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709 Peptidyl IL-2/15Rβγc-restricted agonists, highly-attenuated and linked to anti-PD-1 antibodies to achieve selectivity and amplified potency in stimulating PD-1high lymphocytes
  1. William Dower1,
  2. Alice Bakker1,
  3. Steven Cwirla1,
  4. Blake Williams1,
  5. Prarthana Joshi1,
  6. Praechompoo Pongtornpipat1,
  7. Michael Needels2 and
  8. Ronald Barrett2
  1. 1Medikine, Menlo Park, CA, United States
  2. 2Medikines, Menlo Park, CA, United States


Background Recent reports demonstrate that directing a “non-alpha” IL-2 mutant to a PD-1high CD8+ stem-like population induces proliferation, and differentiation into a highly functional cytotoxic phenotype. We previously reported small synthetic peptides, unrelated to IL-2 or IL-15, that bind IL-2/15Rβγc to induce receptor signaling. These peptides do not bind IL-2Rα and are therefore IL-2/15Rβγc-restricted agonists. We now describe fusion of potency-attenuated peptide agonists to an anti-PD-1 antibody (α-PD-1) to achieve selective targeting to PD-1high lymphocytes, and enhanced potency of IL-2R agonists acting in Cis with α-PD-1 binding.

Methods Peptidyl IL-2/15Rβγc agonists with attenuated potency due to weakened binding to either IL-2/15Rβ or γc were fused to the C-termini of both heavy chains of an α-PD-1 IgG and expressed in CHO cells. The fusion proteins retained PD-1 binding affinity comparable to the α-PD-1; and were evaluated for potency of IL-2Rβγc-dependent STAT5 phosphorylation in TF-1β cells (with undetectable PD-1 expression), and in TF-1β-derived lines expressing varying levels of PD-1. The fusion proteins were also assessed for Rβγc stimulation of CD8+ cells treated with anti-CD3 and anti-CD28 to induce elevated PD-1 expression.

Results An analysis of pembrolizumab (Pem) fused to MDK1169, a potent IL-2Rβγc agonist, showed a 15-fold increase in potency in TF-1β/PD-1+ cells. This served as an initial demonstration of the PD-1-directed, cis-acting mechanism; but the potency of MDK1169 in this construct (~500pM, EC50 pSTAT5 induction) is too high (relative to the affinity of Pem for PD-1) to achieve a more substantial selectivity for PD-1+ cells. To improve selectivity, fusions of α-PD-1 to peptide agonists with potencies as weak as 1uM on TF-1β cells were constructed. Some of these fusion proteins exhibited up to 100-fold increase in potency when tested on TF-1β/PD-1high compared to parental TF-1β cells; and addition of an excess of α-PD-1 blocked this gain in potency in the PD-1high cells. When tested on CD8+ cells activated to express elevated PD-1 levels, potency of the PD-1-directed agonists correlated with PD-1 expression.

Conclusions The malleability of peptidyl agonists makes them useful for optimizing antibody-targeted cis-acting agonists designed to produce minimal activity on non-targeted cells and high potency at targeted cells. IL-2/15Rβγc agonists directed by PD-1 binding to a stem-like highly cytotoxic tumor infiltrating CD8+ population may have useful anti-tumor applications.

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