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• W3214936723 abstract "•Single dose of AIP immunotherapy inflames the tumor microenvironment (TME)•This sensitizes tumors to immune checkpoint blockade•NK cells and macrophages destruct tumors and recruit cDC1 and lymphocytes to the TME•NK cells and macrophage facilitate rapid tumor vasculature remodeling Treatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB. Treatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB. Immune checkpoint blockade (ICB) therapies based on the administration of antibodies against the T cell inhibitory molecules cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1, or its ligands) have been approved for the treatment of a variety of malignancies alone or in combination (Antonia et al., 2017Antonia S.J. Villegas A. Daniel D. Vicente D. Murakami S. Hui R. Yokoi T. Chiappori A. Lee K.H. de Wit M. et al.Durvalumab after Chemoradiotherapy in Stage III Non-Small-Cell Lung Cancer.N. Engl. J. Med. 2017; 377: 1919-1929Crossref PubMed Scopus (1834) Google Scholar; Hellmann et al., 2018Hellmann M.D. Ciuleanu T.-E. Pluzanski A. Lee J.S. Otterson G.A. Audigier-Valette C. Minenza E. Linardou H. Burgers S. Salman P. et al.Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden.N. Engl. J. Med. 2018; 378: 2093-2104Crossref PubMed Scopus (1547) Google Scholar; Hodi et al., 2010Hodi F.S. O’Day S.J. McDermott D.F. Weber R.W. Sosman J.A. Haanen J.B. Gonzalez R. Robert C. Schadendorf D. Hassel J.C. et al.Improved survival with ipilimumab in patients with metastatic melanoma.N. Engl. J. Med. 2010; 363: 711-723Crossref PubMed Scopus (10123) Google Scholar; Reck et al., 2016Reck M. Rodríguez-Abreu D. Robinson A.G. Hui R. Csőszi T. Fülöp A. Gottfried M. Peled N. Tafreshi A. Cuffe S. et al.Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer.N. Engl. J. Med. 2016; 375: 1823-1833Crossref PubMed Google Scholar; Socinski et al., 2018Socinski M.A. Jotte R.M. Cappuzzo F. Orlandi F. Stroyakovskiy D. Nogami N. Rodríguez-Abreu D. Moro-Sibilot D. Thomas C.A. Barlesi F. et al.Atezolizumab for First-Line Treatment of Metastatic Nonsquamous NSCLC.N. Engl. J. Med. 2018; 378: 2288-2301Crossref PubMed Google Scholar). However, in most tumors, ICB benefits a minority of patients, and only a subset of responders exhibit durable responses (Gao et al., 2016Gao J. Shi L.Z. Zhao H. Chen J. Xiong L. He Q. Chen T. Roszik J. Bernatchez C. Woodman S.E. et al.Loss of IFN-γ Pathway Genes in Tumor Cells as a Mechanism of Resistance to Anti-CTLA-4 Therapy.Cell. 2016; 167: 397-404.e9Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar; George et al., 2017George S. Miao D. Demetri G.D. Adeegbe D. Rodig S.J. Shukla S. Lipschitz M. Amin-Mansour A. Raut C.P. Carter S.L. et al.Loss of PTEN Is Associated with Resistance to Anti-PD-1 Checkpoint Blockade Therapy in Metastatic Uterine Leiomyosarcoma.Immunity. 2017; 46: 197-204Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar; Sade-Feldman et al., 2017Sade-Feldman M. Jiao Y.J. Chen J.H. Rooney M.S. Barzily-Rokni M. Eliane J.-P. Bjorgaard S.L. Hammond M.R. Vitzthum H. Blackmon S.M. et al.Resistance to checkpoint blockade therapy through inactivation of antigen presentation.Nat. Commun. 2017; 8: 1136Crossref PubMed Scopus (336) Google Scholar; Zaretsky et al., 2016Zaretsky J.M. Garcia-Diaz A. Shin D.S. Escuin-Ordinas H. Hugo W. Hu-Lieskovan S. Torrejon D.Y. Abril-Rodriguez G. Sandoval S. Barthly L. et al.Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma.N. Engl. J. Med. 2016; 375: 819-829Crossref PubMed Scopus (1536) Google Scholar). To better understand the underlying mechanisms driving these variations in response, substantial work has been carried out to identify features of the tumor microenvironment (TME) that correlate with long-term survival and predict responses to ICB (Le et al., 2015Le D.T. Uram J.N. Wang H. Bartlett B.R. Kemberling H. Eyring A.D. Skora A.D. Luber B.S. Azad N.S. Laheru D. et al.PD-1 Blockade in Tumors with Mismatch-Repair Deficiency.N. Engl. J. Med. 2015; 372: 2509-2520Crossref PubMed Scopus (5044) Google Scholar; Sade-Feldman et al., 2018Sade-Feldman M. Yizhak K. Bjorgaard S.L. Ray J.P. de Boer C.G. Jenkins R.W. Lieb D.J. Chen J.H. Frederick D.T. Barzily-Rokni M. et al.Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma.Cell. 2018; 175: 998-1013.e20Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar; Snyder et al., 2014Snyder A. Makarov V. Merghoub T. Yuan J. Zaretsky J.M. Desrichard A. Walsh L.A. Postow M.A. Wong P. Ho T.S. et al.Genetic basis for clinical response to CTLA-4 blockade in melanoma.N. Engl. J. Med. 2014; 371: 2189-2199Crossref PubMed Scopus (2611) Google Scholar; Tumeh et al., 2014Tumeh P.C. Harview C.L. Yearley J.H. Shintaku I.P. Taylor E.J.M. Robert L. Chmielowski B. Spasic M. Henry G. Ciobanu V. et al.PD-1 blockade induces responses by inhibiting adaptive immune resistance.Nature. 2014; 515: 568-571Crossref PubMed Scopus (3595) Google Scholar; Yuen et al., 2020Yuen K.C. Liu L.-F. Gupta V. Madireddi S. Keerthivasan S. Li C. Rishipathak D. Williams P. Kadel 3rd, E.E. Koeppen H. et al.High systemic and tumor-associated IL-8 correlates with reduced clinical benefit of PD-L1 blockade.Nat. Med. 2020; 26: 693-698Crossref PubMed Scopus (66) Google Scholar). One factor associated with outcome is the number of infiltrating T cells prior to or during early treatment (Chen et al., 2016Chen P.-L. Roh W. Reuben A. Cooper Z.A. Spencer C.N. Prieto P.A. Miller J.P. Bassett R.L. Gopalakrishnan V. Wani K. et al.Analysis of Immune Signatures in Longitudinal Tumor Samples Yields Insight into Biomarkers of Response and Mechanisms of Resistance to Immune Checkpoint Blockade.Cancer Discov. 2016; 6: 827-837Crossref PubMed Scopus (484) Google Scholar; Tumeh et al., 2014Tumeh P.C. Harview C.L. Yearley J.H. Shintaku I.P. Taylor E.J.M. Robert L. Chmielowski B. Spasic M. Henry G. Ciobanu V. et al.PD-1 blockade induces responses by inhibiting adaptive immune resistance.Nature. 2014; 515: 568-571Crossref PubMed Scopus (3595) Google Scholar), introducing the notion of “hot” (inflamed and highly infiltrated) and “cold” (immunosuppressive and non-infiltrated) tumors (Galon et al., 2006Galon J. Costes A. Sanchez-Cabo F. Kirilovsky A. Mlecnik B. Lagorce-Pagès C. Tosolini M. Camus M. Berger A. Wind P. et al.Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.Science. 2006; 313: 1960-1964Crossref PubMed Scopus (4069) Google Scholar). Those patients responding to ICB most often exhibit a “hot” tumor phenotype. These findings have led to an intensive search for combination treatments that could convert “cold” tumors into the “hot” lymphocyte-infiltrated state, under the assumption that such co-treatments would increase the proportion of ICB-responding patients. To date, a variety of immunotherapeutic strategies have been studied that provide some level of benefit when combined with ICB treatment in preclinical models or small clinical studies (Carmi et al., 2015Carmi Y. Spitzer M.H. Linde I.L. Burt B.M. Prestwood T.R. Perlman N. Davidson M.G. Kenkel J.A. Segal E. Pusapati G.V. et al.Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity.Nature. 2015; 521: 99-104Crossref PubMed Scopus (113) Google Scholar; Ribas et al., 2017Ribas A. Dummer R. Puzanov I. VanderWalde A. Andtbacka R.H.I. Michielin O. Olszanski A.J. Malvehy J. Cebon J. Fernandez E. et al.Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy.Cell. 2017; 170: 1109-1119.e10Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar), but in general the synergies discovered have been modest and the immunological ruleset to promote ICB responsiveness remains poorly defined. Recently, we identified a potent four-component immunotherapy comprised of an anti-tumor antibody (“A”), half-life-extended interleukin-2 (“I”), anti-PD-1 (“P”), and a peptide vaccine (“V,” with the combination treatment abbreviated as AIPV hereafter), which cured a majority of animals in several difficult-to-treat transplanted and genetically engineered mouse models (GEMMs) of cancer (Moynihan et al., 2016Moynihan K.D. Opel C.F. Szeto G.L. Tzeng A. Zhu E.F. Engreitz J.M. Williams R.T. Rakhra K. Zhang M.H. Rothschilds A.M. et al.Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.Nat. Med. 2016; 22: 1402-1410Crossref PubMed Scopus (263) Google Scholar). Mechanistically, this treatment was dependent not only on CD8+ T cells but also several subsets of innate immune cells, including macrophages and natural killer (NK) cells, suggesting the importance of effectively engaging adaptive and innate immune responses against tumors in tandem. However, clinical translation of this therapy is complicated by its multicomponent nature and the need for multiple tumor-specific therapeutic components (i.e., the “A” and “V”). Motivated by the striking changes induced in the TME following a single dose of AIPV therapy, we hypothesized that a treatment simultaneously activating innate and adaptive responses might prime tumors to respond to treatment with ICB alone. Here, we tested AIPV and its subcombinations as a single-dose combination treatment prior to ICB and discovered that a simplified regimen combining a pan-tumor integrin-targeting antibody surrogate molecule with long half-life IL-2 and anti-PD-1, administered once, triggered remodeling of the TME, induction of rapid tumor cell death, and initiated T cell priming in tumor-draining lymph nodes. Mechanistically, these changes were mediated primarily by NK cells and macrophages, which induced rapid tumor antigen release to dendritic cells (DCs), mediated immune infiltration, and promoted vascular normalization. These findings suggest that a relatively simple combination treatment may be capable of substantially increasing responsiveness to checkpoint blockade, by exploiting coordinated functions of innate immune cells together with T cell functionality in the TME. We previously reported that weekly-administered AIPV therapy induced dramatic changes in the TME, leading to eradication of large established tumors in a range of syngeneic cancer models (Moynihan et al., 2016Moynihan K.D. Opel C.F. Szeto G.L. Tzeng A. Zhu E.F. Engreitz J.M. Williams R.T. Rakhra K. Zhang M.H. Rothschilds A.M. et al.Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.Nat. Med. 2016; 22: 1402-1410Crossref PubMed Scopus (263) Google Scholar). To obtain greater insight into the kinetics of changes to the TME following treatment with this combination therapy, we carried out Luminex-based analysis of cytokine and chemokine levels in tumors at early time points after AIPV administration in the B16F10 melanoma model. We used the anti-TYRP1 antibody TA99 as the “A” component and a peptide vaccine against the TRP2180–188 peptide as the “V” component (Figure 1A). Strikingly, we found that a broad range of pro-inflammatory cytokines were upregulated within 1 day of treatment, including interferon γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-12 (IL-12) as well as key chemokines including CCL3/4, CCL5, CXCL9, and CXCL10 (Figure 1B; Figure S1A). CD4+ and CD8+ T cells, NK cells, conventional type 1 dendritic cells (cDC1s) and cDC2s, NK cells, and neutrophils were all recruited to the tumor over the course of 6 days following a single dose of AIPV, and the CD8/Treg ratio shifted dramatically (Figures S1B and S1C). We previously studied AIPV administered as a multi-dose regimen over 5 weeks (“full AIPV” regimen; Figure 1C). Encouraged by the ability of a single dose of AIPV to inflame relatively cold B16F10 tumors within a few days, we evaluated whether a single dose of AIPV followed by clinically approved ICB with anti-PD-1 and anti-CTLA-4 could be effective (“1X AIPV + ICB”; Figure 1C). Strikingly, single-dose AIPV administered to animals bearing large ∼40–50 mm2 tumors followed by PD-1 and CTLA-4 blockade (hereafter, ICB) led to tumor regressions comparable with the “full” AIPV regimen, with complete responses in 80% of animals (Figures 1D and 1E). ICB alone had no impact on overall survival in this setting, and 1X AIPV without subsequent ICB dosing or treatments applying 1X AIPV followed by anti-PD-1 alone or anti-CTLA-4 alone were much less effective than AIPV followed by combined checkpoint blockade (Figures 1D and 1E). The full AIPV regimen elicited high levels of TRP2-specific T cells (the antigen targeted by the “V” component) after repeated dosing, and induced antigen spreading to epitopes expressed by B16F10 cells other than TRP2 (Figures S1D and S1E). 1X AIPV therapy elicited much weaker TRP2-directed T cell responses, but elicited equivalent levels of T cell responses against other B16F10 epitopes (Figures S1D and S1E). Hence, a single dose of the AIPV combination was sufficient to render intractable B16F10 tumors susceptible to ICB. We next sought to determine whether this combination 1X AIPV “priming” treatment could be made simpler and more amenable for pan-tumor treatment. We first replaced the melanoma-specific TA99 antibody with a recently described pan-tumor integrin-binding protein 2.5F-Fc, an engineered integrin-targeting cysteine knot peptide fused to a mouse IgG2c Fc domain (Van Agthoven et al., 2019Van Agthoven J.F. Shams H. Cochran F.V. Alonso J.L. Kintzing J.R. Garakani K. Adair B.D. Xiong J.-P. Mofrad M.R.K. Cochran J.R. Arnaout M.A. Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1.Structure. 2019; 27: 1443-1451.e6Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar; Kimura et al., 2009Kimura R.H. Levin A.M. Cochran F.V. Cochran J.R. Engineered cystine knot peptides that bind alphavbeta3, alphavbeta5, and α5β1 integrins with low-nanomolar affinity.Proteins. 2009; 77: 359-369Crossref PubMed Scopus (0) Google Scholar; Kwan et al., 2017Kwan B.H. Zhu E.F. Tzeng A. Sugito H.R. Eltahir A.A. Ma B. Delaney M.K. Murphy P.A. Kauke M.J. Angelini A. et al.Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses.J. Exp. Med. 2017; 214: 1679-1690Crossref PubMed Scopus (25) Google Scholar; Moore et al., 2013Moore S.J. Hayden Gephart M.G. Bergen J.M. Su Y.S. Rayburn H. Scott M.P. Cochran J.R. Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma.Proc. Natl. Acad. Sci. USA. 2013; 110: 14598-14603Crossref PubMed Scopus (48) Google Scholar) (Figure 1F). This antibody surrogate recognizes α5β1 and multiple αv-containing integrins that are overexpressed in many human and mouse tumors, and it stained B16F10 melanoma, TC-1 HPV E6/E7-expressing tumor cells (Lin et al., 1996Lin K.Y. Guarnieri F.G. Staveley-O’Carroll K.F. Levitsky H.I. August J.T. Pardoll D.M. Wu T.C. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen.Cancer Res. 1996; 56: 21-26PubMed Google Scholar), the YUMM1.7 melanoma cell derived from the BrafV600E/Ptenfl/fl GEMM (Meeth et al., 2016Meeth K. Wang J.X. Micevic G. Damsky W. Bosenberg M.W. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.Pigment Cell Melanoma Res. 2016; 29: 590-597Crossref PubMed Scopus (94) Google Scholar), and KP lung cancer cells generated from the LSL-KrasG12D/+/p53fl/fl GEMM (Figure 1F). We tested 1X AIPV using this revised “A” component as well as each of the triple- and two-component subcombinations of the regimen for their ability to prime B16F10 tumors for eradication by ICB. These experiments revealed that one dose of AIP or AIV prior to ICB was as effective as AIPV for curing large B16 tumors, while IPV or two-component treatments were substantially less effective (Figure 1G). Animals cured by 1X AIPV, AIP, AIV, or IPV followed by ICB had protective memory and rejected tumor rechallenge (Figure 1H). As the “V” remains a tumor-specific treatment component, we opted to focus on 1X AIP as a combination for further study. In the B16F10 model, the 1X AIP + ICB combination therapy generated substantially stronger tumor-specific T cell responses than ICB alone or in mice left untreated by 1-week post dosing (Figure S1F). A broad array of inflammatory cytokines and chemokines were induced one day following AIP treatment using the 2.5F-Fc antibody surrogate, comparable with AIPV therapy (Figure S2A). 1X AIP followed by ICB also elicited high rates (80%–100%) of complete cures of large established TC-1 and YUMM1.7 flank tumors and orthotopic KP lung tumors, where ICB alone was either only partially effective (KP) or completely ineffective (TC-1 and YUMM1.7) (Figures 1I and 1J; Figure S2B). We further assessed the efficacy of the 1X AIP treatment in an autochthonous BrafV600E/Ptenfl/fl melanoma model that better mimics the etiology and progression of human melanoma. In this model, tamoxifen applied to the skin of mice induces expression of Cre in melanocytes, which in turn activates expression of mutant Braf, Pten deletion, and expression of a defined H-2Kb-restricted CD8 T cell neoepitope SIYRYYGL (designated as BP-SIY). This tumor model exhibits modest T cell infiltration and limited responses to ICB (Spranger et al., 2015Spranger S. Bao R. Gajewski T.F. Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity.Nature. 2015; 523: 231-235Crossref PubMed Scopus (1320) Google Scholar), and expression of the SIY neoepitope does not alter tumor growth or the TME compared with parental BrafV600E/Ptenfl/fl tumors (Spranger et al., 2017Spranger S. Dai D. Horton B. Gajewski T.F. Tumor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell Therapy.Cancer Cell. 2017; 31: 711-723.e4Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). Twenty-five days post tamoxifen application, we initiated the 1X AIP +ICB therapy and found that the treatment significantly controlled tumor growth, while untreated lesions progressed to large tumor masses (Figure 1K). A limited exploration of alternate clinically relevant components in the priming regimen suggested that the anti-PD-1 in AIP can be replaced with anti-CTLA-4, whereas switching “A” or “I” with anti-CTLA-4 elicited decreased therapeutic efficacy depending on the tumor model (Figures S2C–S2F). Many combination immunotherapies suffer from unacceptable toxicities. However, 1X AIP treatment induced no significant changes in animal behavior, weight loss, or significant acute elevation of serum liver enzymes (Figures S2G and S2H). Altogether, 1X AIP appears to be an effective and safe combination treatment for enhancing responsiveness to ICB in multiple tumor models. The development of anti-tumor T cell responses led us to assess the role of T cells and DCs in the response to 1X AIP therapy in the B16F10 model. Depletion of CD3+ T cells, CD4+ cells, or CD8+ cells all led to drastic reductions in therapeutic efficacy (Figure 2A; Figure S3A). Treatment of Batf3−/− mice lacking cross-presenting DCs required for CD8+ T cell priming extended survival of tumor-bearing animals, but none rejected their tumors (Figure 2A). We next sought to determine whether AIP treatment induces tumor antigen release and acquisition by DCs. One day post treatment with AI, IP, AP, or ICB, the number of apoptotic tumor cells was doubled compared with untreated tumors, but tumor cell death was increased 3-fold following treatment with AIP, and these cells exposed calreticulin that promotes phagocytosis (Chao et al., 2010Chao M.P. Jaiswal S. Weissman-Tsukamoto R. Alizadeh A.A. Gentles A.J. Volkmer J. Weiskopf K. Willingham S.B. Raveh T. Park C.Y. et al.Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47.Sci. Transl. Med. 2010; 2: 63ra94Crossref PubMed Scopus (398) Google Scholar) (Figures 2B and 2C). Using B16F10 cells expressing the stable fluorescent protein ZsGreen to enable tracking of antigen acquisition by DCs (Matz et al., 1999Matz M.V. Fradkov A.F. Labas Y.A. Savitsky A.P. Zaraisky A.G. Markelov M.L. Lukyanov S.A. Fluorescent proteins from nonbioluminescent Anthozoa species.Nat. Biotechnol. 1999; 17: 969-973Crossref PubMed Scopus (1455) Google Scholar), we found a substantial increase in the level of tumor antigen uptake in intratumoral CD103+ DCs within 1 day of AIP treatment compared with untreated or ICB-treated tumors (Figure 2D). These cells also had higher expression of the costimulatory receptor CD86 and lymph node homing receptor CCR7 (Figures 2E and 2F). Comparing responses of ZsGreen-B16F10 tumors treated with a single dose of AIP versus IP, we found that the striking antigen uptake by cDC1s occurred only in the presence of the 2.5F-Fc antibody surrogate (Figure 2G). Adoptive transfer of pmel TCR-transgenic T cells that recognize the melanoma antigen gp100 into tumor-bearing mice just prior to therapy revealed that AIP induced antigen presentation and activation of tumor-specific T cells in TDLNs by 1 day following treatment (Figures 2H–2J). These data indicate that a single dose of AIP is able to rapidly induce tumor antigen release, which is captured by activated DCs to initiate T cell priming in TDLNs. We hypothesized that rapid tumor cell death induced following AIP administration was mediated at least in part by FcR-expressing macrophages and NK cells responding to the “A” component. Notably, the therapeutic efficacy of 1X AIP was greatly reduced when either NK cells or macrophages, but not neutrophils, were depleted prior to treatment (Figure 3A; Figure S3B). The lack of contribution from neutrophils is consistent with their limited infiltration into the tumors (Figure S3C), in contrast with what we observed with full AIPV treatment (Moynihan et al., 2016Moynihan K.D. Opel C.F. Szeto G.L. Tzeng A. Zhu E.F. Engreitz J.M. Williams R.T. Rakhra K. Zhang M.H. Rothschilds A.M. et al.Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.Nat. Med. 2016; 22: 1402-1410Crossref PubMed Scopus (263) Google Scholar). As NK1.1 upregulation by macrophages has been reported (Steiger et al., 2015Steiger S. Kuhn S. Ronchese F. Harper J.L. Monosodium Urate Crystals Induce Upregulation of NK1.1-Dependent Killing by Macrophages and Support Tumor-Resident NK1.1+ Monocyte/Macrophage Populations in Antitumor Therapy.J. Immunol. 2015; 195: 5495-5502Crossref PubMed Scopus (6) Google Scholar), we evaluated the expression of NK1.1 and F4/80 in tumor-infiltrating macrophages and NK cells, respectively, to confirm that the depleting antibodies used here were not potentially removing unintended cellular targets. We found minimal expression of NK1.1 on macrophages or F4/80 on NK cells and no significant difference between treated and untreated conditions (Figure S3D). Similarly, F4/80 has been reported to be expressed on activated CD8+ T cells in some conditions (Lin et al., 2003Lin Y. Roberts T.J. Sriram V. Cho S. Brutkiewicz R.R. Myeloid marker expression on antiviral CD8+ T cells following an acute virus infection.Eur. J. Immunol. 2003; 33: 2736-2743Crossref PubMed Scopus (55) Google Scholar), but we observed no expression of F4/80 on tumor-infiltrating CD8+ T cells in treated or untreated tumors (Figure S3D) (Lin et al., 2003Lin Y. Roberts T.J. Sriram V. Cho S. Brutkiewicz R.R. Myeloid marker expression on antiviral CD8+ T cells following an acute virus infection.Eur. J. Immunol. 2003; 33: 2736-2743Crossref PubMed Scopus (55) Google Scholar). Furthermore, neither CD1d-restricted NK T (NKT) cells nor “late” NK cell depletion (3 days post AIP treatment) had a significant impact on the efficacy of 1X AIP therapy (Figure 3A; Figure S3E). Depletion of NK cells or macrophages, but not T cells or neutrophils, also led to a statistically significant reduction in immediate tumor cell death following AIP (Figures 3B and 3C). 1X AIP induced major histocompatibility complex class I (MHC class I) and PD-L1 upregulation on cancer cells within 1 day, a response that was substantially reduced if NK cells or macrophages were depleted (Figures 3D and 3E). We next assessed whether rapid NK cell- and macrophage-dependent tumor-cell killing was mediated by the antibody surrogate. Introduction of a D265A mutation in the Fc domain of 2.5F-Fc to ablate binding of the molecule with FcγRs (Baudino et al., 2008Baudino L. Shinohara Y. Nimmerjahn F. Furukawa J. Nakata M. Martínez-Soria E. Petry F. Ravetch J.V. Nishimura S. Izui S. Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions.J. Immunol. 2008; 181: 6664-6669Crossref PubMed Google Scholar) led to a loss of rapid tumor cell death following AIP and substantial reductions in treatment efficacy (Figure 3F; Figure S3F). The upregulation of MHC class I and PD-L1 on cancer cells was also lost when D265A or LALA-PG mutations were introduced in the 2.5F-Fc antibody surrogate (Figures S3G and S3H).Treatment with 2.5F-Fc bearing LALA-PG mutations in the Fc domain to block both FcγR binding and complement activation (Lo et al., 2017Lo M. Kim H.S. Tong R.K. Bainbridge T.W. Vernes J.-M. Zhang Y. Lin Y.L. Chung S. Dennis M.S. Zuchero Y.J.Y. et al.Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice.J. Biol. Chem. 2017; 292: 3900-3908Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) did not further blunt efficacy, suggesting complement does not play a major role. FcγRIII is a key activating FcγR that mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells (Mandelboim et al., 1999Mandelboim O. Malik P. Davis D.M. Jo C.H. Boyson J.E. Strominger J.L. Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity.Proc. Natl. Acad. Sci. USA. 1999; 96: 5640-5644Crossref PubMed Scopus (194) Google Scholar) and is also expressed on macrophages (Kinder et al., 2015Kinder M. Greenplate A.R. Strohl W.R. Jordan R.E. Brezski R.J. An Fc engineering approach that modulates antibody-dependent cytokine release without altering cell-killing functions.MAbs. 2015; 7: 494-504Crossref PubMed Scopus (0) Google Scholar). FcγIIIR deficiency completely abolished the therapeutic efficacy of 1X AIP treatment, further supporting a requirement for the Fc domain of 2.F-Fc and ADCC for the response to treatment (Figure 3G). To test the importance of FcRs on macrophages and NK cells specifically, we used an adoptive transfer approach, with antibody-mediated depletion of endogenous NK cells/macrophages from tumor-bearing Fcgr3−/− mice followed by intravenous (i.v.) transfer of wild-type (WT) NK cells or macrophages just before 1X AIP treatment (Hagemann et al., 2008Hagemann T. Lawrence T. McNeish I. Charles K.A. Kulbe H. Thompson R.G. Robinson S.C. Balkwill F.R. “Re-educating” tumor-associated macrophages by targeting NF-kappaB.J. Exp. Med. 2008; 205: 1261-1268Crossref PubMed Scopus (583) Google Scholar; Tai et al., 2013Tai L.-H. Souza C. Makrigiannis A. Auer R. Natural Killer Cell Transfer Assay.Bio.-Protocol. 2013; 3: e864Google Scholar) (Figure 3H). Adoptive transfer of either WT NK cells or macrophages into Fcgr3−/− mice restored the response rate to 1X AIP treatment to 50%–60%, indicating an essential role for functional FcγRs on NK cells and macrophages in the first few days of the treatment (Figure 3G). CD8α antibody-mediated depletion showed a trend toward some impact on tumor antigen uptake by CD103+ DCs, which could be an indirect effect of removing CD8α+ DCs by the antibody, but transient intratumoral depletion of T cells (Besançon et al., 2017Besançon A. Baas M. Goncalves T. Valette F. Waldmann H. Chatenoud L. You S. The Induction and Maintenance of Transplant Tolerance Engages Both Regulatory and Anergic CD4+ T cells.Front. Immunol. 2017; 8: 218Crossref" @default.
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• W3214936723 date "2021-11-01" @default.
• W3214936723 modified "2023-10-16" @default.
• W3214936723 title "Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade" @default.
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