BACKGROUND 1. Gillies SD, Reilly EB, Lo KM, Reisfeld RA. Antibody-targeted interleukin 2 stimulates T-cell killing of autologous tumor cells. PNAS. 1992,89;1428-32. 2. Morris ZS, Guy EI, Francis DM, Gressett MM, Werner LR, Carmicheal LL, Yang RK, Armstrong EA, Huang S, Navid F, Gillies SD, Korman A, Hank JA, Rakhmilevich AL, Harari PM, Sondel PM. In situ tumor vaccination by combining local radiation and tumor-specific antibody or immunocytokine treatments. Cancer Res. 2016,76;3929-41. 3. Charych DH, Hoch U, Langowski JL, Lee SR, Addepalli MK, Kirk PB, Sheng D, Liu X, Sims PW, VanderVeen LA, Ali CF, Chang TK, Konakova M, Pena RL,Kanhere RS, Kirksey YM, Ji C, Wang Y, Huang J, Sweeney TD, Kantak SS, Doberstein SK. NKTR-214, an engineered cytokine with biased IL2 receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models. Clinc Canc Res. 2016,22;680-90. 4. Bentebibel SE, Bernatchez C, Haymaker C, Hurwitz M, Hwu P, Sznol M, Tannir N, Conley A, Kluger H, Aung S, Imperiale M, Tagliaferri M, Fanton C, Iaucci E, Zalevsky J, Hoch U, Diab A. The novel IL-2 cytokine immune agonist NKTR-214 harnesses the adaptive and innate immune system for the treatment of solid cancers. SITC. 2017, P77 RT alone and NKTR-214 alone slow tumor growth but do not, on average, result in tumor regression and disease-free animals IV NKTR-214 in combination with 12 Gy RT results in a stronger anti- tumor response than IV or IT IL2 in combination with 12 Gy RT The immunologic memory achieved with RT + IV NKTR-214 is more robust than the immunologic memory achieved with RT + IT IL-2 We hope to show, via flow cytometry, that an increase in CD8:T reg ratio is seen at the tumor when NKTR-214 is used with RT Immune depletion studies are currently ongoing Further evaluation of RT + NKTR-214 + K322A (a tumor specific antibody) is required before a conclusion can be drawn about the ability of a tumor specific mAb to synergize with RT + NKTR-214 RESULTS CONCLUSIONS AND FUTURE DIRECTIONS REFERENCES AND ACKNOWLEDGEMENTS NKTR-214 in combination with radiation therapy produces a potent in situ vaccine for B78 melanoma. Alexander Pieper 1 , Alexander Rakhmilevich 1 , Jacob Slowinski 1 , Amy K. Erbe 1 , Jacquelyn Hank 1 , Zachary Morris 1 , Deborah Charych 2 , Paul M. Sondel 1 1 University of WI-Madison, WI; 2 Nektar Therapeautics San Francisco, CA; RESULTS (CONTINUED) METHODS molecules resulting in a more diverse T-cell receptor repertoire. RT alone rarely generates an effective in situ vaccination due, in part, to poor persistence of activated tumor-specific lymphocytes. We have found that by combining RT with intratumoral (IT) injections of tumor-targeted immunotherapy we can create an enhanced immune response capable of eradicating tumors. Preclinical data have shown that one dose of local RT combined with five injections of intratumoral IC (IT IC), starting five days after RT, can work synergistically to cause tumor regression and disease-free animals in a single syngeneic melanoma tumor model. Not only are most mice receiving RT+IT IC cured of their tumor burden, but they also exhibit immunologic memory that is demonstrated by their ability to reject a second challenge with melanoma cells. This immunologic memory demonstrates we can create an in situ vaccine using RT+IT IC 2 . NKTR-214 circumvents the limitations of IL-2: Using IL2 in a treatment regimen does come with consequences, however. It has a short half-life in vivo and patients must be infused continuously with the cytokine in order to achieve a therapeutic dose. IL2 can activate anti-tumor immune cells (i.e. NK cells, CD4+ T cells, CD8+ T cells). Immune inhibitory T regulatory (T reg ) cells, which are present in the tumor microenviroment and used by tumors to evade an immune response, are also activated by IL2. Finally, IL2 has dose limiting toxicity that can be fatal. Nektar Therapeutics has circumvented these IL2 limitations with NKTR-214 by attaching 6 polyethylene glycol (PEG) molecules to the IL2 molecule. The location of the PEG molecules results in receptor bias against the IL2R portion of the IL2 receptor and towards the IL2R portion of the receptor 3 . This bias allows NKTR-214 to better activate anti-tumor immune cells like NK cells, CD4+ T cells, and CD8+ T cells, while not activating immune inhibitory T reg cells. This leads to increased CD8/Treg ratios in human and murine tumors. Furthermore, the PEGylation makes NKTR-214 a prodrug that becomes more active as PEG molecules fall off in vivo, which increases the half-life and maximum tolerated dose of the drug. Cell Culture B78 cells were grown in culture with RPMI-1640 media supplemented with 10% FBS, 2mM glutamax, and 1% Pen/Strep. Cells were grown at 37°C in an environment of 5% CO 2 . Tumor Inoculation, Drug Treatment, Tumor Monitoring 7-8 week old female C57BL/6 mice were injected subcutaneously (s.c.) with 2 million B78 melanoma cells in 0.1mL PBS on the R flank. Once average tumor volumes reached 100-150mm 3 (4-5 wks), mice were randomized and treated with 12 Gy external beam RT on day 0. Lead shields were used to protect the spleen and other vital organs, such that only the tumors were dosed with RT. Depending upon their cohort, mice were dosed with intravenous or intratumoral IL2 (150,000 U per dose) on days 5-9 or intravenous NKTR-214 (0.8 mg/kg per dose, corresponding to 256,000 untis of IL2/dose) on days 5, 13, 22. Tumors were measured with calipers biweekly, and tumor volumes were calculated using the following formula: V=A 2 x (0.5)B with A and B being the short and long dimensions of the tumor respectively. All mice with complete response (CR) were rechallenged with a second B78 tumor. 2 million B78 cells in 0.1mL PBS were injected s.c. on the L abdomen. Statistical Analysis Average tumor growth curves were statistically analyzed using the linear mixed-effects model. Chi-squared analysis was done to compare complete response rates and B78 rechallenge rejection rates across different groups. Log-rank test for survival curve analysis. In all the above figures, p<0.05 : *, p<0.01 : **, p <0.001 : *** C57BL/6 mice Tumor Implantation 2 million B78 Cells Euthanize 20 mm Day 0 12 Gy RT Day 5 6 7 8 9 I.V/I.T. IL2 150,000 U I.V. NKTR 0.8 mg/kg Day 14 Day 23 IV NKTR-214 is Synergistic with Radiation to Cause Significant B78 Melanoma Tumor Regression and Disease Free Mice IV NKTR-214 in Combination with Radiation Causes Greater B78 Melanoma Tumor Regression than IT IL2 with Radiation IV NKTR-214 in Combination with Radiation Results in Prolonged Survival Compared to IT IL2 in Combination with Radiation Purpose : We wanted to investigate the prospect of using NKTR-214 (an engineered IL2 agonist) as a replacement for IL2 in our current in situ vaccine treatment regimen. IV NKTR-214 in Combination with Radiation Cures More Mice and Results in a Stronger Memory than “Naked” IL2 with Radiation Thank you to the UW MSTP for their support and guidance throughout these years in their program. Also, thank you to the members of the Sondel Lab who have provided excellent mentorship. And lastly, thank you to the UW Cellular and Molecular Biology Department for their travel fund award. IV NKTR-214 in Combination with Radiation Causes Greater B78 Melanoma Tumor Regression than IV IL2 with Radiation A. B. C. D. E. Targeted immunotherapies, in combination with radiation therapy, are effective anti-cancer immunotherapy regimens: We have helped develop the use of a tumor-specific immunocytokine (IC) to treat different forms of cancer. An IC is a monoclonal antibody (mAb) specific for a tumor surface antigen with two IL2 molecules attached to the constant region of the antibody. This combination leads to a tumor targeted innate immune Tumor Cell IL-2 IL-2 Receptor T Cell or NK Cell GD2 Antigen hu14.18-IL2 Hu14.18-IL2 a genetically engineered fusion protein linking IL2 to hu14.18 mAb S. Gillies and R. Reisfeld PNAS 89:1428, 1992 Melanoma or Neuroblastoma response and concentration of IL2 at the tumor microenvironment 1 . Radiation therapy (RT) is a tumor-targeted treatment that can be used to enhance immunotherapy, through its ability to increase the release of local pro-inflammatory cytokines, cause immunogenic cell death, and increase neo-antigen exposure on MHC-I
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BACKGROUND
1. Gillies SD, Reilly EB, Lo KM, Reisfeld RA. Antibody-targeted interleukin 2 stimulates T-cell killing ofautologous tumor cells. PNAS. 1992,89;1428-32.
2. Morris ZS, Guy EI, Francis DM, Gressett MM, Werner LR, Carmicheal LL, Yang RK, Armstrong EA,Huang S, Navid F, Gillies SD, Korman A, Hank JA, Rakhmilevich AL, Harari PM, Sondel PM. In situtumor vaccination by combining local radiation and tumor-specific antibody or immunocytokinetreatments. Cancer Res. 2016,76;3929-41.
3. Charych DH, Hoch U, Langowski JL, Lee SR, Addepalli MK, Kirk PB, Sheng D, Liu X, Sims PW,VanderVeen LA, Ali CF, Chang TK, Konakova M, Pena RL, Kanhere RS, Kirksey YM, Ji C, Wang Y, HuangJ, Sweeney TD, Kantak SS, Doberstein SK. NKTR-214, an engineered cytokine with biased IL2receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models. ClincCanc Res. 2016,22;680-90.
4. Bentebibel SE, Bernatchez C, Haymaker C, Hurwitz M, Hwu P, Sznol M, Tannir N, Conley A, Kluger H,Aung S, Imperiale M, Tagliaferri M, Fanton C, Iaucci E, Zalevsky J, Hoch U, Diab A. The novel IL-2cytokine immune agonist NKTR-214 harnesses the adaptive and innate immune system for thetreatment of solid cancers. SITC. 2017, P77
RT alone and NKTR-214 alone slow tumor growth but do not, on average,result in tumor regression and disease-free animalsIV NKTR-214 in combination with 12 Gy RT results in a stronger anti-tumor response than IV or IT IL2 in combination with 12 Gy RTThe immunologic memory achieved with RT + IV NKTR-214 is morerobust than the immunologic memory achieved with RT + IT IL-2We hope to show, via flow cytometry, that an increase in CD8:Treg ratio isseen at the tumor when NKTR-214 is used with RTImmune depletion studies are currently ongoingFurther evaluation of RT + NKTR-214 + K322A (a tumor specific antibody)is required before a conclusion can be drawn about the ability of a tumorspecific mAb to synergize with RT + NKTR-214
RESULTS
CONCLUSIONS AND FUTURE DIRECTIONS
REFERENCES AND ACKNOWLEDGEMENTS
NKTR-214 in combination with radiation therapy produces a potent in situ vaccine for B78 melanoma.
Alexander Pieper1, Alexander Rakhmilevich1, Jacob Slowinski1, Amy K. Erbe1, Jacquelyn Hank1, Zachary Morris1, Deborah Charych2, Paul M. Sondel11University of WI-Madison, WI; 2Nektar Therapeautics San Francisco, CA;
RESULTS (CONTINUED)
METHODS
molecules resulting in a more diverse T-cell receptor repertoire. RT alonerarely generates an effective in situ vaccination due, in part, to poorpersistence of activated tumor-specific lymphocytes. We have found that bycombining RT with intratumoral (IT) injections of tumor-targetedimmunotherapy we can create an enhanced immune response capable oferadicating tumors. Preclinical data have shown that one dose of local RTcombined with five injections of intratumoral IC (IT IC), starting five days afterRT, can work synergistically to cause tumor regression and disease-freeanimals in a single syngeneic melanoma tumor model. Not only are mostmice receiving RT+IT IC cured of their tumor burden, but they also exhibitimmunologic memory that is demonstrated by their ability to reject a secondchallenge with melanoma cells. This immunologic memory demonstrates wecan create an in situ vaccine using RT+IT IC2.
NKTR-214 circumvents the limitations of IL-2: Using IL2 in a treatmentregimen does come with consequences, however. It has a short half-life invivo and patients must be infused continuously with the cytokine in order toachieve a therapeutic dose. IL2 can activate anti-tumor immune cells (i.e. NKcells, CD4+ T cells, CD8+ T cells). Immune inhibitory T regulatory (Treg) cells,which are present in the tumor microenviroment and used by tumors toevade an immune response, are also activated by IL2. Finally, IL2 has doselimiting toxicity that can be fatal. Nektar Therapeutics has circumvented theseIL2 limitations with NKTR-214 by attaching 6 polyethylene glycol (PEG)molecules to the IL2 molecule. The location of the PEG molecules results inreceptor bias against the IL2R𝛼𝛼 portion of the IL2 receptor and towards theIL2R𝛽𝛽𝛽𝛽 portion of the receptor3. This bias allows NKTR-214 to better activateanti-tumor immune cells like NK cells, CD4+ T cells, and CD8+ T cells, whilenot activating immune inhibitory Treg cells. This leads to increased CD8/Tregratios in human and murine tumors. Furthermore, the PEGylation makesNKTR-214 a prodrug that becomes more active as PEG molecules fall off invivo, which increases the half-life and maximum tolerated dose of the drug. Cell Culture
B78 cells were grown in culture with RPMI-1640 media supplemented with 10% FBS, 2mM glutamax, and 1%Pen/Strep. Cells were grown at 37°C in an environment of 5% CO2.
Tumor Inoculation, Drug Treatment, Tumor Monitoring
7-8 week old female C57BL/6 mice were injected subcutaneously (s.c.) with 2 million B78 melanoma cells in0.1mL PBS on the R flank. Once average tumor volumes reached 100-150mm3 (4-5 wks), mice wererandomized and treated with 12 Gy external beam RT on day 0. Lead shields were used to protect the spleenand other vital organs, such that only the tumors were dosed with RT. Depending upon their cohort, micewere dosed with intravenous or intratumoral IL2 (150,000 U per dose) on days 5-9 or intravenous NKTR-214(0.8 mg/kg per dose, corresponding to 256,000 untis of IL2/dose) on days 5, 13, 22. Tumors were measuredwith calipers biweekly, and tumor volumes were calculated using the following formula: V=A2 x (0.5)B with Aand B being the short and long dimensions of the tumor respectively. All mice with complete response (CR)were rechallenged with a second B78 tumor. 2 million B78 cells in 0.1mL PBS were injected s.c. on the Labdomen.
Statistical Analysis
Average tumor growth curves were statistically analyzed using the linear mixed-effects model. Chi-squaredanalysis was done to compare complete response rates and B78 rechallenge rejection rates across differentgroups. Log-rank test for survival curve analysis. In all the above figures, p
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