WO2021155132A1 - De novo stable, modular pd-1 binding proteins and oligomeric variants - Google Patents

Abstract

Disclosed herein are PD-1 binding polypeptides, fusion proteins and oligomers thereof, and methods for using them to treat cancer and/or autoimmune disease.

Description

De Novo Stable, Modular PD-1 Binding Proteins and Oligomeric Variants

Cross Reference

This application claims priority to U.S. Provisional Patent Application Serial No. 62/967093 filed January 29, 2020, incorporated by reference herein in its entirety.

Federal Funding Statement:

This invention was made with government support under Grant No. U01 GM094665, awarded by the National Institutes of Health. The government has certain rights in the invention

Sequence Listing Statement:

A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on January 12, 2021, having the file name “19-2448-PCT_Sequence-Listing_ST25.txt” and is 82 kb in size.

Background

PD-1 expressed on activated T cells inhibits T cell function and proliferation to prevent an excessive immune response, and disease can result if this delicate balance is shifted in either direction. Tumor cells often take advantage of this pathway by over- expressing the PD-1 ligand PD-L1 to evade destruction by the immune system. Alternatively, if there is a decrease in function of the PD-1 pathway, unchecked activation of the immune system and autoimmunity can result.

Summary

In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 82-90, or selected from SEQ ID NOS: 85-90, In one embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, or all 17 following residues are invariant: C1, C3, C5, G21, C26, K28, L30, E32, C33, Q35, N37, P38, G39, A40, 144, Q45, and C46. In another embodiment, amino acid substitutions relative to the reference polypeptide are selected from those listed in Table 1. In a further embodiment, beta strand residues in the polypeptide are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding residues in the reference amino acid sequence. In another embodiment, at least 75%, 80%, 85%, 90%, 91 %, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid changes in the polypeptide relative to the reference polypeptide occur in the alpha helix and/or loop. In various embodiments, the polypeptide is capable of binding to human PD-1 at the PD-L1 interface and/or is capable of binding to murine PD-1 at the PD-L1 interface; and/or the polypeptide is capable of binding to human PD-1 at the PD-L1 interface with a K_d of -100 nM or less, and/or is capable of binding to murine PD-1 at the PD-L1 interface with a Ka of ~100 nM or less. In another embodiment, the polypeptide comprises at least 2 or 3 disulfide bonds. In other embodiments, the polypeptide may be linked to one or more tumor treating agents, tumor targeting agents, autoimmunity treating, or tissue-targeting agents. In a further embodiment, the polypeptide or construct further comprises an oligomerization domain; in a non-limiting embodiment, the oligomerization domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 2-22, 25-29, 31-32, 34-36, 38-41, 43-45, 47, 49-55, and 58-79. In various embodiments, the polypeptide or construct is a dimer, trimer, tetramer, pentamer, or hexamer. In a further embodiment, the polypeptide or construct comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS:91 -98. In another embodiment, the disclosure provides compositions comprising 2, 3, 4, 5, 6, or more copies of the polypeptide or construct of any embodiment or combination of embodiments disclosed herein.

In various other aspects, the disclosure provides nucleic acids encoding the polypeptide, construct, or composition of any embodiment or combination of embodiments disclosed herein; vectors comprising the nucleic acid of the disclosure operatively linked to a promoter, or host cells comprising the polypeptide, construct, composition, nucleic acid, and/or the vector of any embodiment or combination of embodiments disclosed herein. In another aspect, the disclosure provides pharmaceutical compositions comprising

(a) the polypeptide, construct, composition, nucleic acid, vector, and/or host cell of any of any embodiment or combination of embodiments disclosed herein; and (b) a pharmaceutically acceptable carrier.

In a further aspect, the disclosure provides methods for treating and or limiting development of a tumor, comprising administering to a subject in need thereof an amount effective to treat or limit development of the tumor of the polypeptide, construct, pharmaceutical composition, nucleic acid, vector, or host cell of any embodiment or combination of embodiments disclosed herein.

In one aspect, the disclosure provides methods for treating or limiting development of an autoimmune disorder, comprising administering to a subject in need thereof an amount effective to treat or limit development of the autoimmune disorder of the polypeptide, construct, composition, pharmaceutical composition, nucleic acid, vector, and/or host cell of any embodiment or combination of embodiments disclosed herein.

In a further aspect, the disclosure provides methods for designing PD-1 binding polypeptides, comprising the steps of any embodiment or combinations of embodiments disclosed herein.

Description of the Figures

Figure 1(a)-(j): Computational design of de novo PD-1 binding miniproteins. (a-c) Binding motifs used for design in complex with murine PD-1. Water molecules placed in crystal structure as are shown as spheres and hydrogen bonds as dashed lines. (a) ‘WDYKY’ binding motif (SEQ ID NO:01) from PD-L2 (PDB 3BP5). (b) ‘ADYKR’ binding motif (SEQ ID NO: 80) from PD-L1 (PDB 3BIK). (c) ‘WDYKR’ hybrid binding motif (SEQ ID NO: 81). (d-f) Motifgraft computational design protocol. (d) A binding motif docked to mPD-1 is aligned to a set of de novo miniprotein scaffolds. (e) A graft is considered successful if both the backbone RMSD between the motif and the scaffold backbone is less than 0.7 A and the rest of the scaffold does not clash with the target. (f) Motif residues are constrained and the rest of the interface is designed to optimize interactions with PD-1. (g-j) Fold-From-Loops computational design protocol. (g) A primary binding fragment is extracted from the crystal structure of mPD-in complex with mPD-L2 (PDB 3BP5). (h) De novo backbones are generated around the PD-L2 binding fragment. (i) Cysteines are incorporated at geometrically compatible positions for disulfide bond formation. (j) Motif residues and disulfide bonds are first constrained, and then Rosetta™ sequence design is performed to stabilize the fold and increase interactions with PD-1.

Figure 2(a)-(h). Loop redesign and affinity maturation towards higher-affinity, cross- reactive PD-1 binder. (a) Original GR918 design binds mPD-1 above background in yeast display assay but not hPD-1, CTLA-4, or the Fc isotype control. (b) The Direct Segment Lookup protocol was used to extend a 4-residue beta hairpin loop (left) by matching neighboring beta strand residues (left) in the miniprotein binder with large, conformationally constrained loops from the PDB. A 9-residue loop (right) was found in a yeast display screen of loop extension designs that showed binding to both mPD-1 and hPD-1. (c) Murine and human PD- 1 binding affinities of all the evolved variants on yeast. (d) Heat map representing the log enrichments for the GR918.3 SSM library selected with 250 nM mPD-1 -Fc-biot (e) The Shannon Entropy of each position derived from the SSM data is mapped onto the model of GR918.3 ranging from conserved positions in black with a minimum value of 0.01 to variable positions in white with a maximum value of 3.11. (f) Interface residues of GR918.3 colored by Shannon Entropy as in (e) overlaid with PD-L2 interface residues in white. (g) Schematic of the yeast display competition assay. Increasing concentrations of hPD-L1 will compete with GR918.3 displayed on yeast for binding to biotinylated mPD-1 if the binding sites are similar. This will result in a decrease in fluorescent signal upon addition of streptavidin-phycoerythrin (SA-PE) by flow cytometry. (h) Flow data of the hPD-L1 yeast competition assay. Background fluorescence of yeast displaying GR918.3 incubated with SA- PE alone was 143 RFU. The same cells labeled with 0.1 μΜ mPD-1 -Fc-biot and SA-PE gave 1482 RFU, and this was reduced to 426 RFU after co-incubation with 1 μΜ hPD-L1.

Figure 3(aMd). Crystal structure and stability of GR918.2. (a) GR918.2 crystal structure aligned to the design model with a backbone RMSD of 1.33 A. (b) At only 5.6 kDa, GR918.2 captures the functional beta sheet interface of the 11.6 kDa binding domain of PD- L2. (c) CD spectra at an initial temperature of 25° C, after incubating at 95° C for 10 minutes, and then after cooling back down to 25° C. (d) Chemical denaturation with GuHCl, monitoring CD signal at 220 nm.

Figure 4(a)-(e). Monomeric and Trimeric PD-MP1 Binds PD-1 In Vitro & on Mammalian Cells. Three independent BLI binding titrations were performed to determine on and off rates for PD-MP1 binding to mPD-1 (a) and hPD-1 (b)- The on rates (km), off rates (k_off), and dissociation constants (K_d) listed are the average and standard deviation from three independent titrations. (c) Monomeric PD-MP1 binds K562 cells expressing mPD-1 in a concentration-dependent maimer, but not wild type K562 cells or cells expressing hPD-1. (d) Monomeric and trimeric PD-MP1 constructs were confirmed to form the correct oligomeric species by gel filtration followed by multi-angle light scattering (MALS). (e) Monomeric and trimeric LGm.2 proteins bind mPD-1/K562 cells with increasing affinities correlating with increasing avidity. LGm.2 monomer has a K_d for mPD-1 on K562 cells of 6 μΜ, and LGm.2 trimer has a K_d of 90 nM.

Figure 5(a)-(c). Agonistic effect of PD-MPl trimers on mouse T cells. (a)

Experiment schematic. Mouse T cells were incubated in plates coated with anti-CD3ε and PD-L1 -Fc (left) or IgG-Fc (right). PD-MPl trimer or empty trimer control were added for three days, then T cell activation status was measured via upregulation of CD69 protein. (b) Histograms and bar graphs showing T cell activation expressed as the percentage of CD69+ T cells. Mouse T cells were added to tissue culture wells coated with either aCD3ε, aCD3ε + IgG-Fc, αCD3ε+ PD-L1 -Fc, or no αCD3ε. Titrations of PD-MPl trimer or empty trimer were added to the wells immediately after. T cells were incubated for three days then analyzed via flow cytometry for CD69 expression levels in CD4+ T cells (top) and CD8+ T cells (bottom). Results shown are from the 0.44 μΜ concentration of each trimer formulation, n = 2. Error bars indicate the mean ± SD. Comparison between groups was calculated using the two-tailed unpaired Student’s /-test. ***P ≤ 0.0001 , **P ≤ 0.001, *P ≤ 0.05. (c) Percent of inhibition of T cell activation (compared to control trimers) induced by PD-MPl trimer in CD4+ T cells (left) or CD8+ T cells (right). Setting the average %CD69+ T cells treated with the empty trimer as baseline activity, the percent inhibition was calculated by subtracting the %CD69+

T cells treated with PD-MPl trimer from the baseline, then dividing the difference by the baseline activity. n = 2, error bars indicate the mean ± SD. Comparison between groups was calculated using the two-tailed unpaired Student’s /-test. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.05.

Figure 6(a)-(c). High-throughput screen of designed, binders. (a) Shape complementarity and interface size of tested sequences. (b) Shape complementarity and binding energy distribution of tested sequences. (c) Hits in yeast display high-throughput screen.

Figure 7(a)-(c). Additional affinity maturation figures. (a) Heat map representing the log enrichments for the GR918.2 SSM library selected with 2 μΜ mPD- 1 -Fc-biot. (b) Titration of mPD-L2 displayed on yeast binding to mPD-1 -Fc-biot with a Kd of 0.6 μΜ. (c) Rosetta™ model of the mutant found in the oligo array pool of loopgraft designs aligned to the original loopgraft design with the mutated residues shown in sticks.

Figure 8(a)-(b). Crystal diffraction statistics and thermal stability. (a) Data collection and refinement statistics for crystal structure. (b) Thermal denaturation of GR918.2, monitoring CD signal at 222 nm. Figure 9(a>(d). In vitro binding. (a) SDS-PAGE of various PD-MP1 constructs. M=Precision Plus Dual Xtra™ protein ladder (BioRad), 1=PD-MP1 monomer (9.7 kDa), 2=Spycatcher™_SB 13 trimer (25.8 kDa), 3=PD-MP1_SB13 trimer (35.5 kDa), (b) Gel filtration of PD-MP1 monomer on a Superdex™ 75 Increase 10/300 GL column (GE Healthcare Life Sciences). (c) Gel filtration of PD-MP 1_SB 13 trimer andSpycatcher™_SB13 unconjugated trimer on a Superdex™ 200 Increase 10/300 GL column (GE Healthcare Life Sciences). (d) mPD-l/K562 and hPD-l/K562 stable cell lines were validated using anti-mPD-l-FITC and anti-hPD-l-FITC antibodies. Detailed Description

All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al.,

1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA),

“Guide to Protein Purification” in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PC.R Protocols: A Guide to Methods and Applications (Innis, et al.

1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2^nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, “about” means +/- 5 % of the recited value.

All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above," and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 82-90.

Residue numbering of the polypeptides of SEQ ID NOS:82-90 are based on the amino acid sequences of SEQ ID NOS:85-90; thus, SEQ ID NOS: 82-84 have no amino acid residue at positions 11-15. The polypeptides have the following secondary structural regions:

Bold residues: Beta strand; Residues 1-6 (beta strand 1); residues 16-20 (beta strand 2), and residues 42-46 (beta strand 3)

Italicized residues: Alpha helix Residues 23-36 (Helix 1)

Underlined residues: Loop Residues 7-15 (Loop 1), residues 21-22 (Loop 2), residues 37-41 (Loop 3)

As described herein, the disclosure provides non-naturally occurring PD-1 binding proteins that specifically binds human and murine PD-1 at the PD-L1 interface, and thus can be used, for example, in cancer immunotherapy and/or as a therapy for treating autoimmunity, particularly when fused to a oligomeric scaffold.

As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Tip; W), tyrosine (Tyr; Y), and valine (Val; V).

In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS:85-90. This embodiment is particularly useful for binding human PD-1 at the PD-L1 interface. In a specific embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of PD-MP1 (SEQ ID NO: 90).

In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, or all 17 following residues are invariant: C1, C3, C5, G21, C26, K28, L30, E32, C33, Q35, N37, P38, G39, A40, 144, Q45, and C46. In various further embodiments, amino acid substitutions relative to the reference polypeptide are selected from those listed in Table 1. In one such embodiment, residues 11- 15 are present

In a further embodiment, beta strand residues in the polypeptides are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding residues in the reference amino acid sequence.

In one embodiment, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid changes in the polypeptides relative to the reference polypeptide occur in the alpha helix (residues 23-36) and/or loop (residues 7-15; 21-22; 37-41).

In another embodiment, the polypeptides are capable of binding to human PD-1 at the PD-L1 interface and/or are capable of binding to murine PD-1 at the PD-L1 interface. In a further embodiment, the polypeptides are capable of binding to human PD-1 at the PD-L1 interface with a K_d of -100 nM or less, and/or is capable of binding to murine PD- 1 at the PD-L1 interface with a K_d of ~100 nM or less.

The polypeptides of the disclosure may comprise 1 or more disulfide bonds. In one embodiment, the polypeptides comprise at least 2 disulfide bonds; in another embodiment, the polypeptides comprise at least 3 disulfide bonds.

PD-1 expressed on activated T cells inhibits T cell function and proliferation to prevent an excessive immune response, and disease can result if this delicate balance is shifted in either direction. Tumor cells often take advantage of this pathway by over- expressing the PD-1 ligand PD-L1 to evade destruction by the immune system. Thus, in another aspect, the disclosure provides constructs comprising a polypeptide according to any embodiment or combination of embodiments disclosed herein, linked to one or more tumor treating agents or tumor targeting agents. As a PD-1 inhibitor, the polypeptide alone or fused to other targeting or treating agents could reactivate exhausted tumor infiltrating lymphocytes (TILs) to treat cancer. Some studies have shown that regulatory T cells (Tregs) have an opposite role in cancer and therefore fusion of a Treg targeting agent to the multimeric PD-1 agonist form of the polypeptide could also treat cancer.

In various non-limiting embodiments, the tumor targeting agents may include, but are not limited to alemtuzumab (such as for leukemia), bevacizumab (such as for brain cancer, cervical cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, and ovarian cancer), cetuximab (such as for colorectal cancer, and head and neck cancer), daratumumab

(such as for multiple myeloma), denosumab (such as for sarcoma), dinutuximab (such as for pediatric neuroblastoma), elotuzumab (such as for multiple myeloma), isatuximab (such as for multiple myeloma), mogamulizumab (such as for non-Hodgkin lymphoma, mycosis fungoides and Sezary syndrome), naxitamab-gqgk (such as for neuroblastoma), necitumumab (such as for lung cancer), obinutuzumab (such as for leukemia and lymphoma), ofatumumab (such as for leukemia), olaratumumab (such as for sarcoma), panitumumab (such as for colorectal cancer), pertuzumab (such as for breast cancer), ramucirumab (such as for colorectal cancer, esophageal cancer, liver cancer, lung cancer, and stomach cancer), rituximab (such as for leukemia and lymphoma), tafasitamab (such as for lymphoma), trastuzumab (such as for breast cancer, esophageal cancer, and stomach cancer), belantamab mafodotin-blmf (such as for advanced multiple myeloma), brentuximab vedotin (such as for lymphoma), enfortumab vedotin (such as for advanced bladder cancer), gemtuzumab ozogamicin (such as for with leukemia), ibritumomab tiuxetan (such as for lymphoma), inotuzumab ozogamicin (such as for leukemia), moxetumomab pasudotox (such as for leukemia), polatuzumab vedotin (such as for lymphoma), sacituzumab govitecan-hziy (such as for breast cancer), trastuzumab demxtecan (such as for HER2-positive breast cancer), trastuzumab emtansine (such as for breast cancer).

In other non-limiting embodiments, the tumor treating agent may comprise one or more of the following therapeutics and can be used, for example, to treat the indication noted:

Bladder cancer: atezolizumab, nivohimab, durvalumab, enfortumab, vedotin-ejfv; Brain cancer: bevacizumab, everolimus;

Breast cancer: everolimus, tamoxifen, toremifene, trastuzumab, anastrozole, exemestane , lapatinib, letrozole, pertuzumab, ado-trastuzumab emtansine, palbociclib, ribociclib, neratinib maleate, abemaciclib, olaparib, talazoparib tosylate, atezolizumab, alpelisib, fam-trastuzumab deruxtecan-nxki .tucatinib, sacituzumab govitecan-hziy, pertuzumab trastuzumab, hyaluronidase-zzxf, pembrolizumab;

Cervical cancer: bevacizumab, pembrolizumab

Colorectal cancer: cetuximab, panitumumab, bevacizumab, ziv-aflibercept , regorafenib, ramucirumab, nivolumab, ipilimumab, encorafenib, pembrolizumab;

Dermatofibrosarcoma protuberans: imatinib mesylate;

Endocrine/neuroendocrine tumors: lanreotide acetate, avelumab, lutetium Lu 177- dotatate, iobenguane I 131;

Endometrial cancer: pembrolizumab, lenvatinib mesylate;

Esophageal cancer: trastuzumab, ramucirumab, pembrolizumab, nivolumab;

Head and neck cancer: cetuximab, pembrolizumab, nivolumab;

Gastrointestinal stromal tumor: imatinib mesylate, sunitinib, regorafenib, avapritinin, ripretinib;

Giant cell tumor: denosumab, pexidartinib hydrochloride;

Kidney cancer: bevacizumab, sorafenib, sunitinib, pazopanib, temsirolimus, everolimus, axitinib, nivolumab, cabozantinib, lenvatinib mesylate, ipilimumab, pembrolizumab, avelumab;

Leukemia: tretinoin, imatinib mesylate, dasatinib, nilotinib, bosutinib, rituxumab, alemtuzumab, ofatumumab, obinutuzumab, ibiutinib, idelalisib, blinatumomab, venetoclax, ponatinib hydrochloride, midostaurin, enasidenib mesylate, inotuzumab ozogamicin, tisagenlecleucel, gemtuzumab ozogamicin, rituximab, ivosidenib, duvelsib, moxetumomab pasudotox-tdfk, glasdegib maleate, gilteritinib, tagraxofiisp-erzs, acalabbmtinib;

Liver and bile duct cancer: sorafenib, regorafenib, nivolumab, lenvatinib mesylate, pembrolizumab, ramucirumab, ipilimumab, pemigatinib, atezolizumab bevacizumab;

Lung cancer: bevacizumab, crizotinib, erlotinib, gefltmib, afatinib dimaleate, certitinib, ramucirumab, nivolumab, pembrolizumab, osimertinib, necitumumab, aleclinib, atezolizumab, brigatinib, trametinib, dabrafenib, durvalumab, dacomitinib, lorlatinib, entrectinib, capmatinib hydrochloride, ipilimumab, selpercatinib, pralsetinib;

Lymphoma: ibritumomab tiuxetan, denileukin diftitox, brentuximab vedotin, rituximab, vorinostat, romidepsin, bexarotene, bortezomib, pralatrexate, ibrutinib, siltuximab, idelalisib, belinostat, obinutuzumab, nivolumab, pembrolizumab, rituximab, copanlisib hydrochloride, axicabtagene ciloleucel, acalabrutinib, tisagenlecleucel, venetoclax, mogamuliumab-kpkc, duvelisib, polatuzumab vedotin-piiq, zanubrutinib, tazemetostat hydrobromide, selinexor, tafasitamab-cxix, brexucabtagene autoleucel;

Malignant mesothelioma: ipilimumab, nivolumab;

Microsatellite instability-high or mismatch repair-deficient solid tumors: pembrolizumab;

Multiple myeloma: bortezomib, carfilzomib, panobinostat, daratumumab, ixazomib citrate, elotuzumab, selinexor, isatuximab-irfc, belantamab mafodotin-blmf;

Myelodysplastic/myeloproliferative disorders: imatinib mesylate, ruxolitinib phosphate, fedratinib hydrochloride;

Neuroblastoma: dinutuximab;

Ovarian epithelial/fallopian tube/primary peritoneal cancers: bevacizumab, olaparib, rucaparib camsyltate, niraparib tosylate monohydrate ;

Pancreatic cancer: erlotinib, everolimus, sunitinib, olaparib;

Plexiform neurofibroma: selumetinib sulfate;

Prostate cancer: cabazitaxel, enzalutamide, abiraterone acetate, radium 223 dichloride, apalutamide, darolutamide, rucaparib camsyltate, olaparib;

Skin: vismodegib, sonidegib, ipilimumab, vemurafenib, trametinib, dabrafenib, pemb rolizumab, nivolumab, cobimetinib, alitretinoin, avelumab, encorafenib, binimetmib, cemiplimab-rwlc, atezolizumab Soft tissue sarcoma: pazopanib, alitretinoin, tazemetostat hydrobromide;

Solid tumors that are tumor mutational burden-high (TMB-H): pembrolizumab; Solid tumors with an NTRK gene fusion: larotrectinib sulfate, entrectinib;

Stomach (gastric) cancer: pembrolizumab, trastuzumab, ramucirumab;

Systemic mastocytosis: imatinib mesylate, midostaurin; and Thyroid cancer: cabozantinib, vandetanib, sorafenib, lenvatinib mesylate, trametinib, dabrafenib, selpercatinib.

In non-limiting embodiments, the one or more tumor treating agents comprise polypeptides, and the construct comprises a fusion protein.

Alternatively, if there is a decrease in function of the PD-1 pathway, unchecked activation of the immune system and autoimmunity can result. Thus, in another aspect, the disclosure provides polypeptides according to any embodiment or combination of embodiments disclosed herein, linked to one or more autoimmunity treating or tissuetargeting agents. As a PD-1 agonist, this molecule could be specifically target to sites of inflammation to suppress autoreactive immune cells and/or treat autoimmunity. For example, one or more polypeptide of the disclosure could be fused to a molecule targeting a specific inflamed tissue, such as the liver in diabetes. In one non-limiting embodiments, the tissue targeting agents may comprise a collagen-binding peptide (CBP).

In other non-limiting embodiments, the autoimmunity treating agent may comprise one or more of the following therapeutics and can be used, for example, to treat the indication noted:

General Immune Suppressants:

Steroids

Examples: Prednisone, methylprednisolone, dexamethasone

Indications for use: Numerous uses in many autoimmune diseases, asthma, urticaria Colchicine

Indications for use : Gout flares, Familial Mediterranean Fever (FMF), other autoinflammatory disorders

Hydroxychloroquine

Indications for use: Lupus, rheumatoid arthritis, malaria, chronic hives and other autoimmune conditions Sulfasalazine

Indications for use: Rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, ulcerative colitis and other autoinflammatory conditions

Dapsone Indications for use: Leprosy and other infections, dermatitis herpetiformis, other autoimmune conditions Methotrexate

Indications for use: Rheumatoid arthritis, many other autoimmune diseases and certain cancers Mycophenolate Mofetil

Indications for use: Used in many other autoimmune diseases and prevention of rejection in solid organ transplantation

Azathioprine Indications for use: Used in many other autoimmune diseases and prevention of transplant rejection

Innate Immunity Anti-IL-1 Biologies Examples: Anakinra, Canakinumab, Rilonacept

Indications for use: Cryopyrin- Associated Periodic Syndromes (CAPS), including Familial Cold Autoinflammatory Syndrome and Muckle-Wells Syndrome, Systemic Juvenile Idiopathic Arthritis (canakinumab), rheumatoid arthritis (anakinra) Anti-TNF Biologies (TNF Inhibitors) Examples: Infliximab, Adalimumab, Golimumab, Etanercept, Certolizumab

Indications for use: Rheumatoid arthritis, plaque psoriasis/arthritis, ankylosing spondylitis, Crohn’s disease, ulcerative colitis

Anti-IL-6 Biologies

Examples: Tocilizumab, Sarilumab Indications for use: Rheumatoid arthritis, giant cell arteritis, juvenile idiopathic arthritis Complement Examples: Eculizumab

Indications for use: Paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (a-HUS)

Adaptive Immunity: B Cells Anti-CD20 Biologies

Examples: Rituximab Indications for use: Non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, vasculitis (such as granulomatosis with polyangiitis and microscopic polyangiitis), autoimmune skin disease B Cell Growth Factor Targeting Biologies Examples: Belimumab Indications for use: Systemic lupus erythematosus

Adaptive Immunity - T Cells

Examples: Cyclosporine

Indications for use: Prevent organ transplant rejection, graft versus host disease, rheumatoid arthritis, psoriasis, chronic urticaria T Cell Co-stimulation and Activation

Examples: Abatacept

Indications for use: Rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis

Adaptive Immunity: Cytokines Anti-IL-17 Biologies

Examples: Secukinumab, Ixekizumab, Brodalumab Indications for use: Psoriasis, psoriatic arthritis, ankylosing spondylitis Anti-IL-23 Biologies Examples: Guselkumab Indications for use: Psoriasis Anti-IL-12/23 Biologies Examples: Ustekinumab Indications for use: Psoriasis, psoriatic arthritis, Crohn’s disease

Anti-IL-5 Biologies

Examples: Mepolizumab, Reslizumab, Benralizumab

Indications for use: Severe eosinophilic asthma, other eosinophilic related disorders

Anti-IL-4/IL-13 Biologies Examples: Dupilumab

Indications for use: Atopic dermatitis, asthma, chronic rhinosinusitis with nasal polyps

Biologies Targeting IgE

Examples: Omalizumab Indications for use: Allergic asthma, Chronic urticaria

Lymphocyte (White Blood Cell) Movement

Examples: Vedolizumab

Indications for use: Inflammatory bowel disease (ulcerative colitis and Crohn’s) Small Molecules

JAK Inhibitors

Examples: Tofacitinib, Upadacitinib, Baricitinib

Indications for use: Rheumatoid arthritis, psoriatic arthritis, ulcerative colitis In one embodiment, the one or more autoimmunity treating agents or tissue targeting agents comprise polypeptides, and the construct comprises a fusion protein.

In another aspect, the disclosure provides the polypeptide or construct of any embodiment or combination of embodiments described herein further comprises an oligomerization domain. As disclosed herein, oligomerization of the polypeptides or constructs disclosed herein provide additional functionality such as PD-1 agonist activity, and are particularly useful, for example, in treating autoimmunity.

Any suitable oligomerization domain may be used. On one embodiment, the oligomerization domain may comprise an Fc domain.

In various non-limiting embodiments, the oligomerization domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 2-22, 25-29, 31-32, 34-36, 38-41, 43-45, 47, 49-55, and 58-79.

These oligomerization domains have been shown in published PCX application WO2017/173356 and US published application 2019-0112345 to be capable of forming homo-oligomers with modular hydrogen bond network-mediated specificity. The name of the oligomerization domains above indicates oligomerization state and topology, and sequences are organized by topology and oligomerization state. The first two characters indicate supercoil geometry: ‘2L’ refers to a two-layer heptad repeat that results in a left- handed supercoil; ‘3L’ refers to a three-layer 11 -residue repeat with a right-handed supercoil; and ‘5L’ refers to untwisted designs with a five-layer 18-residue repeat and straight helices (no supercoiling), where “layer” in this context is the number of unique repeating geometric slices, or layers, along the supercoil axis. The middle two characters indicate the total number of helices, and the final two indicate symmetry. Thus, “2L6HC3” denotes a left- handed, six-helix trimer with C3 symmetry. Underlined residues are optional.

In a specific embodiment, the oligomerization domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 19.

In this embodiment the resulting oligomers may have any suitable oligomerization state. In various non-limiting embodiments, the polypeptides or constructs are dimers, trimers, tetramers, pentamers, or hexamers.

In one embodiment, the polypeptide or constructs of the disclosure comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS :91-98.

Spytagged, Monomeric PD-MP1

Spytag (Bold) — optional (if present, can be any sequence for conjugation to a scaffold) (X2) Myc Tag (Bold and underlined) — optional (if present, can be any detection tag) (X4)

PD-MP 1 (PD-1 Binder) (Italicized) Residues in parentheses are linkers (XI, X3, and X5) and are optional and, when present, may be any suitable linker >Spytag_Myc_PD-MP 1

Dimeric PD-MP 1

Myc Tag (Bold) — optional (if present, can be any detection tag) (XI) His Tag (Italicized) — optional (if present, can be any purification tag) (X2)

Tev Cleavage Site (Italicized and underlined) — optional (if present, can be cleavage site to, for example, remove purification tag) (X3)

AnklC2-G3 (de novo dimer, oligomerization domain) (Bold and underlined)

PD-MP 1 (Bold, italicized, and underlined) Residues in parentheses are linkers and are optional and, when present, may be any suitable linker (X4)

> Myc His Tev ank 1 C2-G3 PD-MP 1

Trimeric PD-MP1 Spytag (Bold) — optional (if present, can be any sequence for conjugation to a scaffold) (X2) Myc Tag (Italicized) — optional (if present, can be any detection tag) (X4)

PD-MP1 (Bold and underlined)

His Tag (In parentheses) — optional (if present, can be any purification tag) (X7)

Spycatcher (Italicized and underlined) — optional (if present, can be any sequence for conjugation to a scaffold) (X9)

Avi Tag (Bold and italicized) — optional (if present, can be any detection tag) (XI 1) 2L6HC3_13 (de novo trimer, oligomerization domain) (Bold, underlined, and italicized) Other residues not annotated are optional and, if present, may be any sequence, such as any amino acid linker sequence (XI, X3, X5, X6, X8, X10, X12)

Trimeric Conjugation Construct

Spycatcher (Italicized and underlined) — optional (if present, can be any sequence for conjugation to a scaffold) (XI) Avi Tag (Bold and italicized) — optional (if present, can be any detection tag) (X3)

2L6HC3_13 (de novo trimer, oligomerization domain) (Bold, underlined, and italicized) ther residues not annotated are optional and, if present, may be any sequence, such as any amino acid linker sequence (X2, X4).

In a specific embodiment, the polypeptide or construct comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 95 (Trimeric PD-MP1)

The polypeptides or scaffolds may be linked to other compounds, such as stabilization compounds to promote an increased half-life in vivo, including but not limited to albumin, PEGylation (attachment of one or more polyethylene glycol chains), HESylation, PASylation, glycosylation, or may be produced as an Fc-fusion or in deimmunized variants, Such linkage can be covalent or non-covalent. For example, addition of polyethylene glycol

(“PEG”) containing moieties may comprise attachment of a PEG group linked to maleimide group ("PEG-MAL") to a cysteine residue of the polypeptide.

The polypeptides or constructs may be disposed on any suitable scaffold, in additional to the oligomers described herein. In these embodiments, the polypeptides or scaffolds may be genetically fused with a scaffold component, may be linked by chemical conjugation, or via any other suitable linkage In various non-limiting embodiments, the disclosure provides compositions comprising 2, 3, 4, 5, 6, or more copies of the polypeptide or construct of embodiment or combination of embodiments herein disposed on a scaffold.

In another aspect the disclosure provides nucleic acids encoding the polypeptide, constructs, or compositions of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded polypeptide, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the disclosure.

In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence. "Expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence.

Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA, In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.

In another aspect, the disclosure provides host cells that comprise the polypeptides, constructs, compositions, nucleic acids and/or expression vectors (i..e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.

In another embodiment, the disclosure provides pharmaceutical compositions comprising: (a) the polypeptide, construct, composition, nucleic acid, expression vector, or host cell of any embodiment or combination of embodiments herein; and

(b) a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the disclosure can be used, for example, in the methods of the disclosure described below. The pharmaceutical composition may comprise in addition to the polypeptide or other active agent of the disclosure (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer.

In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysofbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

The polypeptides, constructs, compositions, nucleic acids, expression vectors, and/or host cells may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use. In one embodiment, the pharmaceutical compositions further comprise one or more additional therapeutics for treating tumors, including but not limited to those described herein and anti- CTLA4 antibodies, including but not limited to ipilimumab. In another embodiment, the pharmaceutical compositions further comprise one or more additional therapeutics for treating autoimmune disorders, including but not limited to those described herein.

In a further aspect, the present disclosure provides methods for treating and/or limiting a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of one or more polypeptides, constructs, compositions, pharmaceutical compositions, nucleic acids, vectors, and/or host cells of the disclosure, to treat and/or limit development of the tumor. When the method comprises treating tumors, the one or more polypeptides, constructs, compositions, pharmaceutical compositions, nucleic acids, vectors, and/or host cells are administered to a subject that has already been diagnosed as having cancer. As used herein, ’’treat" or "treating" means accomplishing one or more of the following: (a) reducing the size or volume of tumors and/or metastases in the subject; (b) limiting any increase in the size or volume of tumors and/or metastases in the subject; (c) increasing survival; (d) reducing the severity of symptoms associated with cancer; (e) limiting or preventing development of symptoms associated with cancer; and (f) inhibiting worsening of symptoms associated with cancer. When the method comprises limiting development tumors, the one or more polypeptides, constructs, compositions, pharmaceutical compositions, nucleic acids, vectors, and/or host cells are administered to a subject that is at risk for tumor development, for example, based on genetic predisposition, family history, symptomology, etc.

The methods can be used to treat or limit development of any suitable tumor, including but not limited to tumors described herein, and/or those arising from colon cancer, melanoma, renal cell cancer, head and neck squamous cell cancer, gastric cancer, urothelial carcinoma, Hodgkin lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma colorectal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non- Hodgkin lymphoma, multiple myeloma, ovarian cancer, cervical cancer, and any tumor types selected by a diagnostic test, such as microsatellite instability, tumor mutational burden, PD- L1 expression level, or the immunoscore assay (as developed by the Society for Immunotherapy of Cancer).

The subject may be any subject that has or is at risk of developing cancer. In one embodiment the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc. In a specific embodiment, the subject is human. In a further aspect, the present disclosure provides methods for treating and/or limiting an autoimmune disorder, comprising administering to a subject in need thereof a therapeutically effective amount of one or more polypeptides, constructs, compositions, pharmaceutical compositions, nucleic acids, vectors, and/or host cells of the disclosure, to treat and/or limit development of the autoimmune disorder. When the method comprises treating an autoimmune disorder, the one or more polypeptides, constructs, compositions, pharmaceutical compositions, nucleic acids, vectors, and/or host cells are administered to a subject that has already been diagnosed as having an autoimmune disorder. As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing severity of the autoimmune disorder in the subject; (b) limiting any increase in severity of the autoimmune disorder in the subject; (c) reducing the severity of symptoms associated with the autoimmune disorder; (d) limiting or preventing development of symptoms associated with the autoimmune disorder; and (e) inhibiting worsening of symptoms associated with the autoimmune disorder When the method comprises limiting development of an autoimmune disorder, the one or more polypeptides, constructs, compositions, pharmaceutical compositions, nucleic acids, vectors, and/or host cells are administered to a subject that is at risk of developing an autoimmune disorder, for example, based on genetic predisposition, family history, symptomology, etc.

The methods can be used to treat or limit development of any suitable autoimmune disorder, including but not limited to rheumatoid arthritis, psoriatic arthritis, dermatitis herpetiformis, ankylosing spondylitis, juvenile idiopathic arthritis, autoimmune skin disease, systemic lupus erythematosus, psoriasis, inflammatory bowel disease (ulcerative colitis and Crohn’s disease), Type I and II diabetes, and multiple sclerosis.

The subject may be any subject that has or is at risk of developing an autoimmune disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc. In a specific embodiment, the subject is human.

In a further aspect, the disclosure provides methods for designing PD-1 binding polypeptides, comprising the steps of any embodiment or combinations of embodiments disclosed in the examples that follow.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within tire scope of the disclosure, as those skilled in the relevant art will recognize. Examples

PD-1 expressed on activated T cells inhibits T cell function and proliferation to prevent an excessive immune response, and disease can result if this delicate balance is shifted in either direction, Tumor cells often take advantage of this pathway by over-expressing the PD-1 ligand PD-L1 to evade destruction by the immune system. Alternatively, if there is a decrease in function of the PD-1 pathway, unchecked activation of the immune system and autoimmunity can result. Here, we present de novo designed hyperstable miniproteins that specifically bind murine PD-1 on human cells, and a trimerized version of the binder that strongly inhibits T cell activation with potential for further development as a synthetic PD-1 agonist for the treatment of autoimmunity.

Using a combination of computational design and experimental approaches, we have developed a 40 residue miniprotein, PD-MPI, that specifically binds murine and human PD-1 at the PD-L1 interface with a Kd of - 100 nM, The 5,6 kDa protein contains three disulfide bonds, making it highly stable to thermal and chemical denaturation, and can be secreted solubly from mammalian cells. The apo crystal structure shows that the binder folds as designed with a backbone RMSD of 1.3 A to the design model. Trimerization of PD-MP1 resulted in a PD-1 agonist that strongly inhibits murine T cell activation. This small, hyperstable PD-1 binding protein is one of the first computationally designed proteins with an all-beta interface, which can be difficult due to the propensity of beta sheets to aggregate, and the trimeric agonist could contribute to treatments for autoimmune and inflammatory diseases.

RESULTS

Computational Design of De Novo PD-1 Binding Proteins

In the crystal structures of murine PD-1 in complex with human PD-L1 and murine PD-L2 we identified a five-residue edge beta strand binding fragment responsible for the majority of the binding energy of these molecules. In both cases, this fragment contains mostly hydrophobic residues at positions 1, 3, and 5 that fit in a pocket on PD-1 and conserved polar residues at positions 2 and 4 that form hydrogen bonds with the backbone of the target (in PD-L2, residues B110 through B 114 from PDB 3BP5 with sequence ‘WDYKY’ (SEQ ID NO:01) (Figure 1 A), and in PD-L1, positions A121 -A125 with sequence

‘ADYKR’(SEQ ID NO: 80) from PDB 3BIK (Figure IB)), While the tryptophan at the first position in the PD-L2 motif filled the pocket on PD-1 better than the PD-L1 alanine at this position, the arginine in the fifth position of PD-L1 is involved in an extensive hydrogen bond network with the target; hence, in addition to the ‘ADYKR’ PD-L1 binding motif (SEQ ID NO: 80) and the ‘WDYKY’ PD-L2 binding motif (SEQ ID NO:01), we also scaffolded the hybrid motif of ‘WDYKR’ (SEQ ID NO:81) (Figure 1C). We built proteins containing these motifs using two approaches as described in the following paragraphs.

In a first computational design approach, the three binding motifs were grafted onto a set of 34,840 de novo designed scaffolds containing three beta strands and a single alpha helix using the Motifgraft™ protocol (Figure ID). The miniprotein scaffolds were 35-41 residues in length and contained 1-4 disulfide bonds for stability. All five residue contiguous segments of each design were superimposed on the motif in complex with PD-1, and such scaffold/PD-1 docks were selected if the backbone RMSD was less than 0.7 A and the scaffold did not clash with PD-1 (Figure IE). Multiple gratis were allowed for the same backbone if there was good alignment at multiple positions along the peptide chain. Sequences were designed for 25,709 selected grafts by constraining the residues in the binding motif and all disulfide bonds and then optimizing interactions with murine PD- 1 using Rosetta™ Monte Carlo sequence design (Figure IF), Designs were filtered for interface size (buried solvent accessible surface area) above 1,100 A, shape complementarity above 0.6, and binding energy below -6 kcal/mol (as computed by Rosetta™) to yield a total of

1,058 Motifgraft™ designs to evaluate experimentally for binding to PD-1.

In the second design approach, the binding motif was used as a seed around which de novo backbones were generated, as in the Fold-From-Loops™ protocol. In contrast to the Motifgraft™ design protocol, we picked just the PD-L2 binding motif as it has the stronger binding affinity of the two native ligands (Figure 1 G). A secondary structure definition (or ‘blueprint’) file was created to designate the length, identity, and register of each secondary structure element to be constructed on either end of the motif. We chose to focus on topologies consisting of a beta sheet containing two 5-residue strands in addition to the motif strand stabilized by a 14-residue alpha helix packing on the side of the sheet not contacting PD-1. Monte Carlo-based fragment assembly was used to generate thousands of backbones with this topology around the motif (Figure 1H). To increase stability, cysteines were added by scanning for pairs of positions that were geometrically compatible with disulfide bond formation (Figure II). As in the grafting approach, the resulting de novo backbones were superimposed onto the motif strand in the PD-1/PD-L2 crystal structure and those that clashed with PD-1 eliminated. The sequences of the non-clashing docked backbones were then designed (Figure 1J) as described above for the grafting approach. Designs were filtered for interface binding energy below - 10 kcal/mol and on the monomer backbone, disulfide geometry, and core packing, resulting in 3,999 final designs for this set. The biggest difference in the designs generated by each protocol was that the Fold-F rom-Loops protocol generally yielded larger interfaces (Figure 6A-B).

Genes encoding all 5,057 designs - 1,058 Motifgraft™ designs and 3,999 Fold-From- Loops™ designs - were ordered as an oligonucleotide array pool and transformed into yeast with a linearized yeast display vector. The yeast display library of designs was first sorted for the displaying population by collecting all FITC^" yeast cells that had been labeled with a FITC-conjugated antibody against a cMyc tag included at the C terminus of the displayed construct. We tested for binding to both human and mouse PD- 1 as the molecules are slightly different by incubating two separate samples of the library with 1 μΜ mPDl-Fc or 1 μΜ hPDl-Fc and collecting cells with binding signal above background. Background signal was determined using a negative control sample incubated with only the secondary fluorophore labels. To distinguish true binders from those that just bound the Fc domain in the PD-1 construct we used for screening, we also carried out a binding sort with 1 μΜ of the Fc isotype control. DNA was extracted from the yeast in the unsorted library as well as all four selected pools. The gene inserts were amplified and a unique barcode for each pool added using qPCR. Next-generation sequencing (NGS) was used to sequence all five barcoded pools (unsorted, displaying, mPD-1 binding, hPD-1 binding, and Fc binding) simultaneously. Raw sequencing reads were filtered by quality score, and then the count and frequency for each sequence in each pool was calculated. Enrichment ratios were generated by dividing the frequency of a given sequence in one of the binding selections by its frequency in the display reference sort. Binding hits were identified as designs that were enriched in either the murine or human PD-1 selections (enrichment ratio above 2) and depleted in the Fc binding selection (enrichment ratio less than 2).

One Motifgraft™ design and three Fold-From-Loops™ designs were found to bind mPD- 1 specifically, and one Motifgraft™ design was found to bind hPD-1 specifically in this high-throughput screen (Figure 6C). While all the Fold-From-Loops™ designs contained the ‘WDYKY’ (SEQ ED NO:01) binding fragment extracted from PD-L2, both Motifgraft™ hits used the ‘ADYKR’ (SEQ ID NO: 80) hybrid motif. Of the filters used, there was no single metric that delineated working designs from non-working designs, Loop Redesign <6 Affinity Maturation Yield Higher Affinity, Cross-Reactive PD-1 Binder

One of the Fold-From-Loops™ designs, GR918, was chosen for further optimization as this design had the highest mPD-1 enrichment in the high-throughput screen. The original design was found to bind mPD-1 on yeast at a Kd of 2,8 μΜ (Figure 2A; Figure 2C), but showed no affinity for hPD-1 (Figure 2A). GR918 did not bind to the Fc isotype control (Figure 2A) or a protein with a similar immunoglobulin fold, CTLA-4 (Figure 2A), Due to the high error rate of the gene synthesis technology employed, the initial high-throughput screen also included several variants of each design ordered. We identified a variant, GR918.2, containing two subsitutions (D7T and Y38E) screened in the original design pool that slightly increased binding to mPD-1 (Figure 2C).

A site saturation mutagenesis (SSM) library containing every single point substitution of GR918.2 was screened for increased binding to mPD-1 on yeast (Figure 7A). Combinations of four mutations identified in the SSM to be beneficial to binding were tested and a triple mutant, referred to here as GR918.3, consisting of Y13F, K22E, and T26K was found to have a K_d of 1.4 μΜ for mPD-1 (Figure 2C), which is approximately three-fold higher than that of mPD-L2, the higher affinity of the two native ligands, on yeast (Figure 7B). However, no detectable binding to hPD-1 was observed with GR918.3, and we were unable to affinity mature GR918 for hPD- 1 binding using any of the yeast display libraries we had created.

To obtain higher affinity binders, we turned to a computational design approach. A 4- residue beta hairpin loop (Figure 2B, left) was observed to be highly tolerant to mutation in the GR918.2 and GR918.3 SSM screens. This region of the binder was in close proximity to several pockets on the side of the PD-1 receptor and we sought to extend the loop such that sidechains could interact with these pockets. The Direct Segment Lookup protocol in Rosetta™ was used to identify 6- to 9-residue conformationally constrained loops observed in the PDB matching the 2 residue terminal segments of the GR918 loop with backbone atom RMSD less than 1.25 A (Figure 2B, left). Matching loops were grafted onto GR918 and the sequences were designed to increase contacts with PD-1 while preserving graft structure via a structure-based PSSM with explicit preservation of proline or glycine residues. We screened 689 loop designs via yeast display for both mPD-1 and hPD-1 binding. A 9-residue loop

(Figure 2B, right) containing five mutations arising from the high gene synthesis error rate (Figure 7C) had binding signal to hPD-1 while maintaining binding to mPD-1. After two rounds of affinity maturation for mPD-1 binding followed by two rounds of hPD- 1 affinity maturation, the final protein binds mPD-1 with a K_d of 0.6 nM and hPD-1 with a Kd of 1.1 nM on yeast (Figure 2C). We refer to this final variant as PD-MP1 for Programmed Cell Death Miniprotein 1.

Design Binds at PD-L1/2 Interface on mPD-1

The beta sheet of GR918 was designed to bind the ligand interface on PD-1 to block PD-L1/2 binding. GR918.3 SSM data (Figure 2D) was consistent with GR918 binding to PD- 1 as anticipated. The Shannon entropy of each position along the peptide is shown in Figure 2E; interface beta-sheet residues are highly conserved while non-interface, solvent- exposed residues on the helix are highly variable, suggesting that the beta strands of GR918.3 form the interface with PD-1 as designed. All cysteine residues involved in the three designed disulfide bonds and the rest of the core residues are highly conserved suggesting that the protein folds as designed.

To assess whether GR918 binds the intended site on PD-1, we performed a competition assay on yeast using soluble PD-L1. Yeast cells displaying GR918.3 were incubated with 0.1 μΜ biotinylated mPDl-Fc in the absence or presence of 1 μΜ unbiotinylated hPD-L1 competitor, then the secondary label streptavidin-phycoerythrin (SAFE) was added and binding assayed by flow cytometry (Figure 2H). If hPD-L1 is binding the same interface on PD-1 as GR918.3, it will compete for binding to the mPD-1 molecules in the sample, decreasing the number of mPDl-Fc-biot molecules bound to GR918.3 on the yeast cell and hence the number of SA-PE molecules bound (Figure 2G), leading to a decrease in tnPD-1 binding signal by flow cytometry. A large decrease in signal was indeed seen with 10-fold excess of hPD-L1 (Figure 2H) suggesting that GR918.3 binds the intended region of PD-1.

Binder Folds as Designed & Is Hyperstable

After the first round of affinity maturation, soluble GR918.2 was expressed using the Daedalus mammalian expression system to confirm the fold, stability, and monomeric state of the protein in vitro. The apo crystal structure at 1.07 A resolution confirms the protein folds as designed with a backbone RMSD of 1.33 A between the crystal structure and the computational model (Figure 3A). All three disulfides are clearly formed between the correct pairs of cysteines and the rest of the core residues are packed as designed. It is notable that GR918.2 captures the functional interface of PD-L2 using a much smaller, idealized protein backbone (Figure 3B).

GR918,2 was found to be hyperstable to both thermal and chemical denaturation. Thermal denaturation was performed by heating the protein from 25' C to 95" C and the secondary structure was monitored using circular dichroism spectroscopy (CD) at 222 rnn. The protein gave a CD spectra characteristic of an alpha-beta fold, and only -20% of the signal was lost at 95° C which was fully regained upon cooling back down to 25° C (Figure 3C). Chemical denaturation was performed in a similar manner, and the protein only lost ~35% of the CD signal at 220 nm in 7 M GuHCl (Figure 3D). This high stability is due to the three disulfide bonds and is not retained upon reduction of these bonds. Monomeric and Trimeric PD-MPI Binds PD-1 In Vitro & on Mammalian Cells

In vitro binding of soluble PD-MPI to murine and human PD-1 was measured using Octet™ Bio-Layer Interferometry (BLI). Soluble PD-MPI bound biotinylated mPD-l-Fc loaded onto Streptavidin Octet™ sensors with a Ka of 103.4 ± 78.9 nM (Figure 4A) and hPD- 1-Fc-biot loaded sensors with a Ka of 111.3 + 59.7 nM (Figure 4B) in this assay. This is 100 to 200-fold higher than the Kd’s on yeast, likely because of residual avidity effects in the flow cytometry experiments.

K562 human stable cell lines expressing murine or human PD-1 were used to test whether soluble PD-MPI could bind PD-1 on mammalian cells. Anti-mPD-1-FITC and Anti- hPD-l-FITC antibodies were used to confirm expression of PD-1 for both transduced cell lines by flow cytometry (Figure 9E), and an anti-myc-FITC antibody was used to detect binding of the myc-tagged monomeric PD-MPI to the cells. Concentration-dependent binding was observed to the K562 cells expressing murine PD-1 (Figure 4C) that was not observed to the wild type K562 cells (Figure 4C), indicating that this interaction is mPD-1 dependent. However, no binding was detected above background levels to the K562 cells expressing human PD-1 (Figure 4C). Given that the design binds hPD-1 in the biolayer interferometry experiments (Figure 4B), the reasons for this lack of binding on cells are not clear, and for the experiments described in the remainder of this paper, we focus on binding to murine PD-1.

To increase the apparent affinity of PD-MPI, we used an earlier loop-grafted variant, LGm.2, to generate a PD-1 binding trimer. The Spycatcher/Spytag™ system was used to covalently attach LGm.2 to the designed homotrimer 2L6HC3_13 (SEQ ID NO: 19). 2L6HC3_13 was expressed and purified from bacteria as a genetic fusion with a Spycatcher™ domain. LGm.2 was expressed and purified with a Spytag™ and Myc-tag from mammalian cells. 2L6HC3_13 _Spycatcher™ was mixed with a slight excess of

Spytag™_LGm.2, and the conjugated product was purified by gel filtration. The purified 2L6HC3 13 PD-MPI was confirmed to form a trimer by multi-angle light scattering (MALS) (Figure 4D). We then titrated both the monomeric and trimeric LGm.2 constructs and measured binding by flow cytometry. The monomeric LGm,2 yielded a K_d of 6 μΜ to mPD- 1 on K562 cells (Figure 4E), while the trimeric LGm.2 protein had a ~ 65-fold boost in apparent affinity with a K_d of 90 nM (Figure 4E). These results indicate that an increased avidity to mPD-1 on mammalian cells can be achieved via oligomerization due to the increase in valency of the PD-1 binding domain. Trimeric PD-MP1 Exhibits Agonistic Activity in Mouse T Cell Activation Assay

PD-MP1 trimer was cultured with T cells during activation to test for PD-1 agonist or antagonist activity (Figure 5A). To test potential PD-1 antagonism, PD-MP1 trimers were added to mouse T cells stimulated with anti-CD3 antibody in the presence of plate-bound mouse PD-L1 -Fc to see if they would block the PD-L1 -driven inhibition of T cell activation. Potential PD-1 agonism was tested by adding trimers to stimulated T cells without PD-L1 -Fc. After three days, activation of both CD4+ and CD8+ T cells was measured via CD69 expression (Figure 5B). As expected, T cells stimulated with an anti-CD3ε antibody alone or with addition of control IgG-Fc showed high levels of T cell activation as indicated by upregulation of CD69, and stimulation of T cells in the presence of PD-L1-Fc elicited lower

CD69 expression.

The results of these studies demonstrate that trimeric PD-MP1 is a PD-1 agonist and not an antagonist. Addition of the PD-MP1 binders considerably reduced the CD69 induction elicited by anti-CD3ε in both CD4 and CD8 T cells compared to empty control trimers. This inhibition of T cell activation is dose dependent (Figure 5C), and stronger than observed with plate-bound PD- L1-Fc. For T cells treated with anti-CD3ε and IgG-Fc, addition of the PD- MP 1 trimers inhibited activation of CD4+ T cells up to 50% and inhibited activation of CD8+ T cells up to 70% more than empty trimers. In comparison, the PD-L1 -Fc led to 40% inhibition in CD4 T cells and 28% in CD8 T cells. The strongest inhibition was in the presence of both the PD-MP1 trimers and PD-L1 -Fc which inhibited activation of both CD4+ T cells and CD8+ T cells up to 90%. Taken together, this indicates that the PD-MP 1 trimer is an agonist for PD-1 that inhibits T cell activation in both CD4 and CD8 T cells.

DISCUSSION

The PD-1 binders described herein are, to our knowledge, the first to be generated with an all beta protein interface. A challenge in designing beta sheet containing interfaces is that, if the target surface is non-polar, the beta strands will have hydrophobic residues both in the interior hydrophobic core of the monomer and on the exterior to interact with the target, and hence there is no hydrophobic/hydrophilic patterning to guide folding. This problem is exacerbated by the propensity of beta strands to aggregation. In accordance with these observations, we found core stabilization through the use of disulfide bonds to be useful for obtaining a soluble PD-1 binder that folds as designed. While the original GR918 design had weak affinity to mPD-1, following affinity maturation and loop extension we obtained a higher affinity, cross-reactive PD-1 binder. The designed PD-1 binding trimers presented here have potential as a PD-1 agonist for the treatment of autoimmune and inflammatory diseases. The designed PD-1 binding trimers decreased T cell activation up to 70% in our studies.

Targeting of a PD1 agonist to sites of inflammation would avoid systemic immunosuppression and be advantageous for organ-specific autoimmune diseases, such as diabetes. A targeted therapeutic could be generated by fusing trimeric PD-MP1 with other small binding domains against relevant tissue-specific markers. Due to its small size, PD- MP 1 would likely have a short serum half-life as it would likely be cleared from the blood through the kidneys very quickly²⁹. If desired, lifetime in circulation could likely be achieved by fusion to an Fc domain or PEG conjugation. However, the short lifetime could be an advantage for a targeted therapeutic programmed to act locally but be cleared quickly if it diffused away from the target site. Additionally, a genetically encodable and readily secretable PD-1 agonist could have a role in CAR-T_reg therapies currently being developed for the treatment of several autoimmune diseases, including asthma and multiple sclerosis. These cell-based platforms have the benefit of being self-sustaining and are targetable to any cell surface antigen. Trimeric PD-MP1 can be genetically encoded as a fusion protein whose gene could be introduced into the engineered cells with an inducible promoter, allowing specific expression and secretion only upon target activation to locally suppress the immune response and reduce inflammation.

MATERIALS & METHODS

Computational Design & Gene Synthesis

The ROSETTA™ protein structure prediction and design software suite²⁰ was used for all design calculations using the Talaris2013 energy function³⁴. The Motifgraft™_’ ³⁵ (Protocol 1) and Fold-From-Loops™' ²¹ (Protocol 2) computational protocols were performed as previously described using the MotifGraft™ and BluePrintBDR™ movers respectively. The Remodel™ mover was used to add disulfide bonds to de novo backbones. Two rounds of design and all-atom minimization using the PackRotamersMover™ and TaskAwareMinMover™ were performed to generate final sequences for the Motifgraft™ designs. The Fold-From-Loops™ backbones were sequence designed using three rounds of the FastDesign™ mover followed by 15,000 rounds of the GenericSimulatedAnnealer™ to optimize the SSprediction™, CavityVolume™, SSShapeComplementarity™, PackStat™, hbond_lr_bb/residue, dslf_fal3/residue, TotalHydrophobic/TotalSasa_Hydrophobic (buried nonpolars), Holes™, Ddg, and Rmsd Rosetta™ filter scores. The Direct Segment™ Lookup protocol was used for loop redesign (Protocol 3). First, the DirectSegmentLookupMover™ was used to graft up to 10-residue loops into GR918 between positions 6 and 11 that matched the backbone of neighboring residues 5-6 and 11-12 with an RMSD of less than 0.75 A. The grafted loops were Cartesian-space minimized using the T askA wareMinMover™ and a structure-based PSSM was obtained using the

SegmentSequenceProfileMover™. Four cycles of FastDesign™ in conjunction with the FavorSequenceProfile™ mover was used to design the grafted loop sequence using the PSSM. Redesigned loop sequences were filtered for ShapeComplementarity™ above 0.65, Ddg below -30, and Sasa above 1500.

Genes encoding all 5,057 initial designs and 689 loop redesigns to be tested were codon optimized for S. cerevisiae ³⁶ and ordered as a single oligo pool from CustomArray™. SSM genes were amplified from Agilent oligo array pools. Combination Libraries were generated from IDT ultramers containing degenerate codons. Genes for all individual mutants to be tested were constructed through oligo assembly using IDT oligos. Yeast Surface Display

Oligo pools were amplified and transformed into yeast as previously described²². Initial testing of all designs and affinity maturation of GR918 was performed using yeast surface display³⁷. For the high-throughput screen, yeast displaying designs were labeled with either 1 μΜ mPD-l-Fc, 1 μΜ hPD-1 -Fc, or 1 μΜ IgG2a Fc (expressed from HEK 293F cells) as well as 5 μΜ biotinylated protein ZZ^3S (expressed from E, coli BL21 cells) which specifically binds Fc and acts as an intermediate label for 1 hour at 23 ^° C. Cells were then washed and labeled with 0.01 mg/ml SA-PE (ThermoFisher) and 0.01 mg/ml anti-cmyc- FITC (Immunology Consultants Laboratory) as a control for display of the proteins on ice for 10 minutes under aluminum foil. Two consecutive rounds of FACS were performed under these labeling conditions and all FITC⁺PE⁺ cells collected using a Sony SH800 cell sorter. A single reference sort of all FITC⁺ displaying cells was also done on the naive library. Sequencing of the FITC⁺ pool and both binding selections was performed on an Illumina MiSeq^TM.

The loop redesigns were similarly screened in high-throughput as a single yeast display pool. One reference sort was done to collect all FITC⁺ displaying cells. Two consecutive sorts were done for mPD-1 binding, labeling with 250 nM for the first sort and 100 nM mPD-1 -Fc-biot for the second sort. 10 μΜ hPD-l-Fc-biot was used for both hPD-1 sorts. All five sorted pools were then deep sequenced. All SSM libraries went through two subsequent rounds of FACS, First, a titration was done to determine a Kd on yeast for the parent sequence. The first sort was labeled with mPD- 1-Fc-biot or hPD-l-Fc-biot at approximately half of the parent K_d, and the top 5% FITC⁺PE⁺ were collected. The second sort was labeled with a 4-fold lower concentration than the first, and the top 1% FITC⁺PE⁺ were collected. Both selections were deep sequenced along with a FITC⁺ reference sort. Enrichment ratios were calculated for each single mutant by dividing the counts in the selected pool by the counts in the reference pool. The Shannon entropy²³ was calculated for each position as previously described.

Combination Libraries containing all possible combinations of beneficial mutations from the SSM screens were sorted to convergence by doing 4-5 consecutive sorts at decreasing target concentration and collecting only the top 0.2-1% FITC⁺PE⁺ cells. Library convergence was monitored by plating the sorted cultures on C-Trp-Ura and Sanger sequencing 24 clones.

Yeast display titrations were conducted by incubating yeast cells displaying the GR918 variant with a range of concentrations of biotinylated mPD-l-Fc or hPD-l-Fc for 1 hour at 23° C. Cells were washed and then incubated with 0,01 mg/ml SA-PE and 0.01 mg/ml anti-cmyc-FITC on ice for 10 minutes under aluminum foil. Two final washes were done prior to measuring FITC and PE fluorescence on a BD Accuri™ C6 flow cytometer.

For the competition assay, yeast displaying GR918.3 were incubated with 0,1 μΜ mPD-l-Fc-biot alone or co-incubated with 1 μΜ hPD-L1 (expressed ini', coli ) for 1 hour at 23° C. After secondary labeling with SA-PE and anti-cmyc-FITC, the cells were analyzed using a BD Accuri™ C6 flow cytometer.

Protein Expression & Oligomer Conjugation

Soluble GR918.2 was produced using the Daedalus mammalian expression system²⁴ by the Molecular Design & Therapeutics core at the Fred Hutchinson Cancer Research

Center. All other GR918 monomeric constructs, including the loop redesigned variants, were cloned with a His-tag and Myc-tag as a Siderocalin (Sen) fusion after an IgK secretion signal sequence into the mammalian expression vector CMVR. PEI transient transfection was used to introduce the vector into Expi293F™ HEK cells (ThermoFisher), The spent media was harvested 3 days post-transfection, and the soluble protein was purified by immobilized metal affinity chromatography (IMAC) using Ni Sepharose Excel™ Resin (GE Life Sciences). Spytagged monomers were cleavage with Tev protease (expressed in E. coli ) followed by another IMAC step removed the Sen fusion and His-tag, and then gel filtration was used as a final polishing step for the cleaved protein on a Superdex 75 Increase 10/300 GL column (GE Healthcare Life Sciences) (Figure 9A Lane 1 and 7B).

The 2L6HC3_13 trimer was cloned with an N-terminal His-tag and Spycatcher TM domain into pET29b(+). The protein was expressed in the E. coli expression strain Lemo21™ (DE3) (New England BioLabs) using IPTG induction and purified via Ni-NTA IMAC followed by gel filtration using a Superdex™ 200 Increase 10/300 GL column (GE Healthcare Life Sciences) (Figure 9A, Lanes 3 and 5; Figure 9D-E).

Trimeric PD-MP1 was generated via Spycatcher/Spytag™ conjugation by incubating the 2L6HC3_13_Spycatcher™ construct with a 10% molar excess of Spytag™_Myc_PD- MP 1 at 23° C for >1 hour. Conjugated product was separated from unconjugated components via gel filtration using a Superdex™ 200 Increase 10/300 GL column (GE Healthcare Life Sciences) (Figure 9A, Lanes 3-6; Figure 9D-E).

Crystallography

Crystal screens were set up using the sitting drop vapor diffusion method by mixing 2.25 mg/ml GR918.22:1, 1:1, or 1:2 with reservoir solution from the 96-well Morpheus TM

Crystallization Screen. Crystals grew in several drops (Morpheus™ Dl, El, FI, HI) within 3 weeks at 18° C. Optimization screens were set up with each of these four conditions at 1:4, 1:2, 1:1, 2: 1, and 4:1 mixtures with 9.85 mg/ml protein solution. Crystals were seen in most of the drops after —24 hours at 18° C. Because the Morpheus™ screen conditions already contain cryoprotectant, the crystals were looped and immediately flash frozen in liquid nitrogen. Diffraction data was collected from a crystal grown in 0.1 M MES/Imidazole pH 6.5, 0.03 M Diethylene Glycol, 0.03 M Triethylene Glycol, 0.03 M Tetraethylene Glycol,

0,03 M Pentaethylene Glycol, 10% PEG 20,000, 20% PEG MME 500.

X ray diffraction data was collected at the Advanced Photon Source, Northeastern Collaborative Access Team, Beamline™ ID-C. Images were processed to 1.07 A in space group C2 using the program XDS³⁹, The structure was solved by molecular replacement using PHASER™ ⁴⁰ in the PHENIX™ software suite⁴¹ and the helix (residues 18-31) from the GR918 design model as a search model, with SHELXE™'⁴² used for model completion. Two copies of GR918 were placed in the asymmetric unit. Coot™_’ ⁴³ and PHENIX™. refine⁴⁴ were used for several rounds of manual building and refinement.

Circular Dichroism

All CD experiments were conducted using an AVIV Model 420 Circular Dichroism Spectrometer. Thermal denaturation was conducted by first performing a wavelength scan from 190-260 run, sampling every 1 nm, at 23° C using 0.23 mg/ml GR918.2 in PBS in a 1 mm quartz cuvette. Then, the CD signal at 220 nm was monitored while heating the sample to 95° C, taking a data point every 2° C after incubating the sample for 30 seconds at the new temperature (Figure 8B). Another wavelength scan was taken at 95° C and again after cooling the sample back down to 23° C. All measurements were also taken with PBS for buffer subtraction.

Chemical denaturation was performed in a similar manner. Initially, a wavelength scan from 190-260 run was taken on 0.05 mg/ml GR918.2 in PBS in the absence of denaturant in a 1 cm quartz cuvette at 23° C. A solution of 8M GuHCl with 0.05 mg/ml GR918.2 was then titrated into the cuvette under constant volume and with stirring, and the CD signal at 220 nm was recorded every 0,25 M up to 7 M. A final wavelength scan was performed of the protein solution with 7 M GuHCl.

Octet Bio-Layer Interferometry (BLI)

50 nM mPD-l-Fc-biot or hPD-1-Fc-biot was loaded onto Streptavidin Biosensors (Forte Bio) for 60 seconds at 25° C. Loaded sensors were washed in Octet Buffer (HBS-EP+, 0.5% dry milk) for 240 seconds and then a baseline was established in fresh Octet™ Buffer for 300 seconds. Loaded sensors were moved to a solution containing PD-MP1 at a range of concentrations between 3 and 6 μΜ and allowed to associate for 500 seconds. Dissociation was done by moving the sensor back to Octet™ Buffer for 1000 seconds. Association rates, dissociation rates, and dissocation constants are listed in Figure 9F. Cell Binding Assays

Stable K562 cell lines constitutively expressing human or murine PD-1 were generated via lentiviral transduction of wild type K562 cells with a DNA construct containing the ectodomain of either murine or human PD-1 followed by an IRES sequence and an iRFP gene. Transduced cells were sorted for iRFP expression to establish stable clonal cell lines expressing high levels of PD- 1. The K562 cell lines were incubated with varying concentrations of soluble myc-tagged PD-MP1 monomer or trimer at 23° C for >1 hour. Cells were washed and then labeled with 0.01 mg/ml anti-cmyc-FITC (Immunology Consultants Laboratory) for 10 minutes on ice under aluminum foil. Cells were washed twice before measuring the FITC signal on a BD LSR II flow cytometer. T cell Activation Assay

Mouse T cells from C57BL/6 female mice were isolated using a Pan T cell Isolation kit II (Milyteni Biotec). T cells were then resuspended to 1x10⁶ cells/ml in warm media then transferred to a 96-well plate pre-coated with 2.5 pg/mL of anti-mouse CD3ε antibody (BD Pharmingen, clone 145-2C11) plus 10 pg/mL of either recombinant mouse PD-L1-Fc (R&D Systems) or recombinant human IgGl-Fc (R&D Systems). PD1-MPI trimers and control trimers were thawed to room temperature and diluted to 5.32 μΜ in warm media. Three-fold serial dilutions were performed in warm media to achieve further titrations, then trimers were added to the T cells and incubated at 37°C, 5% CO2 for three days. After incubation, cells were washed in PBS and stained with a fixable live/dead stain (APC-ef780, Invitrogen) for 15 minutes on ice. Cells were washed in staining buffer (PBS + 0.5% BSA) and resuspended in a T cell activation antibody cocktail (CD3 -PerCP-Cy5,5, BD Pharmingen; CD8-FITC, Biolegend; CD4-APC, Invitrogen; CD69-BV421, BD Horizon) on ice for 30 minutes. Cells were then washed in staining buffer and analyzed using BD FACSymphony™ A3 Flow Cytometer, FACSDiva™ software, FlowJo™ v 10.6.1, and Graphpad™ Prism v7.

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PROTOCOLS

_

_

_ _

_ _

_ _

Protocol 2: Fold-From-Loops Scripts, (a) RosettaScripts™ xml file to generate c/e novo backbones around motif fragment using the specified blueprint files, and then build disulfide bonds into those backbones, (b) RosettaScripts™ xml file adding PD-1 to the generated de novo backbones, constraining the motif residues, and then doing several rounds of sequence design on the designed peptide, (c) RosettaScripts™ xml to optimize the designed binder sequence, (d) RosettaScripts™ xml to filter final designs on several monomer metrics.

(b

<

Protocol 3: Direct Segment Lookup™ Scripts, (a) RosettaScripts™ xml file running the DirectSegmentLookup™ mover and generating a PSSM for the grafted loops, (b) RosettaScripts™ xml file for sequence design and filtering of grafted loops.

Claims

We claim

1. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS:82- 90.

2, The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS:85-90. 3, The polypeptide of claim 1 or 2, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15

16, or all 17 following residues are invariant: C1, C3, C5, G21, C26, K28, L30, E32, C33, Q35, N37, P38, G39, A40, 144, Q45, and C46.

4. The polypeptide of any one of claims 1 -3, wherein amino acid substitutions relative to the reference polypeptide are selected from those listed in Table 1 5. The polypeptide of claim 4, wherein residues 11 - 15 are present.

6. The polypeptide of any one of claims 1-5, wherein beta strand residues in the polypeptide are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding residues in the reference amino acid sequence.

7. The polypeptide of any one of claims 1-6, wherein at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid changes in the polypeptide relative to the reference polypeptide occur in the alpha helix and/or loop,

8. The polypeptide of any one of claims 1 -7, wherein the polypeptide is capable of binding to human PD-1 at the PD-L1 interface and/or is capable of binding to murine PD-1 at the PD-L1 interface. 9. The polypeptide of claim 8, wherein the polypeptide is capable of binding to human

PD-1 at the PD-L1 interface with a K_d of -100 nM or less, and/or is capable of binding to murine PD-1 at the PD-L1 interface with a K_d of ~100 nM or less.

10. The polypeptide of any one of claims 1 -9, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of PD-

MP1 (SEQ ID NO:90).

11. The polypeptide of any one of claims 1-10, comprising at least 2 disulfide bonds.

12. The polypeptide of claim 11, comprising 3 disulfide bonds.

13. A construct comprising the polypeptide of any one of claims 1-12 linked to one or more tumor treating agents or tumor targeting agents.

14. The construct of claim 13, wherein the one or more tumor treating agents comprise polypeptides, and wherein the construct comprises a fusion protein. 15. A construct comprising the polypeptide of any one of claims 1-12 linked to one or more autoimmunity treating or tissue-targeting agents.

16. The construct of claim 15, wherein the one or more autoimmunity treating agents comprise polypeptides, and wherein the construct comprises a fusion protein.

17. The polypeptide or construct of any one of claims 1-16, further comprising an oligomerization domain.

18. The polypeptide or construct of claim 17, wherein the oligomerization domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 2-22, 25-29, 31-32, 34-36, 38-41, 43-45, 47, 49- 55, and 58-79.

19. The polypeptide or construct of claim 17, wherein the oligomerization domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 19. 20. The polypeptide or construct of any one of claims 17-19, wherein the polypeptide or construct is a dimer, trimer, tetramer, pentamer, or hexamer.

21. The polypeptide or construct any one of claims 1-20, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 91-98.

22. The polypeptide or construct any one of claims 1-20, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

95. 23. The polypeptide or construct of any of the preceding claims, wherein the polypeptide or construct is linked to other compounds, such as stabilization compounds to promote an increased half-life in vivo.

24. A composition comprising 2, 3, 4, 5, 6, or more copies of the polypeptide or construct of any one of claims 1-23.

25. A nucleic acid encoding the polypeptide, construct, or composition of any one of claims 1-24.

26. A vector comprising the nucleic acid of claim 25 operatively linked to a promoter.

27. A host cell comprising the polypeptide, construct, or composition of any one of claims 1 -24, the nucleic acid of claim 25, and/or the vector of claim 26.

28. A pharmaceutical composition comprising

(a) the polypeptide, construct, composition, nucleic acid, vector, and/or host cell of any one of claims 1-27; and

(b) a pharmaceutically acceptable carrier. 29. The pharmaceutical composition of claim 28 further comprising one or more additional therapeutics for treating tumors, including those disclosed herein and/or anti- CTLA4 antibodies, including but not limited to ipilimumab.

30. The pharmaceutical composition of claim 28 further comprising one or more additional therapeutics for treating autoimmune disorders, including but not limited to those disclosed herein.

31. A method for treating and or limiting development of a tumor, comprising administering to a subject in need thereof an amount effective to treat or limit development of the tumor of the polypeptide, construct, pharmaceutical composition, nucleic acid, vector, and/or host cell of any of the preceding claims. 32. A method for treating or limiting development of an autoimmune disorder, comprising administering to a subject in need thereof an amount effective to treat or limit development of the autoimmune disorder of the polypeptide, construct, composition, pharmaceutical composition, nucleic acid, vector, or host cell of any of the preceding claims. 33. A method for designing PD- 1 binding polypeptides, comprising the steps of any embodiment or combinations of embodiments disclosed herein.