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  • Antigen specificity can be irrelevant toimmunocytokine efficacy and biodistributionAlice Tzenga,b, Byron H. Kwana,b, Cary F. Opelb,c, Tejas Navaratnac, and K. Dane Wittrupa,b,c,1

    Departments of aBiological Engineering and cChemical Engineering and bKoch Institute for Integrative Cancer Research, Massachusetts Institute ofTechnology, Cambridge, MA 02139

    Edited by Ira Pastan, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved February 9, 2015 (received for reviewAugust 20, 2014)

    Cytokine therapy can activate potent, sustained antitumor re-sponses, but collateral toxicity often limits dosages. Althoughantibodycytokine fusions (immunocytokines) have been designedwith the intent to localize cytokine activity, systemic dose-limitingside effects are not fully ameliorated by attempted tumor targeting.Using the s.c. B16F10 melanoma model, we found that a nontoxicdose of IL-2 immunocytokine synergized with tumor-specific anti-body to significantly enhance therapeutic outcomes compared withimmunocytokine monotherapy, concomitant with increased tumorsaturation and intratumoral cytokine responses. Examination ofcell subset biodistribution showed that the immunocytokine associ-ated mainly with IL-2Rexpressing innate immune cells, with morebound immunocytokine present in systemic organs than the tumormicroenvironment. More surprisingly, immunocytokine antigenspecificity and Fc receptor interactions did not seem necessaryfor therapeutic efficacy or biodistribution patterns because immu-nocytokines with irrelevant specificity and/or inactive mutant Fcdomains behaved similarly to tumor-specific immunocytokine.IL-2IL-2R interactions, rather than antibodyantigen targeting, dic-tated immunocytokine localization; however, the lack of tumor target-ing did not preclude successful antibody combination therapy.Mathematical modeling revealed immunocytokine size as anotherdriver of antigen targeting efficiency. This work presents a safe,straightforward strategy for augmenting immunocytokine efficacy bysupplementary antibody dosing and explores underappreciated factorsthat can subvert efforts to purposefully alter cytokine biodistribution.

    immunocytokine | biodistribution | immunotherapy | antibody | IL-2

    Cytokines constitute a class of small immunomodulatory pro-teins, many of which possess tumoricidal properties usefulfor cancer immunotherapy (1, 2). One of the earliest successfulcytokine therapies, IL-2, which has antitumor functions thatinclude the activation of natural killer (NK) and cytotoxic T cells,can induce durable remissions in 510% of patients with meta-static melanoma and renal cell carcinoma, malignancies with poorprognoses (3). However, IL-2s potent immunostimulatory abili-ties often cause serious side effects, including life-threateningvascular leak syndrome, during systemic IL-2 administration. Intheory, cytokine side effects might be mitigated by localizing cyto-kine activity to tumor tissues. One approach for tumor targeting hasbeen to link cytokines to antibodies specific for tumor-associatedantigens, generating immunocytokines (1, 2). Treatment with IL-2immunocytokines has proven to be superior to treatment withequivalent antibody and cytokine given as separate agents (47).Nonetheless, systemic dose-limiting side effects have been ob-served after IL-2 immunocytokine administration, despite its ex-pected tumor localization (8, 9).Based on previous findings that antibody-dependent cell-

    mediated cytotoxicity (ADCC) plays an important role in immu-nocytokine efficacy (2), we hypothesized that IL-2 immunocytokineefficacy could be enhanced without introducing toxicity by theadministration of additional tumor-specific antibody. We showin a syngeneic solid tumor model that IL-2 immunocytokine in-deed synergized with antitumor antibody to significantly prolong

    survival. Although the antibody component has generally beenexpected to mediate immunocytokine localization, we show insteadthat the IL-2 moiety entirely governed biodistribution, explainingour unexpected observation that immunocytokines recognizing ir-relevant antigen performed equivalently to tumor-specific immu-nocytokines when combined with antibody. Mathematical modelingcomplemented our experimental results and highlights molecularsize as another determinant of tumor-targeting outcome.

    ResultsGeneration and Characterization of IL-2 Immunocytokines. Cytokinefusion to the IgG light-chain rather than the heavy-chain C ter-minus yields constructs that are more stable and possess greatereffector functions (10, 11). To generate immunocytokines of thisformat, we fused murine IL-2 to full-length mouse IgG2a mol-ecules with variable regions specific for either TRP1 antigen ex-pressed by murine melanomas or CEA, a human tumor marker,to produce TA99-IL2 and sm3E-IL2, respectively (Fig. 1A andDataset S1). The purified proteins were predominantly homog-enous monomers (Fig. S1A) and showed the expected masses onSDS/PAGE (Fig. S1B). As confirmed by flow cytometry, bothimmunocytokines retained the ability to bind simultaneously tocell surface antigens and antiIL-2 antibody (Fig. S1C). Assess-ment of simultaneous binding function also revealed excellentthermal stability and reasonable proteolytic stability for the twoconstructs (Fig. S1D). Moreover, the immunocytokines exhibitedsimilar in vitro specific activity to WT IL-2 as measured by IL-2dependent CTLL-2 cell proliferation (Fig. 1B). When injectedinto mice, TA99-IL2 and sm3E-IL2 displayed plasma half-livesof 1114 h (Fig. 1C and Table S1), considerably longer than thatof WT IL-2, which is on the order of minutes (12). Both the

    Significance

    Cytokines (potent immunostimulatory proteins) exert powerfulantitumor effects but often cause severe whole-body in-flammation when used as cancer therapies. Contrary to the cur-rent paradigm that fusion to antitumor antibodies can constraincytokine activity to tumors, we have found that, for some im-munocytokines incorporating the cytokine IL-2, the cytokine moi-ety overrides antibody-mediated targeting, localizing the fusionprotein to IL-2 receptor-expressing cells rather than tumor cells.Although the IL-2 immunocytokines did not selectively home totumors, they persisted longer in circulation than free IL-2, suchthat a nontoxic immunocytokine dose could synergize with tumor-specific antibody to cure mice with aggressive solid tumors.

    Author contributions: A.T. and K.D.W. designed research; A.T., B.H.K., C.F.O., and T.N.performed research; A.T., B.H.K., C.F.O., T.N., and K.D.W. analyzed data; and A.T. and K.D.W.wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: wittrup@mit.edu.

    This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplemental.

    33203325 | PNAS | March 17, 2015 | vol. 112 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1416159112

    http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplemental/pnas.1416159112.sd01.docxhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplemental/pnas.201416159SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplemental/pnas.201416159SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplemental/pnas.201416159SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplemental/pnas.201416159SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplemental/pnas.201416159SI.pdf?targetid=nameddest=ST1http://crossmark.crossref.org/dialog/?doi=10.1073/pnas.1416159112&domain=pdfmailto:wittrup@mit.eduhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416159112/-/DCSupplementalwww.pnas.org/cgi/doi/10.1073/pnas.1416159112

  • immunocytokines larger size and the presence of the antibodyFc region, allowing recycling through the neonatal Fc receptor,likely account for this extended plasma persistence. Injection ofimmunocytokines but not antibody also resulted in splenomegaly,providing additional evidence of bioactivity (Fig. S1E).

    Combination Therapy Using Immunocytokine and Tumor-SpecificAntibody Provides Synergistic Antitumor Responses. We tested theantitumor efficacy of TA99-IL2 by administering it to C57BL/6mice bearing established syngeneic B16F10 melanoma tumors,which express TRP1. To circumvent IL-2 toxicity, we used an im-munocytokine dosing regimen that did not induce systemic toxicityas measured by weight loss (Fig. S2A). Despite significantly delayingtumor growth compared with PBS, TA99-IL2 was insufficient forprolonged tumor control (Fig. 2A and Fig. S2B). We hypothesizedthat the lack of durable remissions could be caused by insufficienttumor saturation with the immunocytokine, because antitumoractivity has been correlated with a therapeutic antibodys abilityto penetrate throughout tumor tissue (13). Because antitumorantibodies such as TA99 are generally far better tolerated thancytokines and can independently elicit tumoricidal immunity (14),we combined our original TA99-IL2 dose with additional TA99murine IgG2a antibody to attain a dosage theoretically predictedto fully saturate the tumors (15). This combination therapy sig-nificantly improved the survival of tumor-bearing mice comparedwith treatment with TA99-IL2 alone, yielding long-term survivors(Fig. 2A and Fig. S2B). The additional antibody did not lead tosystemic toxicity (Fig. S2A), although normal melanocytes alsoexpress TRP1. Interestingly, when mice were treated with equi-molar WT murine IL-2 in lieu of TA99-IL2 concurrently withTA99, survival was similar to that of PBS-treated controls (Fig.2A an