KY1043, a novel PD-L1 IL-2 immunocytokine directed towards ...
1
In vitro generation of tumour-reactive T cells and analysis of tumour cell killing Targeting of immunocytokine to PD-L1 increases efficacy KY1043 non-targeted immunocytokine Start of culture CD4 + and CD8 + T cells, and A375 tumour cells were co-cultured for 5 days in the presence of KY1043 or a non- targeted immunocytokine with equivalent IL-2 activity. Images were captured using the Incucyte S3™ system. Live tumour cells are indicated by red nuclei. Caspase 3/7-mediated cell death is indicated by green fluorescence. Increased tumour cell death was observed with KY1043, compared with the non-targeted immunocytokine. (a) (b) CD4 + and CD8 + T cells from seven different donors were co-cultured with tumour cells (1:1:1 ratio) for 4 days in the presence of an unmodified anti-PD-L1 antibody, non-targeted immunocytokine and KY1043 at 10 μg/ml (a) and images collected using the Incucyte S3™ system. Live red cell counts over time were used to calculate the area under curve (AUC). Reduction in AUC was calculated by comparing responses to test molecules with the response to an isotype control. In (b), KY1043 was compared to the combination of anti-PD-L1 antibody and equimolar concentration of IL-2 in six donors. * non-parametric one-way ANOVA and Dunn’s multiple comparison test KY1043 in vitro Introduction • IL-2 therapy has been approved for use in certain cancers since 1989. • The major limitation with this therapy is tolerability, with many patients experiencing grade 3 or higher adverse events. • Approaches to improve this have focused on engineering molecules with selectivity towards the dimeric IL-2 receptor (CD122 and CD132, IL-2Rβ chain and common γ chain, respectively). • This form of the receptor complex is expressed on peripheral naïve T cells as well as memory T cells and NK cells, potentially leading to unproductive stimulation of irrelevant cell populations and an increased possibility of systemic toxicity. • KY1043 is a highly differentiated immunocytokine consisting of a neutralising anti-PD-L1 antibody, fused via its light chains to an attenuated IL-2, in which the balance of signalling has been adjusted to favour the trimeric form of the IL-2R, which contains CD25 (IL-2Rα). • We hypothesise that reduced binding to circulating cells will improve targeting to PD-L1 + cells in the tumour and/or lymphoid tissues while also targeting IL-2 to antigen-experienced T cells that express CD25 in the tumour microenvironment. Activity of KY1043 on the dimeric IL-2R (βγ) is highly attenuated (a) (b) In a proliferation assay, the EC 50 of KY1043 was similar to that of rhIL-2 on IL-2R αβγ expressing TF1 cells (a), but approximately 300-fold lower on bg expressing cells (b). Blockade of CD25 reduces KY1043 activity to that seen on the TF1 bg cells, demonstrating the importance of CD25 binding for KY1043 function KY1043 neutralises PD-L1/PD-1 and PD-L1/CD80 interactions similarly to the parental antibody (a) (b) The IC 50 of KY1043 and an unmodified PD-L1 antibody were comparable for the neutralisation of PD-L1/PD-1 (a) and PD-L1/CD80 interactions (b), in an ELISA-based neutralisation assay. This demonstrates that fusion of IL-2 to the light chains of an antibody does not affect ability of the antibody to bind and neutralise PD-L1. #P625 KY1043, a novel PD-L1 IL-2 immunocytokine directed towards CD25, delivers potent anti-tumour activity in vitro and in vivo Cassandra H Van Krinks 1 , Timothy Malcolm 1 , Morgane Lecointre 1 , Richard Brown 1 , Hanif Ali 1 , Hannah Craig 1 , Dirk Zahn 1 , Siobhan O’Leary 1 , Lucy Hepburn 1 , Rachael Kimber 1 , Nikole Sandy 1 , Alla Fane-Dremucheva 1 , Jamie Campbell 1 , Volker Germaschewski 1 , Stephen D Gillies 2 , Matthew McCourt 1 * 1 Kymab Ltd, The Bennet Building, Babraham Research Campus, Cambridge CB22 3AT, U.K. * email: [email protected]. 2 Provenance Biopharmaceuticals, Carlisle, MA KY1043 induces a potent, dose dependent anti-tumour response in vivo KY1043 generates a tumour specific memory response KY1043 induces strong CD8 + T cell proliferation in the tumour microenvironment KY1043 leads to a large expansion of T reg in the peripheral lymphoid tissue • Human PD-L1 KI mice were implanted with human PD-L1 KI MC38 cells • Mice were treated with KY1043 from day 8, receiving two further doses on day 10 and 13 • At 1 mg/kg of KY1043, 3 of 8 animals rejected the tumour challenge • At 3 mg/kg, 6 of 8 animals survived • All mice survived at 10 mg/kg • No significant weight loss was observed • Mice that survived a tumour challenge with MC38 cells following KY1043 treatment were re-challenged at day 56 with the same tumour or B16 F10 melanoma cells • MC38 tumours were rejected without further treatment, while the melanoma tumours were not, suggesting that KY1043 generates a tumour specific memory response hPD-L1 MC38 tumour samples were analysed by flow cytometry following a single dose of KY1043 at 10 mg/kg. KY1043 induces CD8 + T cell proliferation (as shown by Ki67) that peaks at 72 hours, resulting in an expansion of CD8 + T cells in the TME, and an increase of the CD8 + T cell:T reg ratio. Tumour draining lymph nodes were analysed by flow cytometry following a single dose of KY1043 at 10 mg/kg. KY1043 induces a spike in CD8 + T cell proliferation at 72 hours, but an even greater expansion of CD4 + CD25 + FoxP3 + T reg . This results in the decrease of the CD8 + T cell:T reg ratio. A similar effect was noted in the spleen. Acknowledgments We gratefully acknowledge the Animal Sciences and In Vivo Core teams for husbandry and assistance with studies, and the Expression and Purification teams for generation of immunocytokines Reference – Stewart et. al. Cancer Immunol Res. 2015 Sep;3(9):1052-62 End of culture • IL-2 fused directly to light chain - constrains cytokine to reduce activity • N-terminal truncation of IL-2 further reduces binding to IL-2R, in particular the bg dimer ➢ “dial-down” of IL-2 activity to improve tolerability • Effector disabled (FcRn binding intact) KY1043 in vivo • KY1043 induces potent T cell activation and can direct highly effective tumour killing in vitro and in vivo. • Maximal tumour cell killing is achieved by targeting PD-L1 in vitro compared to untargeted immunocytokine • Peripheral expansion of T reg is not detrimental to tumour control in vivo • These results challenge the dogma that selective binding to dimeric (CD122/CD132) IL-2R is required for tumour control • KY1043 - a CD25 (IL-2Rα) directed immunocytokine may provide an advantageous therapeutic-index for the treatment of cancer • KY1043 is currently in development at Kymab alin Das uou olu Das uou olu DL Conclusion ous ous das D lls of D alin aDL D lls in uou ous ous das D ll in uou alin aDL D ll aio in uou ous ous das D ll in dL alin aDL D ll aio in l nod ous ous das o D s of D alin aDL s in l nod ous ous das D i lls of D alin aDL D i lls in l nod ous ous das D i lls of D alin aDL D i lls in uou https://www.kymab.com/media/uploads/files/SITC_2019.pdf * (p=0.0406)
Transcript of KY1043, a novel PD-L1 IL-2 immunocytokine directed towards ...
In vitro generation of tumour-reactive T cells and analysis of tumour cell killing
Targeting of immunocytokine to PD-L1 increases efficacy
KY1043 non-targeted immunocytokine
Start of
culture
CD4+ and CD8+ T cells, and A375 tumour cells were co-cultured for 5 days in the presence of KY1043 or a non-
targeted immunocytokine with equivalent IL-2 activity. Images were captured using the Incucyte S3™ system.
Live tumour cells are indicated by red nuclei. Caspase 3/7-mediated cell death is indicated by green fluorescence.
Increased tumour cell death was observed with KY1043, compared with the non-targeted immunocytokine.
(a) (b)
CD4+ and CD8+ T cells from seven different donors were co-cultured with tumour cells (1:1:1 ratio) for 4 days in the
presence of an unmodified anti-PD-L1 antibody, non-targeted immunocytokine and KY1043 at 10 µg/ml (a) and
images collected using the Incucyte S3™ system. Live red cell counts over time were used to calculate the area
under curve (AUC). Reduction in AUC was calculated by comparing responses to test molecules with the
response to an isotype control. In (b), KY1043 was compared to the combination of anti-PD-L1 antibody and
equimolar concentration of IL-2 in six donors. * non-parametric one-way ANOVA and Dunn’s multiple comparison test
KY1043 in vitroIntroduction
• IL-2 therapy has been approved for use in certain cancers since 1989.
• The major limitation with this therapy is tolerability, with many patients experiencing grade 3 or higher
adverse events.
• Approaches to improve this have focused on engineering molecules with selectivity towards the dimeric IL-2receptor (CD122 and CD132, IL-2Rβ chain and common γ chain, respectively).
• This form of the receptor complex is expressed on peripheral naïve T cells as well as memory T cells and
NK cells, potentially leading to unproductive stimulation of irrelevant cell populations and an increased
possibility of systemic toxicity.
• KY1043 is a highly differentiated immunocytokine consisting of a neutralising anti-PD-L1 antibody, fused via
its light chains to an attenuated IL-2, in which the balance of signalling has been adjusted to favour the
trimeric form of the IL-2R, which contains CD25 (IL-2Rα).
• We hypothesise that reduced binding to circulating cells will improve targeting to PD-L1+ cells in the tumour
and/or lymphoid tissues while also targeting IL-2 to antigen-experienced T cells that express CD25 in the
tumour microenvironment.
Activity of KY1043 on the dimeric IL-2R (βγ) is highly attenuated
(a) (b)
In a proliferation assay, the EC50 of KY1043 was similar to that of rhIL-2 on IL-2R αβγ expressing TF1 cells (a),
but approximately 300-fold lower on bg expressing cells (b). Blockade of CD25 reduces KY1043 activity to that
seen on the TF1 bg cells, demonstrating the importance of CD25 binding for KY1043 function
KY1043 neutralises PD-L1/PD-1 and PD-L1/CD80 interactions similarly to the parental antibody
(a) (b)
The IC50 of KY1043 and an unmodified PD-L1 antibody were comparable for the neutralisation of PD-L1/PD-1
(a) and PD-L1/CD80 interactions (b), in an ELISA-based neutralisation assay. This demonstrates that fusion of
IL-2 to the light chains of an antibody does not affect ability of the antibody to bind and neutralise PD-L1.
#P625
KY1043, a novel PD-L1 IL-2 immunocytokine directed towards CD25, delivers potent anti-tumour activity in vitro and in vivoCassandra H Van Krinks1, Timothy Malcolm1, Morgane Lecointre1, Richard Brown1, Hanif Ali1, Hannah Craig1, Dirk Zahn1, Siobhan O’Leary1, Lucy Hepburn1, Rachael Kimber1, Nikole Sandy1, Alla Fane-Dremucheva1, Jamie Campbell1, Volker Germaschewski1, Stephen D Gillies2, Matthew McCourt1*1 Kymab Ltd, The Bennet Building, Babraham Research Campus, Cambridge CB22 3AT, U.K. * email: [email protected]. 2 Provenance Biopharmaceuticals, Carlisle, MA
KY1043 induces a potent, dose dependent anti-tumour response in vivo
KY1043 generates a tumour specific memory response
KY1043 induces strong CD8+ T cell proliferation in the tumour microenvironment
KY1043 leads to a large expansion of Treg in the peripheral lymphoid tissue
• Human PD-L1 KI mice were implanted with
human PD-L1 KI MC38 cells
• Mice were treated with KY1043 from day 8,
receiving two further doses on day 10 and
13
• At 1 mg/kg of KY1043, 3 of 8 animals
rejected the tumour challenge
• At 3 mg/kg, 6 of 8 animals survived
• All mice survived at 10 mg/kg
• No significant weight loss was observed
• Mice that survived a tumour challenge with
MC38 cells following KY1043 treatment were
re-challenged at day 56 with the same tumour
or B16 F10 melanoma cells
• MC38 tumours were rejected without further
treatment, while the melanoma tumours were
not, suggesting that KY1043 generates a
tumour specific memory response
hPD-L1 MC38 tumour samples were analysed by flow cytometry following a single dose of KY1043 at 10 mg/kg. KY1043 induces CD8+ T cell
proliferation (as shown by Ki67) that peaks at 72 hours, resulting in an expansion of CD8+ T cells in the TME, and an increase of the CD8+ T
cell:Treg ratio.
Tumour draining lymph nodes were analysed by flow cytometry following a single dose of KY1043 at 10 mg/kg. KY1043 induces a spike in
CD8+ T cell proliferation at 72 hours, but an even greater expansion of CD4+ CD25+ FoxP3+ Treg. This results in the decrease of the CD8+ T
cell:Treg ratio. A similar effect was noted in the spleen.
Acknowledgments We gratefully acknowledge the Animal Sciences and In Vivo Core teams for husbandry and assistance with studies, and the Expression and Purification teams for generation of immunocytokines
Reference – Stewart et. al. Cancer Immunol Res. 2015 Sep;3(9):1052-62
End of
culture
• IL-2 fused directly to light chain - constrains cytokine to reduce
activity
• N-terminal truncation of IL-2 further reduces binding to IL-2R,
in particular the bg dimer
➢ “dial-down” of IL-2 activity to improve tolerability
• Effector disabled (FcRn binding intact)
KY1043 in vivo
• KY1043 induces potent T cell activation and can direct highly effective
tumour killing in vitro and in vivo.
• Maximal tumour cell killing is achieved by targeting PD-L1 in vitro
compared to untargeted immunocytokine
• Peripheral expansion of Treg is not detrimental to tumour control in vivo
• These results challenge the dogma that selective binding to dimeric
(CD122/CD132) IL-2R is required for tumour control
• KY1043 - a CD25 (IL-2Rα) directed immunocytokine may provide an
advantageous therapeutic-index for the treatment of cancer
• KY1043 is currently in development at Kymab
alin
Da s
u ou olu
Da s
u ou o
lu
D L
Conclusion
ou s ou s da s
D lls of D
alin a D L
D lls in u ou
ou s ou s da s
D ll in u ou
alin a D L
D ll a io in u ou
ou s ou s da s
D ll in dL
alin a D L
D ll a io in l nod
ou s ou s da s
o
D s
of D
alin a D L
s in l nod
ou s ou s da s
D i lls of
D
alin a D L
D i lls in l nod
ou s ou s da s
D i lls of D
alin a D L
D i lls in u ou
https://www.kymab.com/media/uploads/files/SITC_2019.pdf
* (p=0.0406)
