Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity

Abstract

Inhibitors that block the programmed cell death-1 (PD-1) pathway can potentiate endogenous antitumor immunity and have markedly improved cancer survival rates across a broad range of indications. However, these treatments work for only a minority of patients. The efficacy of anti-PD-1 inhibitors may be extended by cytokines, however, the incorporation of cytokines into therapeutic regimens has significant challenges. In their natural form when administered as recombinant proteins, cytokine treatments are often associated with low response rates. Most cytokines have a short half-life which limits their exposure and efficacy. In addition, cytokines can activate counterregulatory pathways, in the case of immune-potentiating cytokines this can lead to immune suppression and thereby diminish their potential efficacy. Improving the drug-like properties of natural cytokines using protein engineering can yield synthetic cytokines with improved bioavailability and tissue targeting, allowing for enhanced efficacy and reduced off-target effects. Using structure guided engineering we have designed a novel class of antibody-cytokine fusion proteins consisting of a PD-1 targeting antibody fused together with an interleukin-21 (IL-21) cytokine mutein. Our bifunctional fusion proteins can block PD-1/programmed death-ligand 1 (PD-L1) interaction whilst simultaneously delivering IL-21 cytokine to PD-1 expressing T cells. Targeted delivery of IL-21 can improve T cell function in a manner that is superior to anti-PD-1 monotherapy. Fusion of engineered IL-21 variants to anti-PD1 antibodies can improve the drug-like properties of IL-21 cytokine leading to improved cytokine serum half-life allowing for less frequent dosing. In addition, we show that targeted delivery of IL-21 can minimize any potential detrimental effect on local antigen-presenting cells. A highly attenuated IL-21 mutein variant (R9E:R76A) fused to a PD-1 antibody provides protection in a humanized mouse model of cancer that is refractory to anti-PD-1 monotherapy. Collectively, our preclinical data demonstrate that this approach may improve upon and extend the utility of anti-PD-1 therapeutics currently in the clinic.

Engineered Cytokines for Cancer and Autoimmune Disease Immunotherapy

“…[ 59 ] IL‐21 has also been fused with PD‐1 blocking antibodies, demonstrating treatment efficacy in humanized cancer models refractory to anti‐PD‐1 monotherapy. [ 60 ] Here, treatment‐associated tumor control and CD8 + T‐cell cytolytic activity were superior to anti‐PD‐1 therapy alone, with comparable levels of PD‐1 inhibition.…”

Fundamentals of T Cell Metabolism and Strategies to Enhance Cancer Immunotherapy

“…Recently two independent reports showed that targeting IL-21 directly to T cells rather than systemic delivery in combination with PD-1 therapy improved tumor immunity. They also showed that systemic delivery of soluble IL-21 did not improve the therapeutic efficacy of PD-1 blockade, however when the anti-PD-1 antibody was fused to IL-21 synergetic improvement in tumor immunity was noticed (139,140). There are currently many efforts to translate the use of single and combinations of these cytokines to expand TIL and CAR products for ACT, as well as novel ways to incorporate cytokine releasing switches in CAR constructs and fusion proteins.…”

The PD-1/PD-L1 Checkpoint in Normal Germinal Centers and Diffuse Large B-Cell Lymphomas

“…On another hand, IL-21 might also become toxic for lymphomas by expanding and enhancing tumour-infiltrating cytotoxic CD8 + cells and NK cells [ 163 , 164 , 165 , 166 ]. Such immunotherapeutic potential of IL-21 to stimulate anti-tumour immune responses is being clinically investigated in different cancers [ 160 , 167 , 168 ].…”

“…Antibody-cytokine conjugated proteins represent another class of biopharmaceuticals in cancer immunotherapy [ 201 ]. Combination therapy of IL-21 with PD-1 or CTLA-4 previously showed efficacy in mouse tumour models [ 202 ], and an engineered IL-21 fused to a PD-1 antibody showed promising results by providing superior anti-tumour immunity than anti-PD-1 alone in preclinical studies [ 167 ].…”