Interleukin-12 (IL-12) is a pleiotropic cytokine that has profound effects on many aspects of cell-mediated responses and can enhance antitumor responses in experimental models. IL-12 has been tested clinically, however, side-effects have limited its use. We are developing an attenuated form of IL-12 whose biological activity could be restricted to sites of tumors by taking advantage of overexpressed tumor proteases that can activate the cytokine. We constructed a panel of fusion proteins (FPs) consisting of IL-12 joined to a specific inhibitor connected by a protease cleavage sequence (cs). We first identified a panel of single-chain Fragment variable (scFv) that bind to 3 independent epitopes on IL-12 and then incorporated them into separate IL-12 FPs containing either a matrix metalloproteinase (MMP) cs or a scrambled (scram) control cs. The intact IL-12 FPs showed attenuation in IL-12 activity compared to free IL-12 in 2 separate in vitro functional assays; proliferation of CTLL-2 and interferon-gamma (IFN-γ) induction by spleen cells. Furthermore, the FP containing the MMPcs showed an increase in biological activity of IL-12 in vitro when cleaved by MMP9. This FP strategy could be applied to other immunomodulators and potentially reduce unwanted side-effects observed with systemic delivery thus improving cytokine immunotherapy strategies.
BalunaR, VitettaES. 1997. Vascular leak syndrome: a side effect of immunotherapy. Immunopharmacology, 37(2–3):117–132.
6.
BrahmerJR, TykodiSS, ChowLQ, HwuWJ, TopalianSL, HwuP, DrakeCG, CamachoLH, KauhJ, OdunsiK, PitotHC, HamidO, BhatiaS, MartinsR, EatonK, ChenS, SalayTM, AlaparthyS, GrossoJF, KormanAJ, ParkerSM, AgrawalS, GoldbergSM, PardollDM, GuptaA, WiggintonJM. 2012. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med, 366(26):2455–2465.
7.
BremerC, TungCH, WeisslederR. 2001. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat Med, 7(6):743–748.
8.
CarrenoBM, MagriniV, Becker-HapakM, KaabinejadianS, HundalJ, PettiAA, LyA, LieWR, HildebrandWH, MardisER, LinetteGP. 2015. Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells. Science, 348(6236):803–808.
9.
ChambersAF, MatrisianLM. 1997. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst, 89(17):1260–1270.
10.
ChenEI, KridelSJ, HowardEW, LiW, GodzikA, SmithJW. 2002. A unique substrate recognition profile for matrix metalloproteinase-2. J Biol Chem, 277(6):4485–4491.
11.
ChinnasamyD, YuZ, KerkarSP, ZhangL, MorganRA, RestifoNP, RosenbergSA. 2012. Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice. Clin Cancer Res, 18(6):1672–1683.
12.
CohenJ. 1995. IL-12 deaths: explanation and a puzzle. Science, 270(5238):908.
13.
Dela CruzJS, HuangTH, PenichetML, MorrisonSL. 2004. Antibody-cytokine fusion proteins: innovative weapons in the war against cancer. Clin Exp Med, 4(2):57–64.
14.
DuraiswamyJ, FreemanGJ, CoukosG. 2013. Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer. Cancer Res, 73(23):6900–6912.
15.
EgebladM, WerbZ. 2002. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer, 2(3):161–174.
16.
EgilmezNK, JongYS, SabelMS, JacobJS, MathiowitzE, BankertRB. 2000. In situ tumor vaccination with interleukin-12-encapsulated biodegradable microspheres: induction of tumor regression and potent antitumor immunity. Cancer Res, 60(14):3832–3837.
17.
FreemanBE, RaueHP, HillAB, SlifkaMK. 2015. Cytokine-mediated activation of NK cells during viral infection. J Virol, 89(15):7922–7931.
18.
FuX, ParksWC, HeineckeJW. 2008. Activation and silencing of matrix metalloproteinases. Semin Cell Dev Biol, 19(1):2–13.
19.
GansbacherB, ZierK, DanielsB, CroninK, BannerjiR, GilboaE. 1990. Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity. J Exp Med, 172(4):1217–1224.
20.
GerberSA, SorensenEW, SedlacekAL, LimJY, SkrombolasD, FrelingerJG, LordEM. 2013. Local expression of interleukin-2 by B16 melanoma cells results in decreased tumour growth and long-term tumour dormancy. Immunology, 138(3):280–292.
21.
GibsonDG, YoungL, ChuangRY, VenterJC, HutchisonCA, 3rd, SmithHO. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 6(5):343–345.
22.
GillisS, FermMM, OuW, SmithKA. 1978. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol, 120(6):2027–2032.
23.
GruppSA, KalosM, BarrettD, AplencR, PorterDL, RheingoldSR, TeacheyDT, ChewA, HauckB, WrightJF, MiloneMC, LevineBL, JuneCH. 2013. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med, 368(16):1509–1518.
24.
GulleyJL, MadanRA, PachynskiR, MuldersP, SheikhNA, TragerJ, DrakeCG. 2017. Role of antigen spread and distinctive characteristics of immunotherapy in cancer treatment. J Natl Cancer Inst, 109(4).
25.
HaidarisCG, MaloneJ, SherrillLA, BlissJM, GaspariAA, InselRA, SullivanMA. 2001. Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens. J Immunol Methods, 257(1–2):185–202.
26.
HanesJ, SillsA, ZhaoZ, SuhKW, TylerB, DiMecoF, BratDJ, ChotiMA, LeongKW, PardollDM, BremH. 2001. Controlled local delivery of interleukin-2 by biodegradable polymers protects animals from experimental brain tumors and liver tumors. Pharm Res, 18(7):899–906.
27.
HankJA, GanJ, RyuH, OstendorfA, StauderMC, SternbergA, AlbertiniM, LoKM, GilliesSD, EickhoffJ, SondelPM. 2009. Immunogenicity of the hu14.18-IL2 immunocytokine molecule in adults with melanoma and children with neuroblastoma. Clin Cancer Res, 15(18):5923–5930.
28.
HeY, LiuXD, ChenZY, ZhuJ, XiongY, LiK, DongJH, LiX. 2007. Interaction between cancer cells and stromal fibroblasts is required for activation of the uPAR-uPA-MMP-2 cascade in pancreatic cancer metastasis. Clin Cancer Res, 13(11):3115–3124.
29.
HeemskerkB, LiuK, DudleyME, JohnsonLA, KaiserA, DowneyS, ZhengZ, SheltonTE, MatsudaK, RobbinsPF, MorganRA, RosenbergSA. 2008. Adoptive cell therapy for patients with melanoma, using tumor-infiltrating lymphocytes genetically engineered to secrete interleukin-2. Hum Gene Ther, 19(5):496–510.
30.
HofmannUB, WestphalJR, Van MuijenGN, RuiterDJ. 2000. Matrix metalloproteinases in human melanoma. J Invest Dermatol, 115(3):337–344.
31.
HojillaCV, MohammedFF, KhokhaR. 2003. Matrix metalloproteinases and their tissue inhibitors direct cell fate during cancer development. Br J Cancer, 89(10):1817–1821.
KessenbrockK, PlaksV, WerbZ. 2010. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell, 141(1):52–67.
34.
KhatriA, HusainiY, RussellPJ. 2007. Murine CTLL-2 cells respond to mIL12: prospects for developing an alternative bioassay for measurement of murine cytokines IL12 and IL18. J Immunol Methods, 326(1–2):41–53.
35.
KoblinskiJE, AhramM, SloaneBF. 2000. Unraveling the role of proteases in cancer. Clin Chim Acta, 291(2):113–135.
MatrisianLM. 1999. Cancer biology: extracellular proteinases in malignancy. Curr Biol, 9(20):R776–R778.
46.
McIntyreJO, SchererRL, MatrisianLM. 2010. Near-infrared optical proteolytic beacons for in vivo imaging of matrix metalloproteinase activity. Methods Mol Biol, 622:279–304.
47.
MescherMF, CurtsingerJM, AgarwalP, CaseyKA, GernerM, HammerbeckCD, PopescuF, XiaoZ. 2006. Signals required for programming effector and memory development by CD8+ T cells. Immunol Rev, 211:81–92.
48.
MoranJP, GerberSA, MartinCA, FrelingerJG, LordEM. 2003. Transfection of the genes for interleukin-12 into the K1735 melanoma and the EMT6 mammary sarcoma murine cell lines reveals distinct mechanisms of antitumor activity. Int J Cancer, 106(5):690–698.
49.
MullenL, AdamsG, FosterJ, VessillierS, KosterM, HauserH, LaywardL, GouldD, ChernajovskyY. 2014a. A comparative study of matrix metalloproteinase and aggrecanase mediated release of latent cytokines at arthritic joints. Ann Rheum Dis, 73(9):1728–1736.
50.
MullenL, RigbyA, SclandersM, AdamsG, MittalG, ColstonJ, FatahR, SubangC, FosterJ, Francis-WestP, KosterM, HauserH, LaywardL, VessillierS, AnnenkovA, Al-IzkiS, PryceG, BoltonC, BakerD, GouldDJ, ChernajovskyY. 2014b. Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-beta. Expert Opin Drug Deliv, 11(1):5–16.
51.
NeutraR, LeviBZ, ShohamY. 1992. Optimization of protein-production by the baculovirus expression vector system in shake flasks. Appl Microbiol Biotechnol, 37(1):74–78.
52.
NguyenQT, OlsonES, AguileraTA, JiangT, ScadengM, ElliesLG, TsienRY. 2010. Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival. Proc Natl Acad Sci U S A, 107(9):4317–4322.
PardollDM. 1995. Paracrine cytokine adjuvants in cancer immunotherapy. Annu Rev Immunol, 13:399–415.
56.
PreskyDH, MinettiLJ, GillessenS, WilkinsonVL, WuCY, GublerU, ChizzoniteR, GatelyMK. 1998. Analysis of the multiple interactions between IL-12 and the high affinity IL-12 receptor complex. J Immunol, 160(5):2174–2179.
57.
PreskyDH, YangH, MinettiLJ, ChuaAO, NabaviN, WuCY, GatelyMK, GublerU. 1996. A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits. Proc Natl Acad Sci U S A, 93(24):14002–14007.
58.
PuskasJ, SkrombolasD, SedlacekA, LordE, SullivanM, FrelingerJ. 2011. Development of an attenuated interleukin-2 fusion protein that can be activated by tumour-expressed proteases. Immunology, 133(2):206–220.
59.
RoseRC, BonnezW, Da RinC, McCanceDJ, ReichmanRC. 1994. Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles. J Gen Virol, 75 (Pt 9):2445–2449.
60.
RoseRC, BonnezW, StrikeDG, ReichmanRC. 1990. Expression of the full-length products of the human papillomavirus type 6b (HPV-6b) and HPV-11 L2 open reading frames by recombinant baculovirus, and antigenic comparisons with HPV-11 whole virus particles. J Gen Virol, 71 (Pt 11):2725–2729.
61.
RosenbergSA. 2001. Progress in human tumour immunology and immunotherapy. Nature, 411(6835):380–384.
62.
RosenbergSA, YangJC, TopalianSL, SchwartzentruberDJ, WeberJS, ParkinsonDR, SeippCA, EinhornJH, WhiteDE. 1994. Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2. JAMA, 271(12):907–913.
63.
SadelainM, BrentjensR, RiviereI. 2013. The basic principles of chimeric antigen receptor design. Cancer Discov, 3(4):388–398.
64.
SahinU, TureciO. 2018. Personalized vaccines for cancer immunotherapy. Science, 359(6382):1355–1360.
65.
SchramaD, ReisfeldRA, BeckerJC. 2006. Antibody targeted drugs as cancer therapeutics. Nat Rev Drug Discov, 5(2):147–159.
66.
SharmaP, AllisonJP. 2015. Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential. Cell, 161(2):205–214.
67.
SheaC, BloedornL, SullivanMA. 2005. Rapid isolation of single-chain antibodies for structural genomics. J Struct Funct Genomics, 6(2–3):171–175.
68.
Simpson-AbelsonMR, PurohitVS, PangWM, IyerV, OdunsiK, DemmyTL, YokotaSJ, LoyallJL, KelleherRJJr., Balu-IyerS, BankertRB. 2009. IL-12 delivered intratumorally by multilamellar liposomes reactivates memory T cells in human tumor microenvironments. Clin Immunol, 132(1):71–82.
69.
SkrombolasD, FrelingerJG. 2014. Challenges and developing solutions for increasing the benefits of IL-2 treatment in tumor therapy. Expert Rev Clin Immunol, 10(2):207–217.
70.
SkrombolasD, WylieI, MaharajS, FrelingerJG. 2015. Characterization of an IL-12 p40/p35 truncated fusion protein that can inhibit the action of IL-12. J Interferon Cytokine Res, 35(9):690–697.
71.
SloaneBF, YanS, PodgorskiI, LinebaughBE, CherML, MaiJ, Cavallo-MedvedD, SameniM, DosescuJ, MoinK. 2005. Cathepsin B and tumor proteolysis: contribution of the tumor microenvironment. Semin Cancer Biol, 15(2):149–157.
72.
TurkBE, HuangLL, PiroET, CantleyLC. 2001. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat Biotechnol, 19(7):661–667.
73.
TzengA, KwanBH, OpelCF, NavaratnaT, WittrupKD. 2015. Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution. Proc Natl Acad Sci U S A, 112(11):3320–3325.
74.
VanderlugtCL, MillerSD. 2002. Epitope spreading in immune-mediated diseases: implications for immunotherapy. Nat Rev Immunol, 2(2):85–95.
WatkinsSK, EgilmezNK, SuttlesJ, StoutRD. 2007. IL-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo. J Immunol, 178(3):1357–1362.
XieBW, MolIM, KeereweerS, van BeekER, QueI, SnoeksTJ, ChanA, KaijzelEL, LowikCW. 2012. Dual-wavelength imaging of tumor progression by activatable and targeting near-infrared fluorescent probes in a bioluminescent breast cancer model. PloS One, 7(2):e31875.
80.
YadavM, JhunjhunwalaS, PhungQT, LupardusP, TanguayJ, BumbacaS, FranciC, CheungTK, FritscheJ, WeinschenkT, ModrusanZ, MellmanI, LillJR, DelamarreL. 2014. Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing. Nature, 515(7528):572–576.
81.
ZhangL, MorganRA, BeaneJD, ZhengZ, DudleyME, KassimSH, NahviAV, NgoLT, SherryRM, PhanGQ, HughesMS, KammulaUS, FeldmanSA, ToomeyMA, KerkarSP, RestifoNP, YangJC, RosenbergSA. 2015. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma. Clin Cancer Res, 21(10):2278–2288.
82.
ZhongYJ, ShaoLH, LiY. 2013. Cathepsin B-cleavable doxorubicin prodrugs for targeted cancer therapy (Review). Int J Oncol, 42(2):373–383.
83.
ZitvogelL, KroemerG. 2012. Targeting PD-1/PD-L1 interactions for cancer immunotherapy. Oncoimmunology, 1(8):1223–1225.
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