Recognition of Class II MHC Peptide Ligands That Contain Β-Amino Acids Ross W

Recognition of Class II MHC Peptide Ligands That Contain β-Amino Acids Ross W. Cheloha, Andrew W. Woodham, Djenet Bousbaine, Tong Wang, Shi Liu, John Sidney, Alessandro Sette, Samuel This information is current as H. Gellman and Hidde L. Ploegh of October 1, 2021. J Immunol published online 7 August 2019 http://www.jimmunol.org/content/early/2019/08/06/jimmun ol.1900536 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2019/08/06/jimmunol.190053 Material 6.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 1, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 7, 2019, doi:10.4049/jimmunol.1900536 The Journal of Immunology

Recognition of Class II MHC Peptide Ligands That Contain b-Amino Acids

Ross W. Cheloha,* Andrew W. Woodham,* Djenet Bousbaine,*,† Tong Wang,‡ Shi Liu,‡ John Sidney,x Alessandro Sette,x,{ Samuel H. Gellman,‡ and Hidde L. Ploegh*

Proteins are composed of a-amino acid residues. This consistency in backbone structure likely serves an important role in the display of an enormous diversity of peptides by class II MHC (MHC-II) products, which make contacts with main chain atoms of their peptide cargo. Peptides that contain residues with an extra carbon in the backbone (derived from b-amino acids) have biological properties that differ starkly from those of their conventional counterparts. How changes in the structure of the peptide backbone affect the loading of peptides onto MHC-II or recognition of the resulting complexes by TCRs has not been widely explored. We prepared a library of analogues of MHC-II–binding peptides derived from OVA, in which at least one a-amino acid residue was replaced with a homologous b-amino acid residue. The latter contain an extra methylene unit in the peptide backbone Downloaded from but retain the original side chain. We show that several of these a/b-peptides retain the ability to bind tightly to MHC-II, activate TCR signaling, and induce responses from T cells in mice. One a/b-peptide exhibited enhanced stability in the presence of an endosomal protease relative to the index peptide. Conjugation of this backbone-modified peptide to a camelid single-domain Ab fragment specific for MHC-II enhanced its biological activity. Our results suggest that backbone modification offers a method to modulate MHC binding and selectivity, T cell stimulatory capacity, and susceptibility to processing by proteases such as those found within endosomes where Ag processing occurs. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/ he immune system recognizes and responds to proteina- through the action of proteases and peptidases (1, 2). The resulting ceous Ags through the production and display of short peptides are then exposed to receptive MHC molecules through a peptide Ags bound to the products of the MHC. Class I coordinated series of trafficking steps that differ for MHC-I T + MHC (MHC-I) products that stimulate CD8 T cells typically and MHC-II (3). Productive binding of peptide to MHC yields display peptides of 8–10 residues in length, derived mostly from peptide–MHC complexes that are transported for display at the intracellular proteins, whereas class II MHC (MHC-II) products surface of the APC. These complexes engage TCR complexes, that stimulate CD4+ T cells display longer peptides, mostly from stimulate TCR signaling, and drive subsequent T cell–mediated proteins originating in extracellular space or their topological immune responses. Each of these steps (proteolysis, MHC bind- by guest on October 1, 2021 equivalents. The loading of peptides onto MHC products requires ing, TCR recognition) requires recognition of the polypeptide the breakdown of the source proteins into peptide fragments Ag or some part of it. Modifications of peptide structure can affect each recognition step required for T cell engagement. Structural alterations that affect recognition by proteases can protect peptides against pro- *Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115; †Massachusetts Institute of Technology, Cambridge, MA 02142; ‡Department of teolysis, which not only generates but can also destroy the ligands x Chemistry, University of Wisconsin-Madison, Madison, WI 53706; Division of presented by MHC-II products. For example, synthetic proteins Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; and {Department of Medicine, University of California San Diego, La Jolla, composed entirely of D-amino acids are not degraded by proteases CA 92161 and cannot be processed for loading onto MHC products. Such ORCIDs: 0000-0001-9871-8333 (R.W.C.); 0000-0003-2683-9544 (D.B.); 0000- proteins therefore fail to induce immune responses that require 0003-4208-5578 (T.W.); 0000-0002-0987-405X (J.S.); 0000-0001-7013- T cell participation (4). Alternatively, the introduction of modifi- 2250 (A.S.); 0000-0001-5617-0058 (S.H.G.); 0000-0002-1090-6071 (H.L.P.). cations that inhibit extracellular proteolysis can enhance the effi- Received for publication May 9, 2019. Accepted for publication July 5, 2019. cacy of peptide vaccines (5). In some cases, endosomal proteolysis This work was supported by National Institutes of Health (NIH) National Institute of can destroy potentially antigenic peptides (6), and in this context, Allergy and Infectious Diseases Contract HHSN272201400045C (to A.S.), NIH Grant R01 GM056414 (to S.H.G.), Lustgarten Foundation Award 388167 (to H.L.P.), inhibition of proteolysis enhances immunogenicity (7, 8). These Melanoma Research Alliance Award 51009 (to H.L.P.), and NIH Grant R01-AI087879 findings suggest that carefully introduced structural modifications (to H.L.P.). R.W.C. was supported in part by a Biotechnology Training Grant from the that forestall destruction of antigenic peptides might enhance National Institute of General Medical Sciences (T32 GM008349) and a Cancer Re- search Institute Postdoctoral Fellowship. A.W.W. was supported by a Beckman Institute immunogenicity (9). Postdoctoral Fellowship. MHC-I and MHC-II bind to an enormously diverse array of Address correspondence and reprint requests to Samuel H. Gellman or Hidde L. Ploegh, peptides. The impact of side chain identities at specific sites on Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706 (S.H.G.) binding to specific allomorphs of MHC products and subsequent or Boston Children’s Hospital, 1 Blackfan Circle, RB06215, Boston, MA 02115 (H.L.P.). E-mail addresses: [email protected] (S.H.G.) or [email protected] recognition by T cells has been extensively analyzed (10, 11). (H.L.P.) The development of compounds with selectivity among these The online version of this article contains supplemental material. allomorphs could be useful to address autoimmune disorders (12). Abbreviations used in this article: DC, dendritic cell; LC/MS, liquid chromatography/ In contrast, relatively little attention has been paid to changes mass spectrometry; MHC-I, class I MHC; MHC-II, class II MHC; MOG, myelin in the peptide backbone (13). Interactions between MHC-II and oligodendrocyte glycoprotein; TFA, trifluoroacetic acid. the peptide backbone are crucial for affinity and stability of Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 the peptide-MHC-II complex (14, 15). Only a few studies have

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900536 2 MHC-II PEPTIDE LIGANDS THAT CONTAIN b-AMINO ACIDS probed the impact of peptide backbone modifications on MHC Materials and Methods binding (16–22), with diverse outcomes. TCR recognition of peptide– General MHC complexes and the ensuing T cell responses are sensitive to Cell lines were routinely tested for cell surface marker expression peptide backbone modifications as well (23–30). and mycoplasma infection. Liquid chromatography/mass spectrometry Replacement of a-residues with b-amino acid counterparts has (LC/MS) was performed on a Waters Xevo TOF system equipped with an been used to probe the importance of peptide backbone structure HPLC-C8 column. Calculations and graphing were performed using in a variety of contexts (31, 32). Each a→b replacement adds a Microsoft Excel and GraphPad Prism 6. EC50 values were generated using carbon atom to the backbone compared with the analogous con- a sigmoidal dose-response with variable slope model (four-parameter model). Statistical significance was evaluated using a two-tailed, paired ventional peptide. Such replacements can retain the original side t test. chain. The incorporation of a b-amino acid residue provides protection against proteolysis, at least for peptide bonds in proximity to Flow cytometry and Abs the site of incorporation (33–35). Carefully designed a/b-peptides For cytofluorimetry, gating was performed on viable cells based on forward (i.e., peptides containing a mixture of a-andb-residues) can mimic scatter and side scatter profiles. Fluorescently labeled Abs were purchased the conformations and biological properties of a-helical peptides, from eBioscience (anti-CD45.1, A20; anti-CD4, RM4-5; anti-CD11c, N418) or BioLegend (anti-CD45.2, 104; anti–I-A/I-E, M5/114.15.2). Prolifer- but the a/b-peptides display greater resistance to proteolysis and ation was assessed using the dye CellTrace Violet (Life Technologies) have prolonged activity in vivo (36–40). according to manufacturer’s instructions. Staining for cell surface markers Only a few studies have probed the impact of incorporating was performed at 4˚C for 30 m. Flow cytometry data were acquired on an b-residues on recognition by the machinery of the immune system; LSRFortessa (Becton Dickinson) instrument and analyzed with the FlowJo MHC-I restricted peptide epitopes have been favored (41–43). In software package (Tri-Star). general, the MHC-I allomorph and TCR involved in recognition of Peptide synthesis Downloaded from a conventional peptide epitope can also recognize analogues of this → Peptides were synthesized as C-terminal carboxylic acids with uncapped epitope that contain one or two a b replacements; however, no N termini on Wang resin (100–200 mesh; EMD Millipore). Amino acids general trend for tolerance to b-residue substitution was established. were activated with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium It is unclear whether certain positions in MHC-I binding epitopes hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) in the are especially tolerant to a→b replacements or whether multiple such presence of N,N-diisopropylethylamine (DIPEA), except for the C-terminal

residue. C-terminal residues were coupled onto resin using 1-(mesitylene-2- http://www.jimmunol.org/ replacements can be incorporated with retention of MHC binding and sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) as a coupling agent. Main chain TCR activation. Epitope analogues containing a b-residue density peptide amino groups were Fmoc-protected. The growing peptide chain was sufficient for robust protection from proteolysis were generally unable deprotected using 20% piperidine in dimethylformamide (DMF). Protected 3 to bind MHC-I or elicit TCR responses (44). b ‐homoamino acids were purchased from PepTech and Chem-Impex. Because the mode of peptide interaction with MHC-I differs After synthesis, peptides were cleaved from the resin, and side chains were deprotected using reagent K (82.5% trifluoroacetic acid [TFA], 5% from that of MHC-II, the impact of a→b modifications on MHC-II phenol, 5% H2O, 5% thioanisole, 2.5% ethanedithiol) for 2 h. The re- epitope recognition may differ from that seen for MHC-I epitopes agent K solution was dripped into cold diethyl ether to precipitate the (10). There are two relevant studies on this subject (45, 46). A deprotected peptide. Peptides were purified via reverse-phase HPLC on a ‐ model peptide derived from histone H4 was the template for a set prep C18 column. Peptide purity was assessed by analytical reverse- phase HPLC (solvent A: 0.1% TFA in water, solvent B: 0.1% TFA in by guest on October 1, 2021 of peptides in which one or two a-residues were replaced with acetonitrile, C18 analytical column (4.6 3 250 mm), flow rate 1 ml/min) 3 aza-b -residue(s). These peptides were then tested for their ability by quantifying the area under the peak corresponding to the desired product. to bind mouse MHC-II (I-Ad and I-Ae) and stimulate proliferation Peptide stocks of known concentrations were prepared by quantifying the or cytokine release by the appropriately specific T cells. Substi- absorption at 214 nm (47). tution was tolerated only at the three residues at the C terminus MHC purification and binding assays of the peptide. The backbone of aza-b3-residues differs from that b d of the b3-residues used in this work (44). The second example Purification of H-2 I-A and I-A MHC-II molecules by affinity chromatography and the performance of assays based on the inhibition of binding concerns analogues of an encephalitogenic peptide derived from of a high affinity radiolabeled peptide to quantitatively measure peptide myelin oligodendrocyte glycoprotein (MOG)33-55 binding to binding were performed as detailed elsewhere (48). Briefly, the mouse I-Ab, in which a single residue known to contact the TCR was B cell lymphoma LB27.4 was used as a source of MHC molecules. A replaced with a b3-residue (46). The analogue of MOG33-35 with high affinity radiolabeled peptide (0.1–1 nM; peptide ROIV, sequence b3-homoPhe at a TCR contact position was not encephalito- YAHAAHAAHAAHAAHAA) was coincubated at room temperature with purified MHC in the presence of a mixture of protease inhibitors and an genic and exerted a protective effect against the induction of inhibitor peptide. Following a 2-d incubation, MHC-bound radioactivity experimental autoimmune encephalitis. Although the mecha- was determined by capturing MHC/peptide complexes on mAb (Y3JP, I-Ab; nism that underlies this protection is unclear, replacement of MKD6, I-Ad)-coated Lumitrac 600 plates (Greiner Bio-One, Frickenhausen, a-Phe with b3-homoPhe clearly altered the nature of the T cell Germany) and measuring bound radioactivity using the TopCount (Packard Instrument, Meriden, CT) microscintillation counter. The concentration of response. These two precedents suggest that replacement of an peptide yielding IC of the binding of the radiolabeled peptide was calcu- a b3 50 -residue with a homologous -residue is tolerated, at least in lated. Under the conditions used, where [label] , [MHC] and IC50 $ some positions, in the context of the recognition of MHC-II [MHC], the measured IC50 values are reasonable approximations of the true restricted peptides. KD values (49, 50). Each competitor peptide was tested at six different In this study we performed a more systematic survey of a→b concentrations covering a 100,000-fold range and in three or more independent experiments. As a positive control, the unlabeled version of replacements with preservation of the native side chain. These the radiolabeled probe was also tested in each experiment. modifications allow us to isolate the impact of backbone alterations. Does the introduction of one or a few homologous Cell lines b-residues impact proteolytic processing in a way that alters The murine A20 B cell lymphoma line presenting I-Ad MHC was acquired immunogenicity in cell-based or animal-based assays? Does from American Type Culture Collection (TIB-208). The murine DO11.10 the placement of b-homologs at specific sites within an MHC- T cell hybridoma line was obtained from Dr. P. Marrack, National Jewish Health Center (Denver, CO). A20 cells were maintained in RPMI 1640 II–restricted peptide provide Ags with altered T cell stimula- with 10% heat-inactivated FBS and 0.05 mM 2-ME. DO11.10 cells were tory behavior? Our experiments were designed to answer these maintained in DMEM supplemented with 10% FBS, 100 U penicillin/50 mg questions. streptomycin per milliliter and 2 mM L-glutamine. B16-FLT3L and primary The Journal of Immunology 3

T cells were maintained in in RPMI 1640 supplemented with 10% FBS, 75 ml dH2O was added and the bacterial suspension was incubated over- 100 U penicillin/100 mg streptomycin per milliliter, 0.05 mM 2-ME, 1 mM night at 4˚C. VHHs were purified from the supernatant by Ni-NTA bead sodium pyruvate, and 2 mM L-glutamine. batch purification, followed by buffer exchange. Sortase A pentamutant was expressed and purified as described (53). Hybridoma stimulation assays Sortase conjugation conditions These assays were performed essentially as previously described (51), with a few exceptions. Each well of a 96-well plate was seeded with 50,000 A20 Sortagging reactions were performed in buffer containing 50 mM Tris, cells and DO.110 cells along with indicated concentrations of peptide in 150 mM NaCl, 5 mM CaCl2 (pH 7.4) with 10% glycerol at 12˚C overnight medium comprised of 50% A20 medium and 50% DO11.10 medium. with a 1 mM concentration of G3-peptide and 20 mM Sortase A 5M (53). After a 24 h incubation at 37˚C in a 5% CO2 atmosphere, supernatant was Sortase A-5M and unreacted VHH-LPETGG-His6 were removed by pas- transferred to a new 96-well plate and analyzed with IL-2 ELISA. All sage over nickel-NTA beads. Unreacted G3 peptide was removed through peptide doses were performed in duplicate wells and all experiments were use of an Amicon Ultra 10k cutoff centrifugal filtration device. The replicated the number of times indicated in figure captions. IL-2 levels identity of the conjugates and their purity was assessed using LC/MS. were assessed using a matched pair ELISA (no. 555148; BD Biosciences). Animal protocols ELISPOT assays with splenocytes All procedures were performed in accordance with institutional guidelines IFN-g ELISPOT assays were performed following published procedures and approved by the Boston Children’s Hospital Institutional Animal Care (52). Briefly, 96-well ELISPOT plates (BD ELISPOT Mouse IFNg ELISPOT and Use Committee (protocol number 16-12-3328). Specific pathogen-free Set; BD Biosciences) were coated with an IFN-g capture Ab (BD female (6–8 wk old) OT-II mice were purchased from The Jackson Biosciences) in PBS overnight at 4˚C, and plates were blocked with Laboratory. Prior to harvesting spleens, animals were vaccinated with complete medium (10% FBS in RPMI 1640 with added sodium pyruvate, OVA (100 mg) and polyinosinic:polycytidylic acid (50 mg) to boost T cell 2-ME, and nonessential amino acids) for 2 h at room temperature. responses. Splenocytes (5 3 104) from OT-II mice were added in duplicate to wells Downloaded from containing indicated concentration of peptide in complete medium or Results medium alone as a negative control. Positive control wells for all experiments contained Cell Stimulation Cocktail (Invitrogen). After plating Our experimental design focused on a model peptide Ag from OVA of the splenocytes, ELISPOT plates were incubated overnight at 37˚C. (OVA323-339, WT; Fig. 1). This peptide and its analogues have After 24 h, plates were washed and incubated with a biotinylated IFN-g been extensively characterized with respect to proteolytic processing, detection Ab (BD Biosciences) for 2 h, followed by streptavidin–HRP (BD MHC-II binding, and TCR activation (54–56). The portion of this Biosciences) for 1 h at room temperature. The plates were developed peptide thought to be primarily responsible for binding to MHC-II http://www.jimmunol.org/ with 3-amino-9-ethyl-carbazole substrate (BD ELISPOT AEC Substrate Set) for 5 min and dried for at least 24 h. Spots were enumerated using and engaging the TCR, also called the core region, spans residues the KS ELISPOT Analysis System (Carl Zeiss). 329–337 (51). We began by synthesizing a set of analogues in which one of the In vitro proliferation assay natural a-residues in OVA323-339 was replaced with a homolo- Dendritic cells (DCs) were purified from spleens and mesenteric lymph gous b-residue (Fig. 1A, Supplemental Fig. 1). Each replacement nodes of wild-type (WT) mice injected with 1 3 106 B16-FLT3L cells → + involved retention of the natural side chain; thus, each a b re- using the Pan Dendritic Cell Isolation Kit (Miltenyi Biotec). Naive CD4 placement simply introduced a methylene group into the peptide T cells were purified following manufacturer’s instructions (Miltenyi backbone. A side chain can be located at either of the two back- Biotech) from the spleen and mesenteric lymph nodes of OT-II mice and by guest on October 1, 2021 labeled with 3 mM CellTrace Violet (ThermoFisher Scientific). Purity was bone positions between the carbonyl and nitrogen. b3-Residues always .98% for both purified DCs and T cells. A total of 1 3 105 DCs have the side chain adjacent to nitrogen, and b2-residues have the 3 5 were incubated with peptide (50 nM) for 1 h before adding 1 10 T cells. side chain adjacent to the carbonyl. It is straightforward to in- Proliferation was assessed using dilution of VioletTrace and analyzed by flow cytometry after 3.5 d of coculture. corporate both types of b-residue into peptides by standard solid- phase synthesis methods. b3-Homologs of proteinogenic a-amino In vivo proliferation assay acids are commercially available, b3-homohistidine (b3-homoHis) TCRabKO mice received 5 3 105 naive OT-II CD4+ T cells, purified being the only exception. Therefore, we prepared analogues of following manufacturer’s instructions (Miltenyi Biotec and Thermo Fisher OVA323-339 containing single a→b3 replacements at 15 of the Scientific). Mice were immunized s.c. 24 h later, using 2 mg of peptide in 17 positions (neither of the His residues was replaced). Fewer PBS, adsorbed in alum (Imject; Thermo Fisher Scientific). Proliferation protected b2-homoamino acids are commercially available. We was assessed by enumerating the total number of OT-II T cells in the → 2 draining lymph nodes (inguinal lymph nodes). thus prepared analogues containing a b replacements at only 8 of the 17 positions in the Ag. Assessment of peptide proteolysis For each of the 22 peptides containing a single a→b replace- d Peptides were incubated with recombinant human cathepsin S (1183- ment, we characterized binding to two allelic products, I-A and CY-010; R&D Systems), preactivated according to manufacturer instruc- I-Ab, of mouse MHC-II, both of which are known to bind WT tions, in pH 4.5 sodium acetate buffer with 5 mM DTT at 37˚C. Proteolysis (51, 57). Many sites of a-to-b substitution were tolerated with was quenched at the indicated time points by the addition of an equal volume of 1% TFA in water (volume to volume ratio). A control experiment respect to binding to both MHC-II allelic products (Table I). showed that addition of TFA prior to addition of peptide (prequenching of Several of the positions in which a-to-b substitution were not protease) prevented degradation of peptide over the timeframe investigated tolerated are sensitive to side chain modification (54, 55) and/or (data not shown). Quenched samples were analyzed by LC/MS. The ion make direct contact with MHC-II (58). In general, a-to-b sub- currents corresponding to intact peptide were used to determine peptide stitutions were tolerated better outside the core of the MHC/ concentration. These values were used to create exponential decay plots and TCR-binding region of the peptide. Certain a-to-b substitutions calculate t1/2, which were modeled using GraphPad Prism 6. abrogated binding to one MHC-II allelic product while spar- Expression and purification of proteins ing binding to the other. For example, introduction of a b2-or 3 b VHHs were expressed using the pHEN6 vector as previously described (53). b -residue at position 330 (330-2, 330) abrogates binding to I-A Plasmids coding for PelB-VHH-LPETGG-His6 were transformed into but not to I-Ad.Conversely,placementofab2-residue at position WK6 Escherichia coli. Bacteria were grown under ampicillin selection at 329 (329-2) abrogates binding to I-Ab but not to I-Ad. Interpretation 37˚C. Protein expression was induced with 1 mM IPTG at 30˚C (overnight). Bacterial pellets were collected and resuspended in TES buffer of these findings in the absence of a structure of these complexes is (50 mM Tris, 650 mM EDTA, 2 M sucrose, 15 ml buffer per liter of difficult because many peptides, including OVA323-339, can bind culture) in preparation for osmotic shock. After incubating for 2 h at 4˚C, to MHC-II in different registers (51). Distinct registers differ in 4 MHC-II PEPTIDE LIGANDS THAT CONTAIN b-AMINO ACIDS

FIGURE 1. Structures of peptide analogs of OVA323-339. (A) Sequences of peptides with a single b-residue in this library. The conventional single-letter code is used to indicate a-residue identity. Colored dots indicate sites of a-to-b replacement; each b-residue bears the side chain of the a-residue indicated by the letter. The number of the peptide analog corresponds to the position of the residue in full-size OVA protein. (B) Sequence of prototype peptide and analogs with two b-residues or an N-terminal triglycine extension. (C) Structures of amino acid residues used in this study.

the residues that are crucial for binding to MHC-II and those that could not be attributed to differences in I-Ad afﬁnity, which was

contact the TCR. At present we have no information on the effect similar for 332 and 332-2. The finding that b-substitution at po- Downloaded from of a→b replacement in terms of MHC-II binding register. sition 336 or 337 was well tolerated is surprising, because these We tested T cell responses to the peptides containing single a→b residues in WT were predicted to be important for TCR engagement replacements in the context of both I-Ab and I-Ad. For I-Ad we and MHC-II binding (51, 54). For peptides that efficiently induced used the murine B cell lymphoma line (A20 cell) as the APC and IL2 release at 10 mM, a more thorough analysis of activity was the OVA323-339–specific T cell hybridoma DO11.10 as the re- conducted (Fig. 2). The dose-response measurements showed that sponder (59). Release of IL2 by DO11.10 was monitored as an several analogues matched WT in potency, and one analogue (338) http://www.jimmunol.org/ indicator of activation (51). We first surveyed T cell hybridoma was slightly more potent. activation through incubation of a mixture of A20 and DO11.10 The OT-II mouse model contains CD4+ T cells that respond cells with a single concentration of most of the peptides (10 mM, strongly to WT (54). OT-II mice on a C57/BL6 background were Supplemental Fig. 1, Table I). Peptides with b-residues outside the used to assess T cell activation by I-Ab restricted peptides. Total peptide core known to bind MHC-II and engage the TCR were unfractionated splenocyte preparations from OVA-boosted OT-II generally efficient at stimulating IL2 production, whereas many mice were used as the source of both APCs and Ag-specific with substitutions in the core region showed low or negligible T cells. T cell activation was measured by recording the number of stimulatory activity. The consequences of a-to-b substitution at IFN-g–releasing cells in an ELISPOT assay. We tested most of the 3 position 332 depended on side chain location: the b -substitution library at a high concentration (10 mM) to compare the efficacies by guest on October 1, 2021 (332) was well tolerated, but the b2-substitution (332-2) was not, of these peptides for induction of IFN-g production (Supplemental with respect to retention of T cell stimulatory activity. This difference Fig. 2, Table I). Most of the peptides with b-substitutions outside

Table I. Binding and T cell activation of peptide analogs of OVA323-339 is largely tolerant of single b-amino acid substitutions

I-Ad Binding, DO11.10 (I-Ad), IL2 Response, I-Ab Binding, OT-II (I-Ab), IFN-g Response, IC50, nM Fold WT 10 mM Peptide IC50, nM Fold WT 10 mM Peptide, ELISPOT WT 2.0 1 +++ 230 1.0 +++ 323 5.1 3 +++ 520 2.2 +++ 323-2 0.73 0.37 +++ 370 1.6 +++ 324 8.0 4 +++ 610 2.6 +++ 325 30 15 +++ 620 2.7 +++ 326 17 8 +++ 350 1.5 +++ 326-2 15 7 ND 24,000 100 ND 327 86 44 +++ 3,500 15 +++ 327-2 92 47 ++ 8,900 38 +++ 329 55 28 + 21,000 89 + 329-2 530 270 + 350 1.5 + 330 7.1 4 ND .25,000 .100 ND 330-2 14 7 — .25,000 .100 ++ 332 100 51 +++ .25,000 .100 — 332-2 94 47 + 4,500 19 ++ 333 171 86 — 950 4.1 — 334 43 22 — .25,000 .100 — 334-2 45 23 — 10,000 43 — 335 11 6 ++ 480 2.1 + 336 6.9 3 +++ 550 2.3 — 337 1.0 0.49 +++ 270 1.1 +++ 337-2 6.7 3 +++ 750 3.2 +++ 338 0.70 0.36 +++ 290 1.2 +++

d b MHC-II binding measurements (I-A and I-A ) represent the mean IC50 values from three independent experiments. Fold WT is the observed IC50 normalized relative to the IC50 of WT peptide. The responses of T cells (DO11.10 or OT-II) induced by peptides are categorized into strong (+++), medium (++), weak (+), or undetectable(—).ND indicates that this peptide was not analyzed in this assay. These categories are deﬁned and the responses are enumerated in Supplemental Fig. 1. The Journal of Immunology 5

FIGURE 2. Dose-response for IL-2 release from DO11.10 cells following peptide treatment. Peptide stimulation and measurement of IL-2 levels was performed as described in Materials and Methods.(A and B) Shown are representative dose-response curves from a single experiment for selected peptides that stimulate strong IL-2 responses in the single dose studies. Both plots in this panel are from the same experiment. WT is included in both plots for comparison. (C) Tabulation of calculated potencies for IL-2 production. Data were ﬁt to a four-parameter model as described in Materials and Methods. The number of independent replicates (n)islisted. Downloaded from

of the peptide core were quite active, whereas peptides with contain single a-to-b replacements induced proliferation of OT-II b-substitutions inside the core were only weakly active or inactive T cells. Purified OT-II T cells were incubated with purified DCs in in this assay. The exceptions to this latter trend were peptides with the presence of each peptide (50 nM). Most peptides efficiently http://www.jimmunol.org/ a-to-b substitution at position 337. Certain analogues exhibited induced proliferation (Supplemental Fig. 2). Only three peptides striking differences in T cell stimulatory activity in the different (332, 336, 334-2) failed to induce proliferation, and each of them models. For example, 336 stimulated DO11.10 T cells but not lacked IFN-g stimulatory activity. Peptides that induced IFN-g OT-II T cells, behavior that does not correlate with binding to the release only weakly still caused robust T cell proliferation (329, two MHC-II alleles (strong in both cases). For peptides that at 330-2). T cell proliferation appears to be a more sensitive test of 10 mM displayed efficacy similar to that of WT for stimulating activation than IFN-g production under the conditions tested. IFN-g release from OT-II T cells, we performed more detailed Based on results obtained with WTanalogues containing a single measurements to estimate potencies (Fig. 3). The EC values of a-to-b replacement, we prepared and tested a new analogue,

50 by guest on October 1, 2021 several analogues were within a few-fold of WT. Analogue 338 designated 2b, that contains two a-to-b replacements (Fig. 1). The was more potent than WT, as observed for IL2 release from incorporation of two b-residues is expected to improve protection DO11-10 cells. against proteolysis relative to a single substitution. The two sites Assessment of T cell proliferation serves as an alternative to the of b-residue incorporation in 2b were chosen based on tolerance measurement of cytokine release for evaluating peptide-induced of single a-to-b substitutions (results for 326 and 337). We chose T cell activation. We tested whether the WT analogues that these sites for substitution, and not sites located closer to the N or

FIGURE 3. Dose-response curve for IFN-g release from OT-II splenocyte preparations following peptide treatment. Splenocyte preparation, treatment and analysis was performed as described in Materials and Methods.(A and B) Shown are representative dose-response curves from a single experiment for selected peptides that stimulate strong IFN-g responses in the single dose studies. Both plots in this panel are from the same experiment. WT is included in both plots for comparison. Error bars correspond to SD from technical replicates performed in that experiment. (C) Tabulation of calculated potencies for IFN-g production. Data were fit to a four-parameter model as described in Materials and Methods. The number of independent replicates (n) is listed. 6 MHC-II PEPTIDE LIGANDS THAT CONTAIN b-AMINO ACIDS Downloaded from FIGURE 4. Characterization of peptide T cell stimulatory activity. (A) Representative dose-response curve and tabulated data for DO11.10 IL2 assays. Data points represent mean 6 SD. Tabulated data are derived from the indicated number of independent replicates (n). Data in this figure are distinct from those in Fig. 2. Data were fit to a four-parameter model as described in Materials and Methods.(B) Results and schematic for in vivo proliferation assays. OT-II T cells were transferred into recipient mice lacking T cells. Recipient mice were treated with peptide (1 mg) or PBS as a negative control. 3.5 d after peptide treatment inguinal lymph nodes were excised and OT-II T cells were enumerated by flow cytometry. Data represent mean 6 SD from three (PBS), four (WT), or five (2b/338) mice, respectively. *p , 0.05 versus PBS, #p , 0.05 versus 2b. http://www.jimmunol.org/

C termini of the peptide (such as 338), in an effort to provide than G3-3b, given that b-amino acid residues are known to protection from proteolysis to the central portion of the peptide. disfavor proteolytic cleavage of nearby backbone amide bonds Peptide 2b was ∼13-fold less potent than WT in the DO11.10 (33, 64). Mass spectrometry showed that the principal cleavage by T cell stimulation assay (Fig. 4A). cathepsin S occurred between residues 328 and 329 (Supplemental To test whether b-residue–containing analogues of the WT Fig. 4B). OVA peptide were active in vivo, we returned to the OT-II mouse Sortagging was used to append either G3-WT or G3-3b site- model. T cells from OT-II mice were transferred into mice that specifically to the C terminus of VHHs that target MHC-II [VHH7 lack a/b T cells (TCRa/b knockout mice). Selected peptides were (53)], CD11b [DC13 (53)], or GFP [GFP, VHH “enhancer” (65)]. by guest on October 1, 2021 then injected into recipient mice, and the proliferation of the Delivery of a peptide Ag to MHC-II–expressing cells by conju- transferred cells was analyzed by determining of the number gation to VHH enhances activation of cognate CD4+ T cells, of transferred T cells in the draining lymph node using flow whereas delivery of Ag to CD11b-expressing cells also promotes cytometry (Fig. 4B). In the absence of stimulatory peptide (PBS activation of CD8+ T cells (53). The GFP-binding VHH serves as control), few OT-II T cells were recovered. Injection of WT OVA a negative control for these experiments. We assessed the VHH- peptide resulted in the recovery of significantly more OT-II T cells, peptide conjugates and the free triglycine-containing peptides and peptide 338 was even more effective in this regard, in line used to make them for their ability to stimulate OT-II T cells using with findings from cell-based assays (Figs. 2, 3). Mice treated with an ELISPOT assay (Fig. 6). G3-WT was less potent than WT, and 2b did not differ significantly from mice that did not receive any G3-3b was only weakly active at the highest concentration tested. peptide. This lack of in vivo T cell stimulatory activity for 2b was The latter observation is in line with the lack of activity of 2b in paralleled by observations made with OT-II cells ex vivo, which showed that 2b only induced weak production of IFN-g according to ELISPOT assays (Supplemental Fig. 3). We sought to overcome the negative impact of b-residue incorporation on T cell activation through directed delivery of peptide to APC. Conjugation of an MHC-binding peptide to proteins derived from the variable domain of camelid H chain– only Abs (VHHs or nanobodies) that target cell surface proteins on APCs enhances the immunogenicity of that peptide under immunostimulatory conditions (53, 60, 61). Site-specific and monovalent functionalization of proteins using catalysis by sortase A (sortagging) is our preferred method to generate such adducts (62). We prepared two peptides to pursue this approach. G3-WT is the analogue of WT, bearing a triglycine extension at the N terminus for sortagging (Fig. 1). G -3b contains the N-terminal 3 FIGURE 5. A peptide with multiple b-residues is resistant to an endo- extension and three a-to-b replacements, including one in the somal protease. Time course of degradation of peptides by cathepsin extension. We examined the susceptibilities of G3-WT and G3-3b S. Peptides were incubated with cathepsin S for the indicated times to proteolysis by cathepsin S, an endosomal protease important and peptide abundance was analyzed by tandem LC/MS as described in for loading internalized Ags onto MHC-II (Fig. 5, Supplemental Materials and Methods. Data were fit to an exponential decay model to Fig. 4A) (63). As expected, G3-WT was degraded more rapidly provide indicated t1/2. Error bars represent SD. The Journal of Immunology 7 Downloaded from

FIGURE 6. Effective stimulation of OT-II T cells by VHHMHC-II-peptide conjugates. (A) Schematic for synthesis of VHH-peptide conjugates using sortase A catalysis. Reactions were performed as described in Materials and Methods.(B and C) Representative dose-response curves for stimulation of http://www.jimmunol.org/ IFN-g release by peptides or VHH-peptide conjugates. OT-II splenocytes were stimulated overnight and IFN-g production was measured using ELISPOT as described in Materials and Methods. Data points are mean 6 SD and curves result from the ﬁtting of four-parameter dose-response model. Data in these two panels are split into (B) active conjugates and (C) weakly active conjugates, both taken from the same experiment. (D) Tabulation of dose-response values for peptides and active VHH-peptide conjugates. Data are from at least three independent experiments with replicates shown in Supplemental Fig. 3. the in vivo OT-II proliferation assay (Fig. 4). Conjugates con- homologous b-residues on MHC-II binding and TCR engagement. sisting of VHHs that target GFP or CD11b and G3-WT were We found that a-to-b replacement was tolerated at many posi- weakly active, and conjugates of these VHHs with G3-3b were tions, mostly those outside the core of the peptide, but at a few

virtually inactive. Because of their weak activity we did not cal- within (Fig. 2, Table I). Nonetheless, amino acid substitutions by guest on October 1, 2021 culate EC50 values. In contrast, conjugates composed of VHH7 outside the core can affect T cell recognition by changing the (which targets MHC-II) and either G3-WT or G3-3b were more register that is predominantly adopted in peptide-MHC-II com- potent than any other compound tested in these assays. This trend plexes (51, 67). Substitutions at certain positions imparted selec- was also observed in assays with DO11.10 cells (Supplemental tivity for binding to different allelic variants of MHC-II (329-2, Fig. 3D, 3E). The in vivo properties of the various VHH-peptide 330-2, 330), whereas others abrogated T cell stimulatory activity adducts remain to be explored, although past work has shown that without altering MHC-II binding (330-2, 336) (Table I). The VHHMHC-II-WT is active in mice (53). demonstration that incorporation of a b-residue can abrogate TCR Discussion engagement without compromising MHC-II binding mirrors past findings with an MHC-II binding peptide derived from MOG (46). These experiments were motivated by the fact that very little is Past work has shown that b-residue incorporation can impart se- known of how b-residue incorporation affects binding to MHC-II lectivity between receptor subtypes (68) and between distinct and engagement of the TCR (30, 45, 46). Past studies have probed the impact of reduced amino acid residues (16, 21–24, 26, 27) or signaling pathways for a single receptor (69). The replacement of N-methylated residues (21, 23) on MHC-II binding and TCR a- with b-residues might therefore represent a general strategy for recognition. Reduced analogues of a-amino acid residues replace creating MHC-II binding peptides with unique functional profiles. the carbonyl group with a methylene group. This modification is Incorporation of b-residue(s) can stabilize peptides against predicted to alter backbone charge, because the amino group, hydrolysis by proteases, at least near the site of modification found in place of the former amide group, should be protonated at (33, 34). Proteolytic processing, predominantly within endosomal physiological pH. In addition, this modification should alter compartments, is an important step in transformation of protein backbone conformational properties. Amide N-methylation is Ags into peptides that can be loaded onto MHC-II (3). Whether predicted to exert a strong effect on backbone conformation, and b-residues can be introduced into MHC-II peptides at a density this change further removes an H-bond donor site. In contrast, sufficient to protect against proteolysis while retaining MHC-II replacement of a-amino acid residues with homologous b-amino binding and the ability to engage the TCR is unknown. In an ef- acid residues may lead to more modest changes, relative to replace- fort to balance proteolytic susceptibility and antigenic activity, we ments involving reduced residues or N-methyl residues (32, 66). combined two b-substitutions that were well tolerated in all assays We prepared a library of analogues of a prototypical MHC-II involving the single a-to-b library. The resulting peptide (2b) binding peptide, OVA323-339, in which a single a-amino acid effectively stimulated DO11.10 T cells (Fig. 4A). An analogue residue was replaced by the b3-homolog or the b2-homolog with a short N-terminal extension containing two Gly and one (Fig. 1). In one case, we examined a double replacement. This b-homoGly residues (G3-3b) was substantially more resistant to study reports systematic evaluation of the impact of incorporating degradation by cathepsin S, an endosomal protease, than the 8 MHC-II PEPTIDE LIGANDS THAT CONTAIN b-AMINO ACIDS

analogous peptide lacking b-residues (G3-WT) (Fig. 5). Because to productively bind to MHC-II at the cell surface to form a full-size proteins are often more stable in the presence of proteases complex that then activates TCR signaling. An alternative and than short peptide fragments, the combination of b-residue in- more likely possibility is that the peptide–VHH–MHC-II complex corporation and protein conjugation may further improve peptide must first be internalized for proteolytic processing, prior to stability. Whether exogenously added peptides that can bind to loading of peptide into the MHC-II binding groove. Although our MHC-II undergo internalization into protease-containing com- results do not differentiate between these possibilities, we note partments prior to loading onto MHC-II or bind directly to that G3-3b contains a b-residue in the area of the peptide that peptide-receptive MHC-II at the cell surface is not known (3). We should be cleaved to release the Ag for recognition by MHC-II. therefore cannot at present prove a direct link between the pro- The b-homoGly residue near the N terminus of G3-3b should teolytic resistance of 2b and the immunological properties of inhibit proteolysis of nearby peptide bonds (Fig. 5), but this effect this backbone-modified peptide but speculate that an important does not seem to be detrimental for stimulating T cell responses connection exists. (Fig. 6). Because b-residues disfavor proteolysis at nearby peptide We find that a-to-b replacements at most positions in an bonds, the a-to-b replacement strategy described here can be MHC-II binding peptide do not abrogate MHC-II or TCR en- readily applied to situations in which sites of cleavage are known. gagement. Because these replacements employ b-homologs of the In some cases, discrete proteolytic processing steps are known to original a-residues, only the peptide backbone is modified in these be important for the excision of peptide epitopes from full size peptides. Our data identify b-residue–containing peptides with a protein Ags (7, 8). Such processing steps could be modulated variety of interesting properties, including discrimination between by the incorporation of b-residues into larger peptides near sites MHC-II allelic variants, MHC-II binding without TCR engage- at which proteolysis occurs. This approach might be useful for ment, enhanced MHC affinity/TCR agonism, and enhanced sta- Downloaded from guiding the proteolytic excision of peptides with potent immu- bility in the presence of a protease. One of our backbone-modified nostimulatory properties, such as cryptic epitopes (6) present in analogues displayed enhanced activity in vivo relative to the index larger proteins. peptide (all a peptide). The in vitro T cell stimulatory activity of The observation that 338 is more potent than WT in all assays is a peptide with two b-residues was substantially enhanced by de- striking. Position 338 is outside of the peptide core of OVA323- livery to MHC-II through conjugation to an MHC-II–specific 339, yet modification at this position enhances MHC-II binding VHH. These properties highlight the utility of backbone-modified http://www.jimmunol.org/ and TCR engagement. The enhanced properties of 338 in MHC-II peptides in interrogating Ag presentation and present a strategy binding assays are unrelated to stability in the presence of pro- for the design of new antigenic peptides with improved properties. teases, as these assays were performed with purified proteins or in the presence of protease inhibitors. A more likely explanation for Acknowledgments the high potency of 338, compared with all the other b-residue– We acknowledge Dr. Philippa Marrack, National Jewish Health Center, containing analogues tested, relates to the identity of the amino Denver, CO, for provision of the DO11.10 hybridoma cell line. acid at this position. Position 338 of OVA323-339 contains the only Gly in this peptide, raising the possibility that spatial dis-

Disclosures by guest on October 1, 2021 placement of the side chain is problematic for a-to-b modifications The authors have no financial conflicts of interest. at non-Gly positions in antigenic peptides. It will be interesting to see whether such modifications at Gly in other antigenic peptides are similarly well tolerated. References 1. Villadangos, J. A., and H. L. Ploegh. 2000. Proteolysis in MHC class II antigen Although 2b proved effective at stimulating DO11.10 cells, it presentation: who’s in charge? Immunity 12: 233–239. failed to stimulate proliferation of OT-II T cells in vivo (Fig. 4) 2. York, I. A., A. L. Goldberg, X. Y. Mo, and K. L. Rock. 1999. Proteolysis and its analogue G -3b did not induce IFN-g release from OT-II and class I major histocompatibility complex antigen presentation. Immunol. 3 Rev. 172: 49–66. splenocyte preparations (Fig. 6). To overcome this lack of activity, 3. Blum, J. S., P. A. Wearsch, and P. Cresswell. 2013. Pathways of antigen pro- we made use of past observations that targeting of antigenic cessing. Annu. Rev. Immunol. 31: 443–473. peptides to APC by conjugation to VHHs of appropriate speci- 4. Dintzis, H. M., D. E. Symer, R. Z. Dintzis, L. E. Zawadzke, and J. M. Berg. 1993. A comparison of the immunogenicity of a pair of enantiomeric proteins. ficity can enhance immunogenicity (53, 60, 61). To achieve this Proteins 16: 306–308. goal we needed to introduce b-amino acids in the context of a full- 5. Croft, N. P., and A. W. Purcell. 2011. Peptidomimetics: modifying peptides in the pursuit of better vaccines. Expert Rev. Vaccines 10: 211–226. length protein. Although the cell’s protein synthesis machinery 6. Medd, P. G., and B. M. Chain. 2000. Protein degradation in MHC class II antigen typically excludes b-amino acids, methods have been developed presentation: opportunities for immunomodulation. Semin. Cell Dev. Biol. 11: to incorporate b-amino acids by reprogramming the translational 203–210. 7. Rodriguez, G. M., and S. Diment. 1995. Destructive proteolysis by cysteine machinery; however, current methods yield relatively modest proteases in antigen presentation of ovalbumin. Eur. J. Immunol. 25: 1823–1827. amounts of product, as mixtures (70). We circumvented this 8. Manoury-Schwartz, B., G. Chiocchia, V. Lotteau, and C. Fournier. 1997. Se- problem by using sortagging to conjugate peptides with b-residues lective increased presentation of type II collagen by leupeptin. Int. Immunol. 9: 581–589. to the C termini of VHHs. We used sortagging to attach G3-WT or 9. Moudgil, K. D., E. E. Sercarz, and I. S. Grewal. 1998. Modulation of the immunogenicity of antigenic determinants by their flanking residues. Immunol. G3-3b to the C termini of VHHs that target MHC-II, CD11b, or Today 19: 217–220. GFP. Conjugation of G3-WT or G3-3b to VHHMHC-II resulted in 10. Rammensee, H. G. 1995. Chemistry of peptides associated with MHC class I and dramatically enhanced T cell stimulatory activity in vitro, whereas class II molecules. Curr. Opin. Immunol. 7: 85–96. conjugation of either peptide to VHH or VHH did not. 11. Sidney, J., B. Peters, N. Frahm, C. Brander, and A. Sette. 2008. HLA class I CD11b GFP supertypes: a revised and updated classification. BMC Immunol. 9: 1. This finding is in line with work showing that conjugation of 12. Fernando, M. M. A., C. R. Stevens, E. C. Walsh, P. L. De Jager, P. Goyette, + antigenic peptides to VHHMHC-II dramatically enhanced CD4 R. M. Plenge, T. J. Vyse, and J. D. Rioux. 2008. Defining the role of the MHC in T cell and Ab responses (53). It is unknown whether MHC-II can autoimmunity: a review and pooled analysis. PLoS Genet. 4: e1000024. 13. Painter, C. A., and L. J. Stern. 2012. Conformational variation in structures of simultaneously bind peptide conjugated to the C terminus of classical and non-classical MHCII proteins and functional implications. Immunol. Rev. 250: 144–157. VHHMHC-II and the VHH itself, either in cis to one and the same 14. McFarland, B. J., J. F. Katz, C. Beeson, and A. J. Sant. 2001. Energetic asymmetry MHC-II, or in trans, to an adjoining MHC-II. If simultaneous among hydrogen bonds in MHC class II*peptide complexes. Proc. Natl. Acad. Sci. binding is possible, then peptide conjugated to VHH might be able USA 98: 9231–9236. The Journal of Immunology 9

15. Arneson, L. S., J. F. Katz, M. Liu, and A. J. Sant. 2001. Hydrogen bond integrity 41.Guichard,G.,A.Zerbib,F.A.LeGal,J.Hoebeke,F.Connan,J.Choppin, between MHC class II molecules and bound peptide determines the intracellular J. P. Briand, and J. G. Guillet. 2000. Melanoma peptide MART-1(27-35) fate of MHC class II molecules. J. Immunol. 167: 6939–6946. analogueswithenhancedbindingcapacitytothehumanclassIhistocom- 16. Benkirane, N., G. Guichard, J. P. Briand, and S. Muller. 1996. Exploration of patibility molecule HLA-A2 by introduction of a beta-amino acid residue: requirements for peptidomimetic immune recognition. Antigenic and immuno- implications for recognition by tumor-infiltrating lymphocytes. J. Med. genic properties of reduced peptide bond pseudopeptide analogues of a histone Chem. 43: 3803–3808. hexapeptide. J. Biol. Chem. 271: 33218–33224. 42.Reinelt,S.,M.Marti,S.De´dier,T.Reitinger,G.Folkers,J.A.L.deCastro, 17. Bergseng, E., J. Xia, C. Y. Kim, C. Khosla, and L. M. Sollid. 2005. Main and D. Rognan. 2001. Beta-amino acid scan of a class I major histocompat- chain hydrogen bond interactions in the binding of proline-rich gluten ibility complex-restricted alloreactive T-cell epitope. J. Biol. Chem. 276: peptides to the celiac disease-associated HLA-DQ2 molecule. J. Biol. Chem. 24525–24530. 280: 21791–21796. 43. Webb, A. I., M. A. Dunstone, N. A. Williamson, J. D. Price, A. de Kauwe, 18. Bianco, A., C. Brock, C. Zabel, T. Walk, P. Walden, and G. Jung. 1998. New W. Chen, A. Oakley, P. Perlmutter, J. McCluskey, M. I. Aguilar, et al. 2005. synthetic non-peptide ligands for classical major histocompatibility complex T cell determinants incorporating beta-amino acid residues are protease resistant class I molecules. J. Biol. Chem. 273: 28759–28765. and remain immunogenic in vivo. J. Immunol. 175: 3810–3818. 19. Bolin, D. R., A. L. Swain, R. Sarabu, S. J. Berthel, P. Gillespie, N. J. S. Huby, 44. Cheloha, R. W., J. A. Sullivan, T. Wang, J. M. Sand, J. Sidney, A. Sette, R. Makofske, L. Orzechowski, A. Perrotta, K. Toth, et al. 2000. Peptide and M. E. Cook, M. Suresh, and S. H. Gellman. 2015. Consequences of periodic peptide mimetic inhibitors of antigen presentation by HLA-DR class II MHC a-to-b(3) residue replacement for immunological recognition of peptide epi- molecules. Design, structure-activity relationships, and X-ray crystal structures. topes. ACS Chem. Biol. 10: 844–854. J. Med. Chem. 43: 2135–2148. 45. Dali, H., O. Busnel, J. Hoebeke, L. Bi, P. Decker, J. P. Briand, M. Baudy-Floc’h, 20. McFarland, B. J., J. F. Katz, A. J. Sant, and C. Beeson. 2005. Energetics and and S. Muller. 2007. Heteroclitic properties of mixed alpha- and aza-beta3- cooperativity of the hydrogen bonding and anchor interactions that bind peptides peptides mimicking a supradominant CD4 T cell epitope presented by nucleosome. to MHC class II protein. J. Mol. Biol. 350: 170–183. Mol. Immunol. 44: 3024–3036. 21. Guichard, G., S. Calbo, S. Muller, P. Kourilsky, J. P. Briand, and J. P. Abastado. 46. McDonald, C. A., N. L. Payne, G. Sun, D. J. Clayton, M. P. Del Borgo, 1995. Efficient binding of reduced peptide bond pseudopeptides to major his- M. I. Aguilar, P. Perlmutter, and C. C. A. Bernard. 2014. Single b3-amino acid tocompatibility complex class I molecule. J. Biol. Chem. 270: 26057–26059. substitutions to MOG peptides suppress the development of experimental 22. Quesnel, A., A. Zerbib, F. Connan, J. G. Guillet, J. P. Briand, and J. Choppin. autoimmune encephalomyelitis. J. Neuroimmunol. 277: 67–76. 2001. Synthesis and antigenic properties of reduced peptide bond analogues 47. Kuipers, B. J. H., and H. Gruppen. 2007. Prediction of molar extinction co- Downloaded from of an immunodominant epitope of the melanoma MART-1 protein. J. Pept. Sci. efficients of proteins and peptides using UV absorption of the constituent 7: 157–165. amino acids at 214 nm to enable quantitative reverse phase high-performance 23. Cotton, J., M. Herve´,S.Pouvelle,B.Maille`re, and A. Meńez. 1998. Pseudopeptide liquid chromatography-mass spectrometry analysis. J. Agric. Food Chem. ligands for MHC II-restricted T cells. Int. Immunol. 10: 159–166. 55: 5445–5451. 24. Bastian, M., J. M. Lozano, M. E. Patarroyo, G. Pluschke, and C. A. Daubenberger. 48. Sidney, J., S. Southwood, C. Moore, C. Oseroff, C. Pinilla, H. M. Grey, and 2004. Characterization of a reduced peptide bond analogue of a promiscuous CD4 A. Sette. 2013. Measurement of MHC/peptide interactions by gel filtration T cell epitope derived from the Plasmodium falciparum malaria vaccine candidate or monoclonal antibody capture. Curr. Protoc. Immunol. Chapter 18: Unit

merozoite surface protein 1. Mol. Immunol. 41: 775–784. 18.3. http://www.jimmunol.org/ 25. de Haan, E. C., E. E. Moret, J. P. A. Wagenaar-Hilbers, R. M. J. Liskamp, and 49. Cheng, Y., and W. H. Prusoff. 1973. Relationship between the inhibition constant M. H. M. Wauben. 2005. Possibilities and limitations in the rational design (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of modified peptides for T cell mediated immunotherapy. Mol. Immunol. 42: of an enzymatic reaction. Biochem. Pharmacol. 22: 3099–3108. 365–373. 50. Gulukota, K., J. Sidney, A. Sette, and C. DeLisi. 1997. Two complementary 26. Calbo, S., G. Guichard, P. Bousso, S. Muller, P. Kourilsky, J. P. Briand, and methods for predicting peptides binding major histocompatibility complex J. P. Abastado. 1999. Role of peptide backbone in T cell recognition. J. Immunol. molecules. J. Mol. Biol. 267: 1258–1267. 162: 4657–4662. 51. Roy, B. M., D. V. Zhukov, and J. A. Maynard. 2012. Flanking residues are 27. Stemmer, C., A. Quesnel, A. Pre´vost-Blondel, C. Zimmermann, S. Muller, central to DO11.10 T cell hybridoma stimulation by ovalbumin 323-339. PLoS J. P. Briand, and H. Pircher. 1999. Protection against lymphocytic choriomeningitis One 7: e47585. virus infection induced by a reduced peptide bond analogue of the H-2Db-restricted 52. Yan, L., A. W. Woodham, D. M. Da Silva, and W. M. Kast. 2015. Functional CD8(+) T cell epitope GP33. J. Biol. Chem. 274: 5550–5556. analysis of HPV-like particle-activated Langerhans cells in vitro. Methods Mol. 28. Andersson, I. E., T. Batsalova, S. Haag, B. Dzhambazov, R. Holmdahl, J. Kihlberg, Biol. 1249: 333–350. by guest on October 1, 2021 and A. Linusson. 2011. (E)-alkene and ethylene isosteres substantially alter the 53. Duarte, J. N., J. J. Cragnolini, L. K. Swee, A. M. Bilate, J. Bader, J. R. Ingram, hydrogen-bonding network in class II MHC A(q)/glycopeptide complexes and A. Rashidfarrokhi, T. Fang, A. Schiepers, L. Hanke, and H. L. Ploegh. 2016. affect T-cell recognition. J. Am. Chem. Soc. 133: 14368–14378. Generation of immunity against pathogens via single-domain antibody-antigen 29. Bianco, A., C. Zabel, P. Walden, and G. Jung. 1998. N-Hydroxy-amide analogues constructs. J. Immunol. 197: 4838–4847. of MHC-class I peptide ligands with nanomolar binding affinities. J. Pept. Sci. 4: 54. Robertson, J. M., P. E. Jensen, and B. D. Evavold. 2000. DO11.10 and OT-II 471–478. T cells recognize a C-terminal ovalbumin 323-339 epitope. J. Immunol. 164: 30. Hart, M., and C. Beeson. 2001. Utility of azapeptides as major histocompat- 4706–4712. ibility complex class II protein ligands for T-cell activation. J. Med. Chem. 44: 55. Sette, A., S. Buus, S. Colon, J. A. Smith, C. Miles, and H. M. Grey. 1987. 3700–3709. Structural characteristics of an antigen required for its interaction with Ia and 31. Horne, W. S. 2011. Peptide and peptoid foldamers in medicinal chemistry. recognition by T cells. Nature 328: 395–399. Expert Opin. Drug Discov. 6: 1247–1262. 56. Michalek, M. T., B. Benacerraf, and K. L. Rock. 1989. Two genetically identical 32. Checco, J. W., and S. H. Gellman. 2016. Targeting recognition surfaces on antigen-presenting cell clones display heterogeneity in antigen processing. Proc. natural proteins with peptidic foldamers. Curr. Opin. Struct. Biol. 39: 96–105. Natl. Acad. Sci. USA 86: 3316–3320. 33. Hook, D. F., P. Bindschadler,Y.R.Mahajan,R.Sebesta,P.Kast,and ¨ 57.Marrack,P.,R.Endres,R.Shimonkevitz,A.Zlotnik,D.Dialynas,F.Fitch, D. Seebach. 2005. The proteolytic stability of ‘designed’ beta-peptides and J. Kappler. 1983. The major histocompatibility complex-restricted an- containing alpha-peptide-bond mimics and of mixed alpha,beta-peptides: tigen receptor on T cells. II. Role of the L3T4 product. J. Exp. Med. 158: application to the construction of MHC-binding peptides. Chem. Biodivers. 1077–1091. 2: 591–632. 58. Scott, C. A., P. A. Peterson, L. Teyton, and I. A. Wilson. 1998. Crystal structures 34. Werner, H. M., C. C. Cabalteja, and W. S. Horne. 2016. Peptide backbone of two I-Ad-peptide complexes reveal that high affinity can be achieved without composition and protease susceptibility: impact of modification type, position, large anchor residues. Immunity 8: 319–329. and tandem substitution. ChemBioChem 17: 712–718. 59. White, J., K. M. Haskins, P. Marrack, and J. Kappler. 1983. Use of I region- 35. Steer, D. L., R. A. Lew, P. Perlmutter, A. I. Smith, and M. I. Aguilar. 2002. Beta- restricted, antigen-specific T cell hybridomas to produce idiotypically specific amino acids: versatile peptidomimetics. Curr. Med. Chem. 9: 811–822. anti-receptor antibodies. J. Immunol. 130: 1033–1037. 36. Horne, W. S., L. M. Johnson, T. J. Ketas, P. J. Klasse, M. Lu, J. P. Moore, and 60. Woodham, A. W., R. W. Cheloha, J. Ling, M. Rashidian, S. C. Kolifrath, S. H. Gellman. 2009. Structural and biological mimicry of protein surface rec- M. Mesyngier, J. N. Duarte, J. M. Bader, J. G. Skeate, D. M. Da Silva, et al. ognition by alpha/beta-peptide foldamers. Proc. Natl. Acad. Sci. USA 106: 2018. Nanobody-antigen conjugates elicit HPV-specific antitumor immune re- 14751–14756. 37. Horne, W. S., M. D. Boersma, M. A. Windsor, and S. H. Gellman. 2008. sponses. Cancer Immunol. Res. 6: 870–880. Sequence-based design of alpha/beta-peptide foldamers that mimic BH3 do- 61.Fang,T.,C.H.M.J.VanElssen,J.N.Duarte,J.S.Guzman,J.S.Chahal, mains. Angew. Chem. Int. Ed. Engl. 47: 2853–2856. J. Ling, and H. L. Ploegh. 2017. Targeted antigen delivery by an anti-class II 38. Johnson, L. M., S. Barrick, M. V. Hager, A. McFedries, E. A. Homan, MHC VHH elicits focused aMUC1(Tn) immunity. Chem. Sci. (Camb.) 8: M. E. Rabaglia, M. P. Keller, A. D. Attie, A. Saghatelian, A. Bisello, and 5591–5597. S. H. Gellman. 2014. A potent a/b-peptide analogue of GLP-1 with prolonged 62. Guimaraes, C. P., M. D. Witte, C. S. Theile, G. Bozkurt, L. Kundrat, A. E. Blom, action in vivo. J. Am. Chem. Soc. 136: 12848–12851. and H. L. Ploegh. 2013. Site-specific C-terminal and internal loop labeling of 39. Cheloha, R. W., A. Maeda, T. Dean, T. J. Gardella, and S. H. Gellman. 2014. proteins using sortase-mediated reactions. Nat. Protoc. 8: 1787–1799. Backbone modification of a polypeptide drug alters duration of action in vivo. 63. Plu¨ger, E. B. E., M. Boes, C. Alfonso, C. J. Schro¨ter, H. Kalbacher, H. L. Ploegh, Nat. Biotechnol. 32: 653–655. and C. Driessen. 2002. Specific role for cathepsin S in the generation of antigenic 40. Checco, J. W., D. F. Kreitler, N. C. Thomas, D. G. Belair, N. J. Rettko, peptides in vivo. Eur. J. Immunol. 32: 467–476. W. L. Murphy, K. T. Forest, and S. H. Gellman. 2015. Targeting diverse protein- 64. Johnson, L. M., W. S. Horne, and S. H. Gellman. 2011. Broad distribution of protein interaction interfaces with a/b-peptides derived from the Z-domain energetically important contacts across an extended protein interface. J. Am. scaffold. Proc. Natl. Acad. Sci. USA 112: 4552–4557. Chem. Soc. 133: 10038–10041. 10 MHC-II PEPTIDE LIGANDS THAT CONTAIN b-AMINO ACIDS

65. Rothbauer, U., K. Zolghadr, S. Tillib, D. Nowak, L. Schermelleh, A. Gahl, 68. Liu, S., R. W. Cheloha, T. Watanabe, T. J. Gardella, and S. H. Gellman. 2018. N. Backmann, K. Conrath, S. Muyldermans, M. C. Cardoso, and H. Leonhardt. Receptor selectivity from minimal backbone modification of a polypeptide 2006. Targeting and tracing antigens in live cells with fluorescent nanobodies. agonist. Proc. Natl. Acad. Sci. USA 115: 12383–12388. Nat. Methods 3: 887–889. 69. Hager, M. V., L. M. Johnson, D. Wootten, P. M. Sexton, and S. H. Gellman. 2016. 66. Johnson, L. M., and S. H. Gellman. 2013. a-Helix mimicry with a/b-peptides. b-arrestin-biased agonists of the GLP-1 receptor from b-amino acid residue in- Methods Enzymol. 523: 407–429. corporation into GLP-1 analogues. J. Am. Chem. Soc. 138: 14970–14979. 67. Levisetti, M. G., A. Suri, S. J. Petzold, and E. R. Unanue. 2007. The insulin- 70. Melo Czekster, C., W. E. Robertson, A. S. Walker, D. So¨ll, and A. Schepartz. specific T cells of nonobese diabetic mice recognize a weak MHC-binding 2016. In vivo biosynthesis of a b-amino acid-containing protein. J. Am. Chem. segment in more than one form. J. Immunol. 178: 6051–6057. Soc. 138: 5194–5197. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

Peptide name Peptide mass calculated [M+H] Peptide mass observed Purity (%) OT-II (I-Ab), IFNγ response DO11.10 (I-Ad), IL2 response WT 1773.9 1773.7 >95 10 μM peptide, ELISpot 10 μM peptide, ELISA 323 1787.9 1787.9 >95 Average SD Mean SD 323-2 1787.9 1788.2 >95 WT 362 45 WT 1.23 0.082 324 1787.9 1788.1 >95 323 351 35 323 1.12 0.064 325 1787.9 1788.3 88 323-2 357 72 323-2 1.22 0.004 326 1787.9 1788.5 92 324 295 107 324 1.26 0.077 326-2 1787.9 1787.6 >95 325 368 51 325 1.31 0.043 327 1787.9 1789.3 >95 326 383 42 326 1.23 0.217 327-2 1787.9 1788.2 >95 326-2 ND 326-2 ND 329 1787.9 1788.7 >95 327 381 80 327 1.18 0.066 329-2 1787.9 1788.1 >95 327-2 343 105 327-2 0.77 0.073 330 1787.9 1788.6 >95 329 106 41 329 0.29 0.070 330-2 1787.9 1787.9 >95 329-2 93 14 329-2 0.28 0.048 332 1787.9 1788.2 92 330 ND 330 ND 332-2 1787.9 1787.8 >95 330-2 177 29 330-2 0.17 0.000 333 1787.9 1788.2 >95 332 22 10 332 1.16 0.064 334 1787.9 1788.2 >95 332-2 198 64 332-2 0.20 0.006 334-2 1787.9 1787.6 77 333 7 1 333 0.13 0.014 335 1787.9 1788.3 >95 334 11 12 334 0.19 0.009 336 1787.9 1788.2 >95 334-2 13 6 334-2 0.15 0.006 337 1787.9 1788.1 >95 335 53 16 335 0.50 0.037 337-2 1787.9 1787.9 >95 336 8 7 336 0.92 0.060 338 1787.9 1788 >95 337 339 56 337 1.18 0.012

G3-WT 1944 1943.2 >95 337-2 320 33 337-2 0.97 0.050 2β 1799.9 1800.1 93 338 399 80 338 1.19 0.046

G3-3β 1986 1985.2 87 Background 0.14 0.003 +++ is < 250 spots +++ is < 0.9 ++ is 100

Supporting Figure 2: OT-II T cell proliferation induced by peptides. (a) Summary of OT-II proliferation in the presence of peptides at a dose indicated (50 nM, unless otherwise indicated). Proliferation was measured by flow cytometry as described in the methods section. Error bars represent standard deviation from replicate 2 or 3 experiments. (b) Representative flow cytometry plots measuring dye dilution of OT-II T cells in response to the peptide listed below the plot.

Supporting Figure 3. Effective stimulation of T cells by VHHMHC-II-peptide conjugates. (a-d) Dose-response curves for stimulation of IFNγ release by peptides or VHH-peptide conjugates. Note that each of these panels have data from independent experiments. OT-II splenocytes were stimulated overnight and IFNγ production was measured using ELISpot as described in the methods section. (e) Representative dose-response curve for stimulation of IL2 release from DO11.10 cells. Data points are mean ± SD and curves result from the fitting of four parameter dose-response model. (f) Tabulation of dose-response values for peptides and active VHH- peptide conjugates. Data are from ≥ 2 independent experiments. Data in this table for WT are identical to those presented in Figure 3.

Supporting Figure 4: Characterization of peptide degradation by cathepsin S. (a) LC/MS spectra total ion chromatographs of peptides at indicated durations of incubation with protease. The peaks corresponding to the intact peptide and the fragments observed, as determined by mass spectra, are indicated in specific panels. Positions of β-residue incorporation are underlined. Retention times of peaks (m) are listed above the corresponding peaks. (b) Position of site of cleavage for each of the peptides tested. The bond cleavage by cathepsin S is indicated by the vertical line. Sites of β-residue incorporation in G3-3β are underlined.