-1 and CTLA-4 Blockade Mediated by MGD019 (A) Simulated multiple-dose PK profiles for the 3, 6, and 10 mg/kg Q3W regimens with observed pre-dose and post-dose data superimposed. Target concentration overlaid as dashed line. (B) MGD019 receptor occupancy for CD4+ T cells collected 21 days after second infusion (green) compared to measured immediately after third infusion (blue) (N = 22). Means and SDs are depicted. (C) Binding of MGD019-competing FACS mAbs to circulating T cells in patients treated with MGD019 before first dose (gray) and 8 (blue) and 22 (green) days later (N = 28). Bars indicate minimum to maximum intervals. (D) Fraction of proliferating CD8+ T cells (N = 6) and regulatory T cells (N = 7) observed in cryopreserved PBMCs of patients treated with 3 (brown), 6 (yellow), and 10 (blue) mg/kg MGD019 collected at the indicated days. Paired t-test with two tailed p value calculation was used. (E) ICOS expression on peripheral blood CD4+ T cells measured before (gray) and 8 days after (blue) first infusion of indicated doses of MGD019 (N = 28) or patients treated with a PD-1 based therapy not containing CTLA-4 blockade (obrtained from an independent study) that serves as a Control (N=4). Bars indicate mean values. (F) Scans of the patient with microsatellite stable (MSS) colorectal cancer (CRC) obtained ~15 weeks after treatment initiation, demonstrating resolution of a 3.0-cm subcarinal lymph node. (G) Waterfall plot of RECIST 1.1 response evaluable patients treated with 3, 6, and 10 mg/kg MGD019. # indicates previous treatment with checkpoint inhibitor and + indicates patients currently staying in the study. See also Figure S6.

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