31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes

<div><p>Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to <em>in vivo</em> proteolytic degradation and its propensity to <em>in vitro</em> aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the <em>in vitro</em> and <em>in vivo</em> potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the <em>in vitro</em> activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.</p> </div

In vitro activity of FGF21 variants.

<p>293T cells stably expressing human βKlotho along with luciferase reporter constructs were treated with FGF21 variants for 6 hours and lysates were collected to measure luciferase activity.</p

The N-terminal Fc fusion (Fc-FGF21) was superior to the C-terminal Fc fusion (FGF21-Fc) in retaining the biological activity of FGF21.

<p>(A–B) Stimulation of Elk1-luciferase reporter activity by FGF21, Fc-FGF21 and FGF21-Fc in 293T cells stably expressing human βKlotho (A) or in CHO cells stably expressing both human βKlotho and human FGFR1c (B); (C) Activation of Erk phosphorylation by FGF21, Fc-FGF21 and FGF21-Fc in human primary adipocytes. All <i>in vitro</i> data represent mean ± SEM, n = 4/concentration group. (D-F) 8-9 week old male <i>db/db</i> (C57/BL6 strain) mice were ip administered with various doses of native FGF21 (D), Fc-FGF21 or FGF21-Fc (E). Blood glucose levels were measured at baseline and 6 h after injection (D–E). % change of blood glucose levels from baseline was plotted as a function of FGF21 molar doses (F). Data are mean ± SEM, n = 9–10 animals per group, ̂ p<0.01; # p<0.001 compared with vehicle. (G–J) The binding activity of FGF21 (G), Fc-FGF21 (H) and FGF21-Fc (I) to human βKlotho determined in a solution equilibrium binding assay on a BIAcore instrument. (J) Quantification of βKlotho binding to the biotinylated FGF21 immobilized on the chip. 100% βKlotho binding is the signal obtained with no FGF21 in the solution.</p

Fc-FGF21(RG) was resistant to degradation in cynomolgus monkeys.

<p>LBMS (MALDI-TOF) analysis of native FGF21 (A), Fc-FGF21 (B) or Fc-FGF21(RG) (C) in male cynomolgus monkeys. FGF21 (10 mg/kg) or Fc-FGF21 variants (23.5 mg/kg) were iv administered in cynomolgus monkeys and blood samples were collected at indicated time points. The mass positions of the parent constructs are indicated by vertical hashed lines. The peak marked with a heavy arrow corresponds to the primary metabolite of FGF21 or Fc-FGF21, which is absent in the Fc-FGF21(RG). Peaks marked with asterisks in all of the spectra correspond to sinapinic acid adducts of primary peaks and are an artifact of the ionization process of MALDI. The MALDI-TOF mass spectra are normalized to the most intense peak in the plotted <i>m/z</i> range.</p

Identification of aggregation-resistant FGF21 mutants. (A–B) FGF21 aggregation was concentration-(A), temperature- (B), and time-dependent (A&B).

<p>The % of high molecular weight species over the total was obtained from size exclusion chromatography (SEC) analysis. (C) Homology model of FGF21 (residues 14–155) with the mutated residues shown in magenta. The leucine to arginine mutation is shown for residue 98 (magenta) and all other mutation sites were shown as native sequence. The FGF21 homology model was prepared using Modeler (Fiser and Sali) in Discovery studio 3.1 (Accelrys) with FGF19 (PDB code: 2P23) used as a template. Image was created using PyMol (Schrödinger, LLC). (D) The aggregation rate of FGF21 mutants. Samples were concentrated at 60±10 mg/ml and stored at 4°C for 1, 6 and 10 days before SEC analysis.</p

In vitro activity of Fc-FGF21(RG) relative to native FGF21 and Fc-FGF21 wild type.

<p>(A) Structural diagram of Fc-FGF21(RG), an E coli-expressed homodimeric Fc-FGF21 fusion protein containing 2 engineered point mutations at positions 98 and 171. 15G indicates the 15 aa polyglycine and polyserine linker. (B) The binding activity of FGF21, Fc-FGF21 and Fc-FGF21(RG) to human βKlotho determined in a solution equilibrium binding assay on a BIAcore instrument. (C) Elk1-luciferase reporter activity in 293T cells treated with FGF1, FGF21, Fc-FGF21 and Fc-FGF21(RG). The cells were stably expressing reporter constructs but no βKlotho. (D) Stimulation of Elk1-luciferase reporter activity by FGF21, Fc-FGF21 and Fc-FGF21(RG) in 293T cells stably expressing human βKlotho along with luciferase reporter constructs. (E) Stimulation of Elk1-luciferase reporter activity by FGF21, Fc-FGF21 and Fc-FGF21(RG) in mouse NIH/3T3 fibroblast cells transiently co-transfected with human βKlotho and human FGFR1c. (F) Stimulation of Erk phosphorylation by FGF21, Fc-FGF21 and Fc-FGF21(RG) in human primary adipocytes. All data represent mean ± SEM, n = 4/concentration group. ND: not detectable. FGF21, closed circle; Fc-FGF21: opened circle; Fc-FGF21(RG): closed triangular and dash line; FGF1: closed square.</p