Nanobody Antigen Conjugates Elicit HPV-Speciﬁc Antitumor Immune Responses · Nanobody–Antigen Conjugates Elicit Anti-HPV Tumor Responses Cancer Immunol Res; 6(7) July 2018 871

Research Article

Nanobody–Antigen Conjugates ElicitHPV-Specific Antitumor Immune ResponsesAndrew W.Woodham1,2, Ross W. Cheloha1,2, Jingjing Ling1,3,Mohammad Rashidian1,2, Stephen C. Kolifrath1, Maia Mesyngier1, Joao N. Duarte4,Justin M. Bader3, Joseph G. Skeate5, Diane M. Da Silva5,6,W. Martin Kast5,6,7, andHidde L. Ploegh1

Abstract

High-risk human papillomavirus-associated cancers expressviral oncoproteins (e.g., E6 and E7) that induce and maintainthe malignant phenotype. The viral origin of these proteinsmakes them attractive targets for development of a therapeuticvaccine. Camelid-derived single-domain antibody fragments(nanobodies or VHHs) that recognize cell surface proteins onantigen-presenting cells (APC) can serve as targeted deliveryvehicles for antigens attached to them. Such VHHs were shownto induce CD4þ and CD8þ T-cell responses against modelantigens conjugated to them via sortase, but antitumorresponses had not yet been investigated. Here, we tested theability of an anti-CD11b VHH (VHHCD11b) to target APCs andserve as the basis for a therapeutic vaccine to induce CD8þ

T-cell responses against HPVþ tumors. Mice immunized with

VHHCD11b conjugated to anH-2Db-restricted immunodominantE7 epitope (E749-57) hadmore E7-specificCD8þT cells comparedwith those immunized with E749-57 peptide alone. These CD8þ

T cells acted prophylactically and conferred protection againsta subsequent challenge with HPV E7-expressing tumor cells.In a therapeutic setting, VHHCD11b-E749-57 vaccination resultedin greater numbers of CD8þ tumor–infiltrating lymphocytescompared with mice receiving E749-57 peptide alone in HPVþ

tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET),which correlated with tumor regression and survival outcome.Together, these results demonstrate that VHHs can serve as atherapeutic cancer vaccine platform for HPV-induced cancers.Cancer Immunol Res; 6(7); 870–80. �2018 AACR.

IntroductionProphylactic vaccination against humanpapillomavirus (HPV)

using capsid protein-based virus-like particles (VLP) elicits pro-tective humoral immunity against subsequent viral exposure andcan prevent HPV-induced malignancies (1). However, for eco-nomic, political, and cultural reasons, not all individuals at riskfor HPV infection receive the vaccine (2, 3). Consequently, trans-mission of HPV and the attendant risk of malignancies remainmajor global concerns (4). HPV-inducedmalignancies are of viralorigin and, as such, are excellent immunological targets because

viral antigens (Ag) are distinct from self-Ags. HPV type 16(HPV16) is the most common oncogenic high-risk HPV strain(5), with its primary drivers of oncogenesis, the early genes E6 andE7, constitutively expressed in HPV-induced tumors (6), makingthem major targets of antitumor vaccination strategies (7).Although prophylactic vaccination with VLP-based vaccines isprotective prior to HPV exposure, established HPVþ tumors willrequire a vaccination approach that elicits early gene product-specific cytotoxic CD8þ T cells to eradicate tumors. Targeteddelivery of Ags to professional antigen-presenting cells (APC)enhances immunity against the delivered Ags through the gener-ation of Ag-specific CD8þ T cells (8, 9). Here, we explored single-domain antibody fragments (also known as nanobodies orVHHs) as a therapeutic vaccine platform to deliver and inducecellular immunity against such VHH-conjugated antigens in anHPV cancer model.

VHHs are derived from the variable region of heavy chain–only antibodies naturally present in the serum of camelids(10). They are the smallest antibody fragments that retain Agspecificity (11). Their small size confers a number of desirableproperties, such as stability, solubility, ease of production andmodification, short circulatory half-lives, and similarity tohuman immunoglobulin V region sequences (12, 13). Wedemonstrated that VHHs specific for immune cell surfaceproteins (i.e., MHC class II, CD36, and CD11b) can deliverconjugated Ags to APCs and elicit an immune response to them(14). An Ag conjugated to the anti-CD11b VHH (VHHCD11b orDC13) raised the strongest CD8þ T-cell response against amodel peptide antigen derived from ovalbumin. CD11b is the

1Program in Cellular andMolecular Medicine, Boston Children's Hospital, Boston,Massachusetts. 2Department of Pediatrics, Harvard Medical School, Boston,Massachusetts. 3Department of Biology, Massachusetts Institute of Technology,Cambridge, Massachusetts. 4Whitehead Institute for Biomedical Research,Cambridge, Massachusetts. 5Department of Molecular Microbiology andImmunology, University of Southern California, Los Angeles, California. 6NorrisComprehensive Cancer Center, University of Southern California, Los Angeles,California. 7Department of Obstetrics and Gynecology, University of SouthernCalifornia, Los Angeles, California.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Authors: Andrew W. Woodham, Boston Children's Hospital, 1Blackfan Circle, 06005A, Boston, MA 02115. Phone: 323-434-6011; E-mail:[email protected]; andHidde L. Ploegh, Phone: 617-919-1613; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0661

�2018 American Association for Cancer Research.

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a subunit of integrin aMb2 (Mac-1), expressed on neutrophils,macrophages, myeloid dendritic cells (DC), NK cells, andsubsets of CD8þ T cells and B cells (15). To conjugate an Agto a specific site on the VHH, we installed a sortase A recog-nition motif near the C-terminus of the VHH and used anenzymatic transacylation reaction to attach a triglycine-equipped peptide (G3-peptide) containing the Ag of interest(16, 17). We thus conjugated an immunodominant HPV type16 E7 Ag (HPV16 E749-57) to VHHCD11b via a sortase A–catalyzed reaction (Fig. 1). This VHH–Ag conjugate was suc-cessful as a therapeutic vaccine in an HPV cancer model in mice.

Materials and MethodsAll procedures were performed in accordancewith institutional

guidelines and approved by the Boston Children's HospitalInstitutional Animal Care and Use Committee (IACUC protocolnumber 16-12-3328).

Antibodies and reagentsRat anti-CD11b-PE (clone M1/70) was purchased from

eBioscience. Anti-mouse H-2Db, H-2Kb, and CD80 were pur-chased from BioLegend. Anti-mouse CD40 (clone 1C10) waspurchased from Southern Biotech. Polyinosinic–polycytidylicacid (Poly(I:C)) and lipopolysaccharide (LPS) from Escherichiacoli (E. coli) were purchased from Sigma-Aldrich.

Mice and cell cultureSpecific pathogen-free female (6–8-week-old) C57BL/6 mice

were purchased from The Jackson Laboratory (000664), andOT-IRag-deficient (Rag�/�) mice were purchased from Taconic Bios-ciences (2334-F). Tumor challenge studies were performed withthe C3.43 HPV16-transformed murine cell line (18, 19). C3.43cells were a gift fromW.Martin Kast, PhD, andweremaintained inIscove's modified Dulbecco's medium (IMDM) supplementedwith 10% heat-inactivated fetal bovine serum (IFS), 50 mmol/L2-mercaptoethanol, and 100U/mLpenicillin/streptomycin. Cells

Figure 1.

VHHCD11b-E749-57 conjugate design, purification, and validation. A, i, VHHCD11b (yellow) was cloned from the VHH genes of an immunized alpaca, expressedwith a C-terminal sortase-recognition motif (LPETG) and purified via FPLC. ii, The E749-57 antigen (green) was synthesized with a G3 motif and was sitespecifically conjugated to VHHCD11b via a sortase A (purple)-mediated reaction. iii, The reaction mixture was purified in two steps. First, Ni-NTA beads wereused to remove his-tag containing sortase A and unreacted VHHCD11b. Second, size exclusion was used to remove unreacted E749-57 antigen. B, Chromatogramsshowing the retention times of different species on a C8 HPLC column of (i) the purified sortase-ready VHH, (ii) the complete reaction mixture, and (iii) thefinal product following two-step purification. C, i–iii, The calculated and observed mass(es) of the primary peaks (at 1.36, 1.43, and 1.43 minutes in the panelsabove, respectively) of the corresponding HPLC chromatograms.

Nanobody–Antigen Conjugates Elicit Anti-HPV Tumor Responses

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used for tumor challenges were cultured no longer than 2 weeksfrom original seed stocks before in vivo injection. The murinedendritic DC2.4 and B-cell lymphoma A20 cell lines wereobtained from the ATCC and maintained in RPMI 1640 supple-mented with 10% IFS, 2 mmol/L glutamine, MEM nonessentialamino acids, 1 mmol/L sodium pyruvate, 50 mmol/L 2-mercap-toethanol, and 100 U/mL penicillin/streptomycin. DC2.4 cellswere characterized as CD11bþH-2DbþH-2KbþCD80þ as assessedby cytofluorimetry (Figs. 2A and 3A). A20 cells did not expressCD11b (Fig. 2A). DC2.4 and A20 cells routinely test positive forknown DC and B-cell markers (i.e., DEC-205 and CD19, respec-tively) as examined by FACS and are passaged no more than5 times from cryopreserved stocks. All cell lines were found to bemouse pathogen free (and Mycoplasma pulmonis free) by IDEXXBioResearch pathogen testing.

Recombinant protein expression, peptide synthesis, andsortase reactions

Recombinant VHHs containing a sortase recognitionmotif and5� his-tag for purificationwere expressed inWK6 E. coli overnightin Terrific Broth at 30�C after isopropyl b-D-thiogalactopyrano-side induction (1 mmol/L) at an OD600 of �0.6 (sequencesavailable in Supplementary Table S1). VHHswere harvested fromthe periplasm by osmotic shock and purified by adsorption toNi-NTA beads (MCLab), followed by size-exclusion chromatog-raphyon a Superdex 7516/600 column (GEHealthcare) in PBSorTris–HCl (50 mmol/L, pH 7.5). VHHs were then concentratedusing centrifugal filters with a 10 kDa molecular weight cutoff(Millipore Sigma, UFC901024). G3-TAMRA and G3-RRAAYRA-HYNIVTF were synthesized as described (20). G3-AAYSIINFEKLwas synthesized at the MIT Biopolymers and Proteomics CoreFacility. Heptamutant Ca2þ-independent sortase A from Staphy-lococcus aureus (SrtAstaph7M) was expressed with a 5� his-tag and

purified following published procedures (20, 21). All sortasereactions were performed in 50 mmol/L Tris–HCl (pH 7.5) for 2hours at 20�C to 25�C as described (20). The reagent concentra-tions were VHH (2 mg/mL or �140 mmol/L), 300 mmol/L nucle-ophile (G3-RRAAYRAHYNIVTF, etc.), and 10 mmol/L SrtAstaph7M.Residual unconjugated VHH and SrtAstaph7M were removed byadsorption to Ni-NTA beads (MCLab). Excess nucleophile wasremoved via centrifugal filters with a 10-kDa molecular weightcutoff (Millipore Sigma, UFC901024). Identity of the final pro-ducts was confirmed by liquid chromatography–mass spectrom-etry (LC-MS; Fig. 1).

In vitro experimentsFor binding experiments, CD11bþDC2.4 cells or CD11b� A20

cells were incubated with different concentrations of TAMRA-labeled VHHCD11b or VHHcont (anti-GFP VHH; ref. 22), or PE-labeled anti-CD11bmAb (M1/70-PE) for 30minutes at 4�C, andwashed. Mean fluorescence intensity (MFI) was assessed by cyto-fluorimetry. For competition binding experiments, DC2.4 cellswere preincubated with unlabeled VHHCD11b (10 mg/mL) for30 minutes at 4�C, and then incubated with VHHCD11b-TAMRA(1 mg/mL) or M1/70-PE for 30 minutes at 4�C. MFI was thenassessed via cytofluorimetry after washing. For in vitro presenta-tion assays, DC2.4 cells were treated with 100 nmol/L OVA257-264

(SIINFEKL), VHHcont-OVA257-264, or VHHCD11b-OVA257-264 for 1hour in complete medium at 37�C and then washed with PBS.Cells were then stimulated with LPS (1 mg/mL) in completemedium overnight at 37�C. The DC2.4 cells were then collectedand seeded onto precoated 96-well enzyme-linked immunosor-bent spot (ELISpot) plates (see ELISpot assay below), towhich105

splenocytes from a RAG�/� OT-I mouse were added and incu-bated for 24 hours at 37�C (1 DC2.4 cell: 20 OT-I splenocytes).DC2.4 cells and OT-I splenocytes are both C57BL/6 derived. The

Figure 2.

Binding of VHHCD11b to CD11bþ cells. A,CD11bþ DC2.4 cells or CD11b� A20 cells wereincubated with TAMRA-labeled VHHCD11b orVHHcont (anti-GFP control VHH). The MFI asassessed by cytofluorimetry was normalizedto the average maximum observed MFI ofVHHCD11b-TAMRA on DC2.4 cells. B, DC2.4cells were preincubated with unlabeledVHHCD11b (10 mg/mL) for 30 minutes at 4�Cand then stained with VHHCD11b-TAMRA orM1/70-PE for 30 minutes at 4�C. MFI wasassessed via cytofluorimetry, and bindingwas normalized to those observed withVHHCD11b-TAMRA (left 2 columns) orM1/70-PE (right 2 columns). C and D, forDC2.4 cells were incubated with differentconcentrations of VHHCD11b-TAMRA (C) orM1/70-PE (D) for 30 minutes at 4� , and theMFI as assessed by cytofluorimetry wasnormalized to the maximum observed MFIfor VHHCD11b-TAMRA or M1/70-PE,respectively. One-site (solid) or two-site(dashed) hyperbolic binding models werethen used to calculate relative KD values. Alldata shownare representative of at least twoindependent experiments performed intriplicate (mean � SD).

Woodham et al.

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number of OVA257-264-specific interferon-g (IFNg)-secreting cellswas then measured via IFNg ELISpot assay (see below).

In vivo experimentsTo evaluate the induction of HPV16 E7-specific T cells, wild-

type C57BL/6 mice were injected intraperitoneally (i.p.) with6 nmol/L VHHCD11b, E749-57, or VHHCD11b-E749-57 plus adju-vant (adjuvant: 50 mg Poly(I:C) þ 50 mg agonistic anti-CD40),or adjuvant only. Fourteen days after vaccination, spleens wereharvested and anti-E749-57-specific CD8þ T cells were enumer-ated by IFNg ELISpot assay. In a similar experiment, mice wereinjected i.p. with 6 nmol/L VHHCD11b-E749-57 or VHHcont (anti-GFP VHH)-E749-57 plus adjuvant, or adjuvant only 14 daysprior to IFNg ELISpot assay. To evaluate the protective effects ofthe induced CD8þ T cells, C57BL/6 mice were injected i.p. withadjuvant alone; adjuvant plus 6 nmol/L E749-57 or VHHCD11b-E749-57 on days –21 and –7; or left untreated (n ¼ 5 per group).Serum was collected on days –14 and –7 to assess anti-VHHresponses (Supplementary Fig. S1). On day 0, mice werechallenged with 3 � 105 C3.43 cells subcutaneously (s.c.).Tumor growth and overall survival were then monitored. Fortherapeutic studies, C57BL/6 mice were challenged with 3 �105 C3.43 cells on day 0, resulting in palpable tumors by day 8,and then randomized. On day 8 and 15, mice received an i.p.injection with adjuvant alone; adjuvant plus 6 nmol E749-57 orVHHCD11b-E749-57; or left untreated (n ¼ 8 per group for theE749-57 and VHHCD11b-E749-57 groups, and n ¼ 4 for theuntreated group). CD8þ tumor-infiltrating lymphocytes (TIL)were evaluated via immunoPET on day 8. To evaluate immu-nological memory, mice with complete tumor regression fromthe E749-57 and VHHCD11b-E749-57 groups in the therapeuticstudy (n ¼ 3 per group) were challenged with 3 � 105 C3.43

cells contralaterally, and then tumor growth and overall sur-vival were monitored.

ELISpot assayIFNg ELISpot assays were performed following published pro-

cedures (23). Briefly, 96-well ELISpot plates (BD ELISPOTMouseIFNg ELISPOT Set, BD Biosciences) were coated with an IFNgcapture antibody (BD Biosciences) in PBS overnight at 4�C andplates were blocked with complete medium for 2 hours atroom temperature. For in vitro presentation experiments, DC2.4cell/OT-I splenocyte cocultures were set up in quadruplicate asdescribed above. Negative control wells contained only DC2.4cells. For in vivo experiments, splenocytes from vaccinated micewere added in triplicate to wells containing the H-2Db-restrictedE749-57 peptide (2 mg/mL) in completemediumormedium aloneas a negative control. Positive control wells for all experimentscontained 1� Cell Stimulation Cocktail (Invitrogen). After sple-nocyte plating, ELISpot plates were incubated overnight at 37�C.After 24 hours, plates were washed and incubated with a bioti-nylated IFNg detection antibody (BD Biosciences) for 2 hours,followed by streptavidin–horse radish peroxidase (BD Bios-ciences) for 1 hour at room temperature. The plates were devel-oped with 3-amino-9-ethyl-carbazole substrate (BD ELISPOTAEC Substrate Set) for 5 minutes and dried for at least 24 hours.Spotswere enumerated using theKSELISpot analysis system (CarlZeiss), and the number of spots in negative control wells wassubtracted from experimental wells to determine the number ofspots above background.

ImmunoPET imaging and analysisRadiolabeling of the anti-CD8 VHH with 89Zr (VHHCD8-

89Zr)was performed as described (Fig. 5D; ref. 24). Specifically,

Figure 3.

Antigen presentation is increased with VHHCD11b. A,DC2.4 cells express CD11b, H-2Kb (MHCI known topresent OVA257-264), and CD80 (costimulatorymolecule)as assessed via cytofluorimetry (light gray histograms).Isotype controls (isotype) are shown as empty dashedhistograms. CD80 and H-2Kb expression was increasedfollowing 24-hour incubation with LPS (dark grayhistograms). B–C, DC2.4 cells were treated as indicatedbefore LPS stimulation, followed by coculture withsplenocytes from a RAG�/� OT-I mouse (1 DC2.4 cell:20 OT-I splenocytes), and the number of IFNg-secretingcells was measured via ELISpot assay. The spotsshown in B are quantified in C (means � SD are shown;�� , P < 0.001) of an experiment performed inquadruplicate, and data are representative of threeindependent experiments.






VHHCD8 was conjugated to G3-desferrioxamine (DFO)-azide viaSrtAstaph7M. The azide group was used to install 20 kDa polyeth-ylene glycol (PEG) moiety (as DBCO-PEG), which improves thequality of the PET signal seenwith VHHCD8 in vivo (24). A volumeof 89Zr4þ stock solution (1.0 mol/L oxalic acid adjusted to pH6.8–7.5using2.0mol/LNa2CO3) corresponding to1.0 to1.5mCiwas added to 200 mL VHHCD8-DFO solution (�2.0 mg of che-lexed PEGylated-VHHCD8-DFO in 0.5 mol/L HEPES buffer, pH7.5) in a 2 mL microcentrifuge tube, and the total volume wasadjusted to 300 mL using 1.0 mol/L oxalic acid. The reactionmixture was incubated for 60 minutes at room temperature withagitation, loaded onto a PD-10 size-exclusion column (GEHealthcare), and eluted with PBS. Radiolabeling of VHHCD11b

with 64Cu (VHHCD11b-64Cu) was performed as described (25). In

brief, VHHCD11b was conjugated to G3-NOTA via SrtAstaph7M.VHHCD11b-NOTA (400 mL; 20 mmol/L in 200 mmol/L NH4OAcbuffer; pH 6.5) was mixed with 75 mL64CuCl2 (�6 mCi in 200mmol/L NH4OAc buffer; pH 6.5). The reaction mixture wasincubated for 20 minutes at room temperature with agitation,loaded onto a PD-10 size-exclusion column (GEHealthcare), andeluted with PBS. PET-CT was performed following publishedprocedures (25). Briefly, mice were anaesthetized using isoflur-ane, injected with 20 to 50 mCi radiolabeled VHH via the tail vain,and imaged by PET-CT 24 hours later using a G8 PET-CT smallanimal scanner (PerkinElmer). PET and CT acquisition was �10and 1.5 minutes, respectively, per animal. Images were recon-structed using the manufacturer's software. Data were furtheranalyzed and quantified using VivoQuant software (inviCROImaging Service and Software). PET signal values were convertedto units of percentage of injected dose per gram by using as inputthe total radioactivity at the time of measurement with thepreprocessing tool. CT scans overlaid with PET signals were usedto generate 3D regions of interest (ROI) to represent certainregions within each mouse (i.e., tumor or lymph node). Onceall ROIswere generated, a tablewas exported containing statisticalinformation, such as mean PET signal or variation for each ROI.TIL and lymph node (LN) intensity was defined as the PET signalobserved from 89Zr-labeled VHHCD8 at the ROI over backgroundPET signal observed in muscle tissue ROI.

Statistical analysesAll statistical analyses were performed on GraphPad Prism

version 7.0 (GraphPad Software Inc.). One- and two-site hyper-bolic binding curves were used to estimate the equilibriumdissociation constants (KD values) with R-square values used asa metric to evaluate the quality of fit of the model to the data.Statistical significance for ELISpot assays, tumor growth curves,and CD8þ TILs was assessed with two-tailed unpaired t tests orANOVA. Survival curves were assessed via the Kaplan–Meiermethod, with statistical significance determined by the log-rank(Mantel–Cox) test. Alternatively, two-tailed Fisher exact tests wereused to assess differences between outcomes among groups.Significance was defined as P < 0.05 for all experiments.

ResultsDesign of VHH-antigen constructs

Mouse models of E6/E7-positive tumors show that HPVAg-specific T cells are few and comparatively poor at homingto and infiltrating tumor tissue, unless the tumor-bearinganimal is vaccinated against agents containing or encoding the

immunodominant E7-derived peptide, E749-57 (RAHYNIVTF;refs. 26, 27). This peptide is recognized by CD8þ T cells inH-2Db

–restricted fashion (18). Therefore, we synthesized theE749-57 peptide as a sortase A-ready nucleophile for conjugationto an anti-CD11b VHH (14). In between the G3 motif at theN-terminus and the E749-57 epitope, two arginines (RR) wereincluded to increase solubility; an AAY linker was added toenhance cross-presentation upon internalization by target APCs(28). The resultant sequence was GGGRRAAYRAHYNIVTF.Although standard genetic means are an obvious alternative tocreate the requisite fusions, the ability to multiplex enzymaticconjugations and extend this approach to multiple peptide orprotein Ags affords flexibility, including the site-specific installa-tion of posttranslational modifications (20, 21) and radioiso-topes for positron emission tomography (PET; ref. 25). A geneticfusion would have to be purified from an appropriate source,where appendage of a C-terminal tag (e.g., a His tag) would bedeployed for affinity enrichment. However, the presence of suchC-terminal extensions would require further processing steps thatwould likely limit the generation of the relevant peptide epitopefor MHC binding, as ER peptidases primarily trim N-terminalresidues (29). Purification tohomogeneity of the desired fusion inthe absence of a tag may not always be possible. Because of theefficiency and specificity of sortase A-catalyzed reactions, wetherefore applied the enzymatic approach, which yields thedesired fusion in yield and purity. Accordingly, VHHCD11b wasexpressed with a C-terminal sortase-recognition motif (LPETG)and 5� his-tag for purification (Fig. 1Ai). The E7 Ag nucleophile(G3-RRAAYRAHYNIVTF) was site specifically conjugated usingsortase A andpurified (Fig. 1Aii–Aiii) as verifiedby LC-MS (Fig. 1Band C). The purified VHH–Ag construct (VHHCD11b-E749-57) wasused in subsequent in vivo vaccination experiments.

VHHCD11b specifically binds to CD11bþ cells with high affinityVHHCD11b binds to the splenocytes of wild-type but not

CD11b–/– mice (14). Here, we show that VHHCD11b binds toCD11bþ DC2.4 cells, but not to CD11b� A20 cells (Fig. 2A).Competition assays with unlabeled VHHCD11b showed that thefluorophore attached to VHHCD11b did not affect its binding toDC2.4 and that VHHCD11b binds an epitope different fromthat recognized by the well-established anti-mouse CD11b mAbM1/70 (Fig. 2B). Data from our binding studies fit substantiallybetter to a two-site binding model compared with a one-sitemodel (R2 ¼ 0.9895 and 0.6182, respectively), notwithstandingmonovalency of VHHCD11b. Paradoxically, M1/70 binding waswell modeled using a one-site binding model (R2 ¼ 0.9949;Fig. 2C and D). As CD11b is a member of the integrin familycapable of undergoing dramatic conformational changes, theseresults may indicate that VHHCD11b recognizes two distinctconformations of CD11b, one with high affinity (KD1 of 0.204� 0.090 nmol/L) and one with moderate affinity (KD2 ¼ 110 �47 nmol/L), whereas M1/70 binds the different conformers withequal affinity.

Cross-presentation of OVA257-264 is increased when conjugatedto VHHCD11b

The efficacy of therapeutic vaccines against HPV-driven cancerslies in their ability to elicit strongCD8þT-cell responses against E6and E7 proteins, which in the case ofmost peptide-based vaccinesrequires internalization, processing, and cross-presentation of theAgs on class I MHC (MHCI; ref. 7). To explore the ability of

Woodham et al.





VHHCD11b to enhance Ag presentation, DCs were treated with anH-2Kb

–restricted ovalbumin peptide (OVA257-264) or an equiva-lent molar amount of peptide conjugated to VHHCD11b

(VHHCD11b-OVA257-264). OVA257-264 was chosen for these experi-ments because no TCR transgenic mice are available that recog-nize HPV antigens to serve as a source of adequate numbers ofE7-specific CD8þ T cells. We measured the expression of relevantsurface markers on the treated DCs after activation with LPS(Fig. 3A) and the corresponding CD8þ T-cell responses followingcoculture of the differentially activatedDCswith splenocytes froman OT-I OVA257-26–specific T-cell receptor (TCR) transgenicmouse (30). Treatment with the VHHCD11b-OVA257-264 conjugateresulted in a greater than 2-fold increase in the number of IFNg-secreting effector CD8þ T cells compared with peptide alone orpeptide conjugated to a nontargeting control VHH (VHHcont;Fig. 3B and C). While exogenously added peptides can binddirectly to cell surface MHCI, proteins when added atequimolar concentrations obviously still require processing andcross-presentation (31). These results show that VHHCD11b-OVA257-264 efficiently entered the cross-presentation pathway.

VHHCD11b-E749-57 vaccination elicits stronger T cell responsesthan E749-57 alone

Vaccination with 30 nmol/L E749-57 peptide with the adjuvantpoly(I:C) and an agonistic mAb to CD40 results in robust HPV-specific T-cell responses (32). Therefore, to assess the ability ofVHHCD11b-E749-57 to enhance HPV-specific T-cell responsesin vivo, the dose of E749-57 peptide and VHHCD11b-E749-57 wasreduced to just 6 nmol/L, corresponding to �100 mg ofVHHCD11b-E749-57 injected per mouse. Mice were injected i.p.with VHHCD11b, E749-57, or VHHCD11b-E749-57 plus adjuvant(poly(I:C) and anti-CD40), or adjuvant only. Two weeks aftervaccination, spleens were harvested and anti-E749-57 specificCD8þ T cells were enumerated via IFNg ELISpot assay. A singlevaccination with VHHCD11b-E749-57 resulted in a 2-fold increasein the number of IFNg-secreting effector CD8þ T cells comparedwith peptide alone (Fig. 4A, left), highlighting the enhanced

CD8þ T-cell response due to nanobody targeting via CD11b.In a similar experiment, mice were injected i.p. withVHHCD11b-E749-57 or E749-57 conjugated to a control VHH (anti-GFP: VHHcont) with adjuvant, or with adjuvant only. The controlVHH–peptide adduct did not elicit a robust anti-E749-57 response,indicating that conjugation of the peptide to a nontargeting VHHdoes not enhance the antigen-specific response (Fig. 4A, right).

Prophylactic vaccination with VHHCD11b-E749-57 conferstumor-growth protection

Wenext evaluatedwhether vaccinationwith VHHCD11b-E749-57conferred protection against a subsequent tumor challenge in vivo,using the cell line C3.43 as the HPV cancer model. C3.43 cells arederived from the C3 line by transformation with a completeHPV16 genome-containing pRSVneo-derived plasmid (18, 19).C3.43 cells retainHPV16E6 andE7 expression, andpeptides fromthem are presented on MHC class I. By day 40, only 1 of 5 miceprimed (day –21) and boosted (day –7) with VHHCD11b-E749-57and adjuvant prior to C3.43 challenge (day 0) had developed atumor. There was therefore a significantly lower tumor burden inthis group compared with that of mice receiving E749-57 plusadjuvant (three mice developed tumors) or adjuvant only (fourmice developed tumors; P ¼ 0.018 and 0.008, respectively; Fig.4B). The single tumor in the VHHCD11b-E749-57 group grew moreslowly than tumors of the E749-57 plus adjuvant and adjuvant-only groups, resulting in survival until day 70 (Fig. 4C).VHHCD11b-E749-57 conferred a significant overall survival benefitcompared with that of the adjuvant-only group (P¼ 0.03; P¼ 0.1between VHHCD11b-E749-57 and E749-57 groups), whereas nodifference in survival was observed between the E749-57 plusadjuvant and adjuvant-only groups (P ¼ 0.8; Fig. 4C).

Therapeutic vaccination with VHHCD11b-E749-57 induces tumorregression in vivo

We next evaluated the therapeutic potential of Ag conjugationto VHHCD11b. Tumor-bearing mice were treated with VHHCD11b-E749-57 and adjuvant, or E749-57 and adjuvant on days 8 and

Figure 4.

VHHCD11b-E749-57 vaccine-induced HPV-specific T-cell responses and VHHCD11b-E749-57 vaccine-induced protection against C3.43 tumor growth in vivo. A,Wild-type C57BL/6 mice were injected i.p. with VHHCD11b, E749-57, or VHHCD11b-E749-57 plus adjuvant (adj. ¼ 50 mg Poly(I:C) þ 50 mg agonistic anti-CD40 Ab),or adj. only (left). Fourteen days after vaccination, spleens were harvested and anti-E749-57–specific CD8þ T cells were enumerated via IFNg ELISpot. In aseparate experiment, mice were injected i.p. with 6 nmol/L VHHCD11b-E749-57 or VHHcont (anti-GFP VHH)-E749-57 plus adj., or adj. only 14 days prior to IFNg ELISpotassay (right). Horizontal bars indicate the average number of spots per group from 1 of 2 experiments (n¼ 3 per group per experiment; �P < 0.05). B–C,Wild-typeC57BL/6 mice were injected i.p. with VHHCD11b, E749-57, or VHHCD11b-E749-57 plus adj., or adj. only on days �21 and �7 or left untreated (n ¼ 5 per group). On day0, mice were challenged with 3 � 105 C3.43 cells. Tumor growth (B) and survival (C) were monitored, and the average group means from 1 of 2 independentexperiments are shown (group means � SEM are shown; � , P < 0.05; �� , P < 0.01).






15 after a subcutaneous challengewith C3.43 cells. Tumor growthand overall survival were monitored. The VHHCD11b-E749-57 plusadjuvant group had a significantly lower tumor burden (7/8miceshowed complete tumor regression) than that of the E749-57 plusadjuvant group (4/8mice had complete regression; P¼0.002; Fig.5A), and the survival benefit of VHHCD11b-E749-57 over E749-57

trended toward significance (P ¼ 0.08, Fig. 5B). No mice in theuntreated group exhibited tumor regression (0/4), and all diedwithin 40 days of challenge; thus, both the VHHCD11b-E749-57 andE749-57 treatment groups provided significant survival benefitsover untreated controls (P < 0.01). After day 40, the tumor in theone tumor-bearing mouse from the VHHCD11b-E749-57 plus

Figure 5.

VHHCD11b-E749-57 vaccine induced responses in tumor-bearing mice in vivo. A–B, Wild-type C57BL/6 mice were challenged with 3 � 105 C3.43 cells on day0 resulting in palpable tumors (�200 mm3) on day 8. Mice were then injected i.p. with E749-57 (n ¼ 8) or VHHCD11b-E749-57 (n ¼ 8) plus adj. on days 8 and 15or left untreated (n ¼ 4). Tumor growth (A) and survival were monitored (means � SEM are shown; �� , P < 0.01 as assessed by an unpaired t test). C, Mice withcomplete tumor regression from E749-57 or VHHCD11b-E749-57 groups in Bwere rechallenged 80 days after the initial challenge with 3� 105 C3.43 cells contralaterallyand survival was monitored (n ¼ 3 per group). D, Schematic of 89Zr-labeled VHHs preparation for immunoPET imaging (DFO: desferoxamine; PEG: polyethyleneglycol). E–F, All tumor-bearing mice from A were injected via the tail vein with VHHCD8-

89Zr on day 15 and CD8þ T cells were imaged in vivo on day 16 byPET-CT on the same day. TIL, draining lymph node (DLN), and contralateral lymph node (CLN) CD8þ T-cell intensities from the PET images were quantified(means�SEMare shown; � ,P<0.05). The ratios of the intensities in theDLN toCLN shown inF (graybars) are plotted according to the right y-axis (shown in gray).G,Tumor growth was stratified by TIL intensity (responder TIL intensity >5 (n ¼ 9); nonresponder <5 (n ¼ 11); means � SEM are shown; �� , P < 0.001). H–J,Representative PET-CT images from the untreated group (H), E749-57 þ adj. group (I), and VHHCD11b-E749-57 þ adj. group (J) from 1 of 2 independent experiments.White arrows in the coronal (left), insets from the coronal (bottom right), and transverse (top right) images indicate the sites of the tumors. Green and yellowarrows indicate the sites of the DLN and CLN, respectively.

Woodham et al.





adjuvant group grew more slowly than those of tumor-bearingmice in the E749-57 plus adjuvant group, resulting in survival untilday 75 (Fig. 5B). Mice with complete tumor regression from boththe E749-57 and VHHCD11b-E749-57 groups were rechallenged onthe opposite flank with C3.43 tumor cells 80 days after the initialtumor challenge. None of these rechallenged mice developedtumors, suggesting that the level of immunological memory wasno different between the two groups (Fig. 5C).

We have developed methods to noninvasively image immunecell subsets in vivo using nanobody-based immuno-PET (Fig. 5D;refs. 24, 25). We used immunoPET to monitor CD8þ TILs in alltumor-bearing mice 15 days after tumor challenge, which was7 days after the first vaccination for the VHHCD11b-E749-57 andE749-57 groups (representative PET images are shownin Fig. 5H–J). We saw greater TIL intensity (see Materials andMethods) in the tumors of VHHCD11b-E749-57-treated mice com-pared with those in untreated mice (Fig. 5E). No obvious differ-ence was seen when comparing the E749-57 and untreated groups.When stratified by TIL intensity as measured by PET (responder>5; nonresponder <5), there was a significantly lower tumorburden in responders than in nonresponders (Fig. 5G), and TILintensity was significantly correlated with survival (P ¼ 0.01),indicating the prognostic value of TILs visualized noninvasivelyby immunoPET. Similarly, we measured the CD8þ T-cell signalintensities in the draining and contralateral lymph nodes (DLNand CLN, respectively) via immunoPET. The CD8þ T-cell signalintensities in both the DLN and CLN were increased in theVHHCD11b-E749-57 group compared with the other groups, witha significant difference observed between the DLN signal inten-sities of the VHHCD11b-E749-57 and E749-57 groups (Fig. 5F). Wehypothesized that the better induction of antigen-specific CD8þ Tcells in the VHHCD11b-E749-57 group as seen by ELISpot (Fig. 4A)would yield ahigherCD8þT-cell signal in theDLNof E7þ tumors,where antigen-specific T cells are expected to accumulate duringan antitumor response. Indeed, we found that the ratio of CD8þ

T-cell signal intensity in the DLN over that in the CLN washighest in the VHHCD11b-E749-57 group (Fig. 5F, gray bars). Wesuggest this is due to the presence of a greater number of antigen-specific T cells.

CD11bþ cells are found in C3.43 tumors in vivoCD11bþ cells have been observed in the skin of transgenic

K14E7 mice that constitutively express E7 in squamous epi-thelium (33). Therefore, we utilized VHHCD11b as an immu-noPET imaging agent in C3.43 tumor-bearing mice to test forthe presence of intratumoral CD11bþ cells. We labeledVHHCD11b with 64Cu and injected it into wild-type mice(Fig. 6A) or mice harboring �200 mm3 (16 days after chal-lenge) C3.43 tumors with or without VHHCD11b-E749-57 treat-ment (mice were treated on day 8). C3.43 tumors were found tobe positive for VHHCD11b-

64Cu staining (Fig. 6B and C).Although we do not know where exactly induction of theanti-E7 response occurs in VHHCD11b-E749-57 vaccinated mice,a pool of Ag delivered to the tumor site may allow residentAPCs to acquire it. If these APCs were to migrate to the draininglymph node, further E7-specific T cells may be recruited fromthat lymph node and participate in the antitumor response.Such localized delivery may be especially important whenantigen levels are limiting. This imaging experiment furtherhighlights the utility of sortase A to conjugate payloads toVHHs, as we used the identical sortase-ready VHH as a delivery

vehicle for the E7 antigen in one setting, and as an immunoPETimaging agent in another.

DiscussionAs HPV DNA testing becomes more widely implemented as a

primary screening method for HPV infection (34), more womenwill test positive for high-risk HPV infection, including those withnormal cytology. More than 15% of women infected with high-risk HPV strains do not initiate effective immune responsesagainst the virus, leaving them at risk for the development oflow-grade cervical intraepithelial neoplastic (CIN1) lesions (35).Although CIN1 is often eliminated by the host's immune system,regression of high-grade lesions (CIN2/3) is far less common.Progression is thus correlated with limited immune responses,increasing the risk for developing invasive cancers (36, 37). Atherapeutic HPV vaccine that can stimulate the immune system toclear established HPV infections is needed, especially for patientswith consecutiveHPVþDNA tests or with CIN1 lesions, for whichthe only treatment option is watchful waiting (38).

We explored whether an HPV vaccine based on anti-CD11bVHH-targeted delivery of an HPV antigen-derived peptide wouldelicit stronger antitumor CD8þ T-cell responses than vaccinationwithpeptide alone.Ourdata support that hypothesis. Specifically,MHC class I presentation of an ovalbumin-derived peptide washighest when conjugated to VHHCD11b, compared with equimo-lar amounts of peptide delivered via a control VHH or as peptidealone. Other conjugates comprising VHHCD11b and a differentpeptide should be handled similarly. The VHHCD11b-E749-57

Figure 6.

ImmunoPET analysis of CD11bþ cells in vivo. Wild-type mice or micechallenged with 3 � 105 C3.43 cells 16 days prior [with or without VHHCD11b-E749-57 treatment (Tx) on day 8] were injected via the tail vein withVHHCD11b-

64Cu and imagedbyPET-CT (n¼4per group). Representative PET-CTimages from (A) wild-type, (B) C3.43 tumor-bearing without Tx, and (C) C3.43tumor–bearing with Tx animals are shown from two independent experiments.Images from A and B were collected on the same day, and C was acquiredon a separate day. White arrows in the coronal (top) and transverse (bottom)images indicate the sites of the tumors.






construct elicited a strong CD8þ T-cell response in vivo, whichafforded prophylaxis against C3.43 tumor growth. VHHCD11b-E749-57 therapeutic vaccination induced TILs as seen in live miceby immunoPET and resulted in tumor regression and increasedsurvival. Considerably less Ag was needed than previouslyreported to achieve strong E749-57-specific responses (i.e. 6 vs.30 nmol/L) when conjugated to VHHCD11b (32). The approachreported here can be extended to the inclusion of multipleepitopes. Collectively, these data demonstrate that targeted deliv-ery of HPV Ags to CD11bþ cells via VHH conjugates is a viablestrategy to elicit effective cellular immune responses against HPV-induced cancers. An extension to other tumor models wouldrequire the availability of a similarly immunodominant peptide,but currently such sequences are not known for other commonlystudied preclinical cancer models, such as murine B16-F10 mel-anoma or MC-38 colon adenocarcinoma.

Other studies have shown that targeted delivery of an Ag toCD11bþ cells can induce strong CD8þ T-cell responses. Specifi-cally, a detoxified form of Bordetella pertussis adenylate cyclasetoxin (CyaA), a ligand of CD11b, has been used to deliver Ags asCyaA fusion products to elicit MHC class I– and MHC class II–restricted T-cell responses, both in vitro and in vivo (39, 40). SuchCyaA–E7 fusions elicit HPV-specific CD8þ T-cell responses andcorresponding antitumor responses in vivo (41). However, themolecular mass of these CyaA fusions is �180 kDa, which maylimit access to tissues harboring appropriateCD11bþpopulationsof APCs. In contrast, the VHH–Ag constructs are �15 kDa,providing superior tissue penetration, even compared with con-ventional antibodies (�150 kDa; refs. 42, 43). Our previous workalready showed that a conventional full-size Ag-modified anti-body was less efficient in eliciting Ag-specific CD4þ T-cellresponses than a VHH conjugated covalently to that same Ag(14). As to the use of CyaA as an alternative delivery vehicle, itsbacterial origin entails the added risk of immunogenicity and thegeneration of CyaA-neutralizing antibodies, which may compro-mise efficacy when a CyaA-based vaccine requires repeatedadministration. VHHs are comparatively poorly immunogenicas well (Supplementary Fig. S1) and can be humanized ifnecessary (44). VHHCD11b binds more tightly to CD11bþ cells(KD value 0.204 � 0.090 nmol/L on DC2.4 cells) than CyaA(KD 9.2� 4.5 and 3.2� 1.9 nmol/L depending on cell type). Ourprevious (14) and current work shows that VHHCD11b-Ag con-structs have their payloads presented on MHCI. Their ease ofexpression, excellent tissue penetration, high affinity, and inter-changeability of VHHs and Ags in VHH–Ag constructs thus createa flexible vaccine platform.

E7 expression in the skin of mice results in expansion ofCD11bþ cells (33). In particular, more CD11bþGr1þ cells werepresent in the skin of transgenic K14E7 mice (33), which consti-tutively express HPV16 E7 in squamous epithelium (45).CD11bþGr1þ cells are defined as myeloid-derived suppressorcells (MDSC) that can show intratumoral immunosuppressiveactivity (46). If sites ofHPV infection and/orHPV-induced cancersare at the same time foci of CD11b expression, then deliveringimmunodominant HPV Ags to these locations under stimulatoryconditionsmay activate local APCs, whichmay also pick up otherHPV Ags in addition to those delivered, thus allowing for epitopespreading. This might occur despite the immunosuppressionassociated with increased CD11bþGr1þ cells, but if and how abalance between the action of stimulatory APCs and suppressiveMDSCs is established in our vaccination protocol will require

further study. We readily visualized the presence of CD11bþ cellsinE7-expressingC3.43 tumors in vivo via immunoPET, but it is notyet known whether these cells are associated with the observedanti-E7 immune responses in VHHCD11b-E749-57 vaccinatedmice.

Spatial resolution and imaging depth by PET is superior tobioluminescence-based in vivo noninvasive imaging methodssuch as the current in vivo imaging systems, a technique not easilytranslatable to a clinical setting (47). Moreover, PET providesquantitative information on the distribution of label (i.e., 89Zr)throughout the entire animal, with a spatial resolution of�1mm.Although two-photon microscopy provides better cellular andtemporal resolution, it is an invasivemethod that requires surgicalintervention, for example, to expose lymph nodes for imaging orto implant a window that allows inspection of the underlyingtissue (48, 49). Multiphoton microscopy does not provide infor-mation on the distribution of label over the entire animal. Wecombined the spatial resolution of PET with the specificity of ananti-CD8 VHH to noninvasively visualize TILs in vivo. We foundthat TILs as seen via immunoPET were prognostic for tumorregression, in agreement with our previous work (24). Thesetechniques could be applied to any number of cancer immuno-therapy animal models and could potentially be translated tohuman cancers to not only provide prognostic information abouttreatment outcomes, but inform physicians whether patients areresponding to immunotherapeutic interventions during a givencourse of treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A.W. Woodham, R.W. Cheloha, J. Ling, J.N. Duarte,J.M. Bader, D.M. Da Silva, H.L. PloeghDevelopment of methodology: A.W. Woodham, R.W. Cheloha, M. Rashidian,J.N. Duarte, J.M. Bader, J.G. SkeateAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.W. Woodham, J. Ling, S.C. Kolifrath, M. Mesyngier,J.N. Duarte, J.G. Skeate, W.M. KastAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.W. Woodham, R.W. Cheloha, J.N. Duarte,D.M. Da Silva, H.L. PloeghWriting, review, and/or revision of the manuscript: A.W. Woodham, J. Ling,M. Mesyngier, J.N. Duarte, J.G. Skeate, D.M. Da Silva, W.M. Kast, H.L. PloeghAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.C. KolifrathStudy supervision: A.W. Woodham, H.L. Ploegh

AcknowledgmentsJ.G. Skeate was supported by the Keck School of Medicine/USC Graduate

School PhD Fellowship. ELISpot plate reading was performed at the USCImmune Monitoring Core Facility supported in part by the National CancerInstitute Cancer Center Support Grant award P30CA014089. A.W.Woodham issupported by the Arnold O. Beckman Postdoctoral Fellowship. R.W. Cheloha issupported in part by funding from the Cancer Research Institute IrvingtonPostdoctoral Fellowship. M. Rashidian is supported by a fellowship from theAmericanCancer Society.W.M. Kast holds theWalter A. Richter Cancer ResearchChair and is supported by NIH R01 CA074397. H.L. Ploegh is supported bythe Lustgarten Foundation (award ID 388167) and the Melanoma ResearchAlliance (award ID 51009).

The authors also thank Jessica Ingram for providing VHHCD11b.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 17, 2017; revised January 19, 2018; accepted May 16,2018; published first May 23, 2018.

Woodham et al.





References1. Wang JW, Roden RB. Virus-like particles for the prevention of human

papillomavirus-associated malignancies. Expert Rev Vaccines 2013;12:129–41.

2. Stokley S, Jeyarajah J, Yankey D, Cano M, Gee J, Roark J, et al. Humanpapillomavirus vaccination coverage among adolescents, 2007–2013, andpostlicensure vaccine safety monitoring, 2006-2014–United States.MMWR Morb Mortal Wkly Rep 2014;63:620–4.

3. Brawner BM, Baker JL, Voytek CD, Leader A, Cashman RR, Silverman R,et al. The development of a culturally relevant, theoretically driven HPVprevention intervention for urban adolescent females and their parents/guardians. Health Promot Pract 2013;14:624–36.

4. Haedicke J, Iftner T. Human papillomaviruses and cancer. RadiotherOncol2013;108:397–402.

5. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV,et al. Human papillomavirus is a necessary cause of invasive cervical cancerworldwide. J Pathol 1999;189:12–9.

6. Stanley MA, Pett MR, Coleman N. HPV: from infection to cancer. BiochemSoc Trans 2007;35:1456–60.

7. Skeate JG, Woodham AW, Einstein MH, Da Silva DM, Kast WM. Currenttherapeutic vaccination and immunotherapy strategies for HPV-relateddiseases. Hum Vaccin Immunother 2016;12:1418–29.

8. Bonifaz LC, Bonnyay DP, Charalambous A, Darguste DI, Fujii S, SoaresH, et al. In vivo targeting of antigens to maturing dendritic cells via theDEC-205 receptor improves T cell vaccination. J Exp Med 2004;199:815–24.

9. FayolleC,OsickovaA,OsickaR,Henry T, RojasMJ, SaronMF, et al.Deliveryof multiple epitopes by recombinant detoxified adenylate cyclase ofBordetella pertussis induces protective antiviral immunity. J Virol2001;75:7330–8.

10. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, HamersC, Songa EB, et al. Naturally occurring antibodies devoid of light chains.Nature 1993;363:446–8.

11. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S.Selection and identification of single domain antibody fragments fromcamel heavy-chain antibodies. FEBS Lett 1997;414:521–6.

12. Muyldermans S.Nanobodies: natural single-domain antibodies. AnnuRevBiochem 2013;82:775–97.

13. Helma J, Cardoso MC, Muyldermans S, Leonhardt H. Nanobodies andrecombinant binders in cell biology. J Cell Biol 2015;209:633–44.

14. Duarte JN, Cragnolini JJ, Swee LK, Bilate AM, Bader J, Ingram JR, et al.Generation of immunity against pathogens via single-domain antibody-antigen constructs. J Immunol 2016;197:4838–47.

15. Arnaout MA. Structure and function of the leukocyte adhesion moleculesCD11/CD18. Blood 1990;75:1037–50.

16. Popp MW, Ploegh HL. Making and breaking peptide bonds: proteinengineering using sortase. Angew Chem Int Ed Engl 2011;50:5024–32.

17. PoppMW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL. Sortagging: aversatile method for protein labeling. Nat Chem Biol 2007;3:707–8.

18. Feltkamp MC, Smits HL, Vierboom MP, Minnaar RP, de Jongh BM,Drijfhout JW, et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by humanpapillomavirus type 16-transformed cells. Eur J Immunol 1993;23:2242–9.

19. Smith KA, Meisenburg BL, Tam VL, Pagarigan RR, Wong R, Joea DK, et al.Lymph node-targeted immunotherapy mediates potent immunity result-ing in regression of isolated or metastatic human papillomavirus-trans-formed tumors. Clin Cancer Res 2009;15:6167–76.

20. Guimaraes CP, Witte MD, Theile CS, Bozkurt G, Kundrat L, Blom AE, et al.Site-specific C-terminal and internal loop labeling of proteins usingsortase-mediated reactions. Nat Protoc 2013;8:1787–99.

21. Theile CS, Witte MD, Blom AE, Kundrat L, Ploegh HL, Guimaraes CP.Site-specific N-terminal labeling of proteins using sortase-mediated reac-tions. Nat Protoc 2013;8:1800–7.

22. Kirchhofer A, Helma J, Schmidthals K, Frauer C, Cui S, Karcher A, et al.Modulation of protein properties in living cells using nanobodies. NatStruct Mol Biol 2010;17:133–8.

23. Yan L,WoodhamAW, Da Silva DM, Kast WM. Functional analysis of HPV-like particle-activated Langerhans cells in vitro. Methods Mol Biol2015;1249:333–50.

24. Rashidian M, Ingram JR, Dougan M, Dongre A, Whang KA, LeGall C,et al. Predicting the response to CTLA-4 blockade by longitudinalnoninvasive monitoring of CD8 T cells. J Exp Med 2017;214:2243–55.

25. Rashidian M, Keliher EJ, Bilate AM, Duarte JN, Wojtkiewicz GR,Jacobsen JT, et al. Noninvasive imaging of immune responses. ProcNatl Acad Sci U S A 2015;112:6146–51.

26. Feltkamp MC, Vreugdenhil GR, Vierboom MP, Ras E, van der Burg SH,ter Schegget J, et al. Cytotoxic T lymphocytes raised against a sub-dominant epitope offered as a synthetic peptide eradicate humanpapillomavirus type 16-induced tumors. Eur J Immunol 1995;25:2638–42.

27. Kanodia S, Da Silva DM, Karamanukyan T, Bogaert L, Fu YX, Kast WM.Expression of LIGHT/TNFSF14 combined with vaccination against humanpapillomavirus Type 16 E7 induces significant tumor regression. CancerRes 2010;70:3955–64.

28. Velders MP, Weijzen S, Eiben GL, Elmishad AG, Kloetzel PM, Higgins T,et al. Defined flanking spacers and enhanced proteolysis is essential foreradication of established tumors by an epitope string DNA vaccine.J Immunol 2001;166:5366–73.

29. Blum JS, Wearsch PA, Cresswell P. Pathways of antigen processing. AnnuRev Immunol 2013;31:443–73.

30. Clarke SR, Barnden M, Kurts C, Carbone FR, Miller JF, Heath WR.Characterization of the ovalbumin-specific TCR transgenic lineOT-I: MHCelements for positive and negative selection. Immunol Cell Biol2000;78:110–7.

31. JoffreOP, Segura E, SavinaA, Amigorena S. Cross-presentation bydendriticcells. Nat Rev Immunol 2012;12:557–69.

32. Barrios K, Celis E. TriVax-HPV: an improved peptide-based therapeuticvaccination strategy against human papillomavirus-induced cancers.Cancer Immunol Immunother 2012;61:1307–17.

33. Damian-Morales G, Serafin-Higuera N, Moreno-Eutimio MA,Cortes-Malagon EM, Bonilla-Delgado J, Rodriguez-Uribe G, et al. TheHPV16 E7 oncoprotein disrupts dendritic cell function and induces thesystemic expansion of CD11b(þ)Gr1(þ) Cells in a transgenic mousemodel. Biomed Res Int 2016;2016:8091353.

34. Abraham J, Stenger M. Cobas HPV test for first-line screening for cervicalcancer. J Community Support Oncol 2014;12:156–7.

35. Giuliano AR, Harris R, Sedjo RL, Baldwin S, Roe D, Papenfuss MR, et al.Incidence, prevalence, and clearance of type-specific human papillomavi-rus infections: the young women's health study. J Infect Dis 2002;186:462–9.

36. Rodriguez AC, Schiffman M, Herrero R, Wacholder S, Hildesheim A,Castle PE, et al. Rapid clearance of human papillomavirus and implica-tions for clinical focus on persistent infections. J Natl Cancer Inst2008;100:513–7.

37. Walker JL, Wang SS, Schiffman M, Solomon D. Predicting absolute risk ofCIN3 during post-colposcopic follow-up: results from the ASCUS-LSILTriage Study (ALTS). Am J Obstet Gynecol 2006;195:341–8.

38. Massad LS, Einstein MH, Huh WK, Katki HA, Kinney WK, Schiffman M,et al. 2012 updated consensus guidelines for themanagement of abnormalcervical cancer screening tests and cancer precursors. Obstet Gynecol2013;121:829–46.

39. Guermonprez P, Khelef N, Blouin E, Rieu P, Ricciardi-Castagnoli P, GuisoN, et al. The adenylate cyclase toxin of Bordetella pertussis binds to targetcells via the alpha(M)beta(2) integrin (CD11b/CD18). J Exp Med2001;193:1035–44.

40. Loucka J, Schlecht G, Vodolanova J, Leclerc C, Sebo P. Delivery of a MalECD4(þ)-T-cell epitope into the major histocompatibility complex class IIantigen presentation pathway by Bordetella pertussis adenylate cyclase.Infect Immun 2002;70:1002–5.

41. Esquerre M, Bouillette-Marussig M, Goubier A, Momot M, GonindardC, Keller H, et al. GTL001, a bivalent therapeutic vaccine againsthuman papillomavirus 16 and 18, induces antigen-specific CD8þ Tcell responses leading to tumor regression. PLoS One 2017;12:e0174038.

42. Smolarek D, Bertrand O, Czerwinski M. Variable fragments of heavy chainantibodies (VHHs): a newmagic bulletmolecule ofmedicine? PostepyHigMed Dosw (Online) 2012;66:348–58.






43. Hassanzadeh-Ghassabeh G, Devoogdt N, De Pauw P,Vincke C, Muylder-mans S. Nanobodies and their potential applications. Nanomedicine(Lond) 2013;8:1013–26.

44. Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S,Conrath K. General strategy to humanize a camelid single-domain anti-body and identificationof a universal humanizednanobody scaffold. J BiolChem 2009;284:3273–84.

45. Herber R, Liem A, Pitot H, Lambert PF. Squamous epithelial hyperplasiaand carcinoma inmice transgenic for the humanpapillomavirus type 16E7oncogene. J Virol 1996;70:1873–81.

46. GabrilovichDI,Nagaraj S.Myeloid-derived suppressor cells as regulators ofthe immune system. Nat Rev Immunol 2009;9:162–74.

47. Zinn KR, Chaudhuri TR, Szafran AA,O'QuinnD,Weaver C, Dugger K, et al.Noninvasive bioluminescence imaging in small animals. ILAR J2008;49:103–15.

48. Dzhagalov IL, Melichar HJ, Ross JO, Herzmark P, Robey EA. Two-photonimaging of the immune system. Curr Protoc Cytom 2012;Chapter 12:Unit12.26.

49. Helmchen F, Denk W. Deep tissue two-photon microscopy. Nat Methods2005;2:932–40.


Woodham et al.




2018;6:870-880. Published OnlineFirst May 23, 2018.Cancer Immunol Res Andrew W. Woodham, Ross W. Cheloha, Jingjing Ling, et al. Immune Responses

Antigen Conjugates Elicit HPV-Specific Antitumor−Nanobody

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Nanobody Antigen Conjugates Elicit HPV-Speciﬁc Antitumor Immune Responses · Nanobody–Antigen Conjugates Elicit Anti-HPV Tumor Responses Cancer Immunol Res; 6(7) July 2018 871