NL-201, a de novo alpha-independent combined IL-2 and IL-15 agonist, enhances the anti-tumor activity of established cancer immunotherapies

NL-201 enhances the activity of immune checkpoint inhibitors in multiple tumor models

NL-201 enhances the activity of ⍺PD-1+⍺CTLA-4 in a melanoma model• Mice bearing B16F10 tumors were treated with ⍺PD-1 + ⍺CTLA-4 (CPI:

10mg/kg; BIWx6 each), NL-201 (275ug/kg; QWx2), or CPI + NL-201. • Tumor volume was measured on Day 12 - the final day in which all mice

remained on study. Tumor growth inhibition by the combination of NL-201 + CPI was significantly (p<0.0001) improved relative to NL-201 or CPI alone.

NL-201 sensitizes tumors to ⍺PD-1 and ⍺PD-L1 therapy• Mice bearing MC38 tumors were treated with vehicle, ⍺PD-1 (10mg/kg;

BIWx6), ⍺PD-L1 (10mg/kg; BIWx6), NL-201 (60µg/kg; QWx2), NL-201 + ⍺PD-1, or NL-201 + ⍺PD-L1.

• Median survival (MS) for mice treated with vehicle was 22 days. MS was not increased with αPD-1 treatment but was increased by 3 days with αPD-L1 treatment. MS was increased by 7 days with low-dose NL-201 treatment, while MS was increased by 17 and 21 days for the combination of NL-201 + αPD-1 and NL-201 + αPD-L1 (resp), an improvement of 10 and 14 days (resp) over NL-201 alone. The increase in MS for the combination of NL-201 and αPD-1 or αPD-L1 is synergistic when compared to each treatment alone.

Carl Walkey, Ryan Swanson, Laurie Tatalick, Kevin Yu, Umut Ulge and Jonathan Drachman; Neoleukin Therapeutics, Inc. Seattle, WA.

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Summary and Future Directions

• Exposure and dose schedule impact the anti-tumor activity of NL-201: PEGylation improves the anti-tumor activity of NL-201 in syngeneic tumor models. Dose schedule may also play an important role in determining anti-tumor activity.

• NL-201 enhances the activity of monoclonal antibodies and checkpoint inhibitors: NL-201 potently stimulates CD8+ T and NK cell proliferation and upregulates PD-1 expression on CD8+ T cells. Preclinical experiments demonstrate enhanced anti-tumor activity when NL-201 is combined with monoclonal antibodies or immune checkpoint inhibitors.

• Evaluation of additional combinations is underway: NL-201 has previously been demonstrated to enhance the anti-tumor activity of CAR-T cells in vivo. Studies evaluating combinations with other established and exploratory cancer therapies are underway. These studies may guide future clinical development.

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IL-2 toxicity is mediated by preferential stimulation of cells expressing CD251

β γ

CD8+ T & NK cells

α β γ

Treg & endothelial

cells

Toxicity &Immunosuppression

IL-2Anti-tumor

Activity

• Recombinant IL-2 (aldesleukin) is an approved cancer immunotherapy that leads to 5-8% durable remission; however, severe toxicity has limited its widespread use.

• IL-2 toxicity is mediated by preferential binding to cells expressing the alpha subunit of the IL-2 receptor (CD25), including endothelial cells and eosinophils. In addition, stimulation of CD25-expressing regulatory T cells (Treg) by IL-2 can be immunosuppressive.

Abstract # 576

• Because NL-201 potently stimulates NK cell proliferation in vitroand in vivo, NL-201 may enhance the antibody-dependent cellular cytotoxicity of tumor targeting antibodies.

• Mice bearing subcutaneous B16F10 tumors were treated with tumor-targeting antibody TA99 (10mg/kg; BIWx6), NL-201 (60µg/kg, 150µg/kg or 375µg/kg; QWx2), or TA99 + NL-201.

• NL-201 + TA99 significantly improved tumor growth inhibition compared to TA99 or NL-201 alone, demonstrating the potential of NL-201 to enhance the activity of a tumor-targeting antibody.

NL-201 enhances the anti-tumor activity of a tumor-targeting antibody 7

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NL-201 treatment upregulates PD-1 expression by CD8+ T cells

• To evaluate the effect of NL-201 stimulation on the expression of the immune checkpoint receptor, PD-1, by lymphocytes, PBMCs from 10 human donors were treated with NL-201 (0-30 ng/mL) before being washed, stained, and analyzed by flow cytometry.

• NL-201 stimulation leads to a concentration-dependent increase in PD-1 expression by CD8+ T cells, consistent with induced proliferation.

• These results suggest that combining NL-201 with a PD-1 inhibitor may overcome immune checkpoint-mediated CD8+ T cell inhibition.

NL-201 vs vehicle: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001

6NL-201 exposure was evaluated in non-human primates

• The pharmacokinetics of NL-201 were evaluated innon-human primates after intravenous administration.

• Plasma NL-201 concentrations remained relatively stable for ~8 hours at all dose levels.

• Beyond 8 hours, non-linear elimination kinetics were observed. This may result from target-mediated disposition driven by the high affinity of NL-201 for the IL-2 receptor.

The potency of lymphocyte stimulation by NL-201 depends on stimulation time

• To understand the impact of stimulation time on lymphocyte proliferation, peripheral blood mononuclear cells (PBMCs) from 3 human donors were exposed to NL-201 for 2, 8, or 24 hours (h) across a wide range of concentrations. Cells were washed and incubated in fresh cell culture media before being stained and evaluated by flow cytometry.

• Increasing incubation time from 2 to 8h led to more potent CD8+ T cell and NK cell stimulation. Between 8 and 24h, incubation time had minimal impact on NL-201 potency.

• These results suggest that NL-201 exposure of ~8h is consistent with potent biological effect on human immune cells.

• Since stimulated lymphocytes migrate to lymphoid tissues by ~12h, continued cytokine exposure beyond 24h may not contribute to greater antitumor activity.

NL-201 exposure is commensurate with potent lymphocyte stimulation5

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• NL-201 was developed from Neo-2/15 (Silva et al. Nature, 2019) by introducing a cysteine residue that is stably conjugated to a 40kDa maleimide-modified PEG to extend blood half-life.

• The tolerability and anti-tumor activity of NL-201 (300 µg/kg, QWx2) was compared to unPEGylated Neo-2/15 (312.5 µg/kg, QDx14) in mice bearing syngeneic CT-26 tumors. Treatment began on Day 10.

• Peak bodyweight loss, a measure of tolerability, was 5.8% for NL-201 vs. 11.9% for Neo-2/15 (not significantly different), demonstrating that weekly NL-201 is at least as well tolerated as daily Neo-2/15. Note that peak bodyweight loss for NL-201 and Neo-2/15 occurred on Days 14 and 24 (respectively).

• NL-201 inhibited tumor growth more effectively than Neo-2/15 (67.2% vs 29.5%) and significantly improved survival.

• These results indicate that NL-201 has improved anti-tumor activity without adversely impacting tolerability as compared to its parent molecule, Neo-2/15.

Extended half-life improves the anti-tumor activity of NL-2013

NL-201 was designed to overcome thelimitations of IL-2 immunotherapy

• NL-201 is more potent and selective than IL-2:NL-201 is ~3-11x more potent than IL-2 on cells lacking CD25 (i.e. CD8+ T cells and NK cells) and ~31x less potent than IL-2 on Tregs.1

• NL-201 demonstrates potential as monotherapy: NL-201 displays robust single-agent activity at tolerated doses in multiple tumor models, including those resistant to checkpoint inhibitors.1

• NL-201 shows minimal immunogenicity in NHPs: ADAs in NHPs after repeated doses of NL-201 were infrequent and did not adversely impact pharmacology or tolerability.1

1: Walkey et al., Cancer Res. 2020

β γ

2• NL-201 is a de novo protein designed to dimerize the beta

and gamma chains of the IL-2 and IL-15 receptors. NL-201 has no binding interface for CD25.

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Treatment vs. vehicle: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001

• Mice bearing subcutaneous CT-26 tumors were treated withNL-201 (500 µg/kg) on the following schedules: QWx2, QWx4, Q2Wx2, or Q3Wx2.

• Weekly NL-201 administration improved tumor growth inhibition compared to less frequent schedules.

• Additional weekly NL-201 administrations beyond 2 weeks did not further enhance anti-tumor activity.

Dose schedule impacts the anti-tumor activity of NL-2014

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VehicleαPD-1αPD-L1NL-201NL-201 + αPD-1NL-201 + αPD-L1

*****

Treatment vs. vehicle: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001

Treatment vs. NL-201: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001