Potency-Reduced and Extended Half-Life IL12 Heterodimeric Fc

Potency-reduced and extended half-life IL12 heterodimeric Fc-fusions exhibit strong anti- tumor activity with potentially improved therapeutic index compared to native IL12 agents SITC 2020 Matthew J. Bernett*, Rajat Varma, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Irene W. L. Leung, Connie Ardila, Katrina Bykova, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais Abstract #564 Introduction Potency-reduced IL12-Fc are engineered for optimal activity and extended serum half-life

. Interleukin-12 (IL12) is a heterodimeric proinflammatory cytokine produced by activated Mechanism and anti-tumor effects of IL12 Design and analytical characterization of IL12-Fc heterodimers antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and IFNγ cytotoxicity of T and NK cells.

. γ T cell Stimulation of these cells by IL12 leads to production of high levels of IFN . These immune- T cell IL12-p40 IL12-p35 stimulating aspects of IL12 are promising for cancer treatment and may help to convert p40 p40-Fc p35-Fc IL12Rβ1/ “cold” tumor immunologically suppressed “cold” tumors into inflamed “hot” tumors. IL12Rβ2 . Preclinical studies in mice revealed that IL12 can have a dramatic effect on shrinking syngeneic “hot” tumor Heterodimeric Fc tumors; however, clinical studies in humans have resulted in severe toxicity and a small region with ablated effector therapeutic window, limiting response rates. p35 NK cell function . Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins IL12 NK cell exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates IFNγ Figure 2. Schematic of IL12-Fc heterodimers and analytical characterization. Monovalent IL12 p35/p40 is attached to through reduction of receptor-mediated clearance. Applying similar principles to IL12, we Xencor’s well-validated heterodimeric Fc domain. The IL12 has been engineered for decreased binding to its receptors in created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency in order to improve Figure 1. IL12 is a heterodimer consisting of p40 and p35 subunits that signals through the STAT4 pathway. IL12 may help order to reduce potency and the Fc domain is modified to eliminate FcγR interactions. The Fc domain may also be modified tolerability, slow receptor-mediated clearance, and prolong half-life compared to native IL12 to turn non-inflamed, cold tumors into inflamed, hot tumors that are amenable to checkpoint inhibitor therapy by inducing with Xtend™ Fc technology to promote longer half-life. IL12-Fc heterodimers can be produced in high yields and are agents. proliferation of NK and T cells and by production of IFNγ. purified using standard methods (protein A and IEX chromatography).

IL12-Fc with reduced in vitro potency were engineered in Potency-reduced IL12-Fc show strong anti-tumor activity and PD response as single-agent and in combination with anti-PD1 order to improve therapeutic index Anti-tumor efficacy at day 33 Tumor volume - Spider plots In vivo pharmacodynamics (PD) + + ) ) Anti-PD1 (3 mg/kg) ) CD25 MFI on CD8 T cells CD8 T cell counts 3 3 Vehicle (PBS) 3 IL12-Fc-v2 (1 mg/kg) )

m p = .0029 m m Engineering potency-reduced IL12-Fc and in vitro activity 3 800 800 800

m 800 m < 0.05 vs. PBS m 40 100000 ( (

*p (

e e p < .0001 700 700 e 700 * * * m m †p < 0.05 vs. anti-PD1 m u u 700 600 600 u 600 l l l o o 3000 + + + 500 500 o

V 500 V

CD8 CD45RA CD25 V

3000 30 10000 r r + - + r o o 400 CD8 CD45RA CD25 IL12-Fc (WT) 600 400 o 400 m m IL12-p40 300 300 m + + + u u 300 CD4 CD45RA CD25 u T T T

Variants 200 200 200 n n + - + n i i 500 i

1000 CD4 CD45RA CD25 † 20 *p < 0.0001 vs. anti-PD1

2000 e e 2000 100 100 e 100 g g * g n n NK cells 0 0 n 0 a a a h 400 h Gamma Delta cells h C C 0 7 14 21 28 0 7 14 21 28 * C 0 7 14 21 28 10 100 1000 300 * Days Post Dose 1 Days Post Dose 1 Days Post Dose 1 CD25 MFI @ Day 14 1000 cell counts @ Day 14 p < .0001 pSTAT4 (MFI) pSTAT4 (MFI) † 200 IL12-Fc-v1 (0.1 mg/kg) 0 10 * PBS Anti-PD1 IL12-Fc-v1 IL12-Fc-v1 IL12-Fc-v2 PBS Anti-PD1 IL12-Fc-v1 IL12-Fc-v1 IL12-Fc-v2 ) )

3 IL12-Fc-v1 (0.1 mg/kg)

3 + anti-PD1 (3 mg/kg) 0 (3 mpk) (0.1 mpk) (0.1 mpk) (1 mpk) (3 mpk) (0.1 mpk) (0.1 mpk) (1 mpk)

0 m 100 800 m

m 800

m + anti-PD1 + anti-PD1 ( (

1 1 1 1 0 0 0 1 1 1 .1 1 0 . 0 0 0 0 1 0 e 0 .0 0 1 . 0 700 e 700 (3 mpk) (3 mpk) 0 .0 0 .0 0 1 0

1 m .0 0 0 m u 0 • NSG-DKO mice 600 u 600 l

0 (mm Change Volume Tumor in l o

IL12-p35 o IL12-Fc (nM) 500

V 500 V +

γ IL12-Fc (nM) serum IFN r • n = 10 per group r PD-1 MFI on CD8 T cells o PBS Anti-PD1 IL12-Fc-v1 IL12-Fc-v1 IL12-Fc-v2 400 o 400 m 300 m

u 300 1000 (3 mpk) (0.1 mpk) (0.1 mpk) (1 mpk) • Q1W dosing u 1500 T T

200 200 * n Figure 3. Left: In vitro activity of WT IL12-Fc was assessed on activated human PBMCs by measuring intracellular pSTAT4 by n * i i

+ anti-PD1 e e 100 100 * * g g * 100 n flow cytometry. Middle: A library of amino acid substitutions at putative IL12-receptor-interface positions (in red) was (3 mpk) n 0 0 a a h h C created. Right: The library was screened for reductions of in vitro potency by pSTAT4 (each curve is an IL12 variant). C 0 7 14 21 28 0 7 14 21 28 1000 10 Days Post Dose 1 Days Post Dose 1

Day 14 1 *p < .0001 vs anti-PD1 In vitro pSTAT4 and MLR activity of lead IL12-Fc variants *p < .0001 vs. PBS Figure 5. Human IL12 is inactive in mice, so in vivo anti-tumor activity was assessed by engrafting pp65-MCF-7 cancer cells into human PBMC engrafted NSG-DKO 500 0.1 recombinant IL12 mice. Mice were dosed weekly (IP) with the indicated dose of test article. Tumor volume, lymphocyte activation/proliferation, and serum IFNγ production were PD-1 MFI @ Day 14 recombinant IL12 (ng/mL) Concentration 3000 10000 IL12-Fc (WT) IL12-Fc (WT) measured over time. Left and middle panels: Potency-reduced IL12-Fc-v1 demonstrated significant anti-tumor activity as a single-agent at 0.1 mg/kg and stronger ) 0 0.01 + IL12-Fc-v2 PBS IL12-Fc-v1 IL12-Fc-v2 PBS Anti-PD1 IL12-Fc-v1 IL12-Fc-v1 IL12-Fc-v2 IL12-Fc-v1 activity when combined with anti-PD1. Potency-reduced IL12-Fc-v2 demonstrated significant anti-tumor activity at a 10x higher dose level of 1 mg/kg. Right panel: 8000 (0.1 mpk) (1 mpk) (3 mpk) (0.1 mpk) (0.1 mpk) (1 mpk) IL12-Fc-v2 Treatment with potency-reduced IL12-Fc results in activation and proliferation of CD8+ T cells, increased PD1 expression, and >200-fold increases in serum IFNγ. + anti-PD1 CD25

- 2000 (3 mpk) 6000 (pg/mL)

γ 4000

CD45RA 1000 pSTAT4 MFI + IFN Potency-reduced IL12-Fc have antibody-like PK in mice 2000 Summary (CD8 100 . IL12 heterodimeric Fc-fusions were engineered with a potency-reduced IL12 in order to improve 0 0 IL12-Fc-v1 Figure 6. The pharmacokinetics (PK) of potency-reduced IL12- 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 . 1 0 0 0 0 0 0 0 . 1 0 0 tolerability, slow receptor-mediated clearance, and prolong half-life in vivo compared to therapeutics . 0 . 0 g/mL) 0 0 .0 0 1 0 0 0 0 .0 0 1 0 Fc-v1 were evaluated in C57BL6/J mice. N = 4 mice were 0 0 1 0 0 .0 0 1 µ 0 . 0 0 .0 0 . 0 .0 0 10 0 0 injected IV with 2 mg/kg IL12-Fc-v1 on Day 0 and drug using native IL12 IL12/IL12-Fc (µg/mL) IL12/IL12-Fc (µg/mL) concentration in serum was measured over time. The estimated . Potency-reduced IL12-Fc demonstrate significant anti-tumor activity concurrent with activation and 1 half-life (t ) is approximately 10 days and similar to that of 1/2 + Figure 4. In vitro activity of rIL12, WT IL12-Fc, and lead potency-reduced IL12-Fc were assessed on activated human PBMCs monoclonal antibodies, indicating that IL12-Fc-v1 has a long proliferation of CD8 T cells, increased PD1 expression, and increased serum IFNγ in mice. by measuring intracellular pSTAT4 by flow cytometry (left) and IFNγ production in a mixed-lymphocyte reaction (MLR) (right). Concentration ( half-life and favorable stability in the absence of TMDD. 0.1 . These results support further testing of potency-reduced IL12-Fc as a potential novel cytokine 0 7 14 21 Study Day therapy in cancer patients. *Contact: [email protected] © 2020 Xencor, Inc., Monrovia, CA 91016 USA