Novel designed binding agents targeting human EpCAM and PDL1 receptors

Abstract

Disclosed herein are polypeptides that bind to epithelial cell adhesion molecule (EpCAM) and programmed cell death ligand 1 (PDL1) receptors, nucleic acids encoding them, and methods of using them in treating or limiting the development of cancer, autoimmune diseases, or inflammation.

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 63 / 336,629, filed April 29, 2022, which is incorporated by reference in its entirety.

[0002] Federal Funding Statement This invention was made with government support under Grant No. HR0011835403 awarded by the Defense Advanced Research Projects Agency, Grant No. R01CA240339-01 awarded by the National Cancer Institute, Grant No. T32GM008268 awarded by the National Institute of General Medical Sciences, and Grant No. 5U19AG065156-02 awarded by the National Institute on Aging. The Government has certain rights in the invention.

[0003] Reference to Electronic Sequence Listing A computer readable form of the Sequence Listing has been submitted with this application via electronic submission and is incorporated herein by reference in its entirety. The Sequence Listing is contained in an XML file created on April 19, 2023, named "22-0544-WO.xml" and is 87,939 bytes in size. [Background technology]

[0004] background Human epithelial cell adhesion molecule (EpCAM) and programmed cell death ligand 1 (PDL1) receptors are involved in various diseases such as cancer, autoimmunity and inflammation. Therefore, therapeutics targeting EpCAM and PDL1 are needed. Although antibodies targeting these receptors exist, the proteins designed by the inventors have unique properties such as rapid pharmacokinetics and high tissue / tumor penetration due to their small size compared to antibodies, and therapeutics made from the inventors' binders may have altered toxicity / efficacy profiles compared to antibodies. Summary of the Invention

[0005] In one aspect, the disclosure provides a polypeptide that binds to a human epithelial cell adhesion molecule (EpCAM) receptor, the polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In one embodiment, interface residues 21, 22, 24, 25, 28, 29, 32, 33, 36, 42, 45, 46, 49, 50, 52, 53, 54, and 57 are selected from the corresponding interface residues present in any one of SEQ ID NOs: 4-18. In another embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 4-18.

[0006] In another aspect, the disclosure provides a polypeptide that binds to a human EpCAM receptor, the polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, interface residues 1, 4, 5, 7, 8, 9, 11, 12, 15, 16, 19, 20, 43, 44, 45, 47, 48, 50, 51, 52, 54, and 55 are selected from interface residues present in an amino acid sequence selected from SEQ ID NOs: 19-21. In another 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 an amino acid sequence selected from SEQ ID NOs: 19-21.

[0007] In a further aspect, the disclosure provides a polypeptide that binds to a human programmed cell death ligand 1 (PDL1) receptor, the polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, interface residues 1, 4, 5, 8, 11, 12, 15, 16, 19, 42, 43, 44, 47, 48, 51, 52, 54, 55, and 56 are selected from interface residues present in an amino acid sequence selected from SEQ ID NOs: 22-30. In another 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 an amino acid sequence selected from SEQ ID NOs: 22-30.

[0008] In another embodiment, the disclosure provides a fusion protein of two, three, four, or more polypeptides according to any embodiment of the disclosure, wherein the polypeptides may be directly linked or may be linked via amino acid linkers.

[0009] The disclosure further provides a nucleic acid encoding a polypeptide of the disclosure; an expression vector comprising a nucleic acid of the disclosure operably linked to suitable control regulatory elements, a host cell comprising the polypeptide, fusion protein, nucleic acid, or expression vector of any embodiment of the disclosure, and a pharmaceutical composition comprising the polypeptide, fusion protein, nucleic acid, expression vector, or host cell of any embodiment; and a pharma- ceutical acceptable carrier.

[0010] The present disclosure also provides a method for treating or limiting the onset of cancer, an autoimmune disease, or inflammation, comprising administering to a subject in need thereof an effective amount of a polypeptide, fusion protein, nucleic acid, expression vector, host cell, or pharmaceutical composition of any of the embodiments herein to treat or limit the onset of the disorder. [Brief description of the drawings]

[0011] [Figure 1]From left to right, binder design models EpcamBind1_cb4, EpcamBind2_cb10, and PDL1Bind_cb6. [Figure 2A] Octet data for EpcamBind1_cb4. EpcamBind1 shows association and dissociation curves with an estimated Kd of 5.6 nM based on kinetic fitting to association and dissociation. Concentrations range from 100 nM (top curve) to 1.56 nM (bottom curve) by 2-fold dilution. [Figure 2B] Octet data for EpcamBind2_cb10. EpcamBind2 shows association and dissociation curves with an estimated Kd of 500 nM based on kinetic fitting to association and dissociation. Concentrations range from 1000 nM (top curve) to 15.6 nM (bottom curve) by 2-fold dilution. [Figure 2C] Octet data for PDL1Bind_cb6. PDL1Bind_cb6 has an estimated Kd of 2 nM based on steady-state analysis. [Diagram 3] Cell binding assay. Binding of control anibodies is shown at the top and binding of engineered binders is shown at the bottom. EpCAMBind1_cterm-cys-AF594 specifically binds to K562 cells engineered to express high levels of EpCAM without significant non-specific binding to EpCAM-knockout K562 cells. Cell binding activity was observed at 50 nM and 500 nM. PDL1Bind1_E39C-AF594 specifically binds to PDL1+SKOV3 cells at 500 nM and 5 μM and slightly non-specifically binds to PDL1− cells at 5 μM, comparable to the non-specific binding seen from the control antibody. [Figure 4] Octet data for gs-linked dimer of EpCAMBind1_cterm_cys. The plot shows the association and dissociation curves, where the binder has an estimated Kd of less than 10 pM based on kinetic fitting to the association and dissociation. The concentrations from the top curve to the bottom curve are 20 nM, 10 nM, 5 nM, 2.5 nM, 1 nM and 0.5 nM. [Diagram 5]Diagram of the sequences and secondary structures of the designs EpcamBind1_cb4 (SEQ ID NO: 5), EpcamBind2_cb10 (SEQ ID NO: 20), and PDL1Bind_cb6 (SEQ ID NO: 23). Alpha helices are shown as boxes, loops as lines, and interface residues are labeled with asterisks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description All references cited are incorporated herein by reference in their entirety. Within this application, unless otherwise indicated, the techniques utilized are based on the techniques described in 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. Deutschcer, ed., (1990) Academic Press, Inc.); PCR 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, 2nd Edition (R.I. Freshney, 1987. Liss, Inc. New York, NY). Protocols for gene transfer and expression (E. J. Murray, ed., The Humana Press Inc., Clifton, NJ), and the Ambion 1998 catalog (Ambion, Austin, TX).

[0013] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0014] As used herein, 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 (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0015] In all embodiments of the polypeptides disclosed herein, any N-terminal methionine residue is optional (ie, the N-terminal methionine residue may be present or absent).

[0016] All embodiments of any aspect of this disclosure can be used in combination unless the context clearly dictates otherwise.

[0017] Unless the context clearly dictates otherwise, throughout the description and claims, words like "comprise", "comprising" and the like are to be construed in an inclusive sense, i.e., "including, but not limited to," rather than an exclusive or exhaustive sense. Words using the singular or plural also include the plural and singular, respectively. Furthermore, the words "herein", "above", "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.

[0018] In a first aspect, the disclosure provides a polypeptide that binds to a human epithelial cell adhesion molecule (EpCAM) receptor, the polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0019] As disclosed in the Examples, the polypeptides of this aspect bind to the human EpCAM receptor. As disclosed in the Examples below, the inventors designed polypeptides according to SEQ ID NO: 1 (exemplary such polypeptides are listed in Table 2) and performed site saturation mutagenesis studies to determine permissible substitutions at each position that reflect the permissible amino acids shown in Table 1. In some embodiments, these polypeptides bind to the extracellular portion of the human EpCAM receptor.

[0020] Table 1 shows the amino acid sequence of SEQ ID NO:1.

[0021] [Table 1] TIFF2025516169000003.tif142132

[0022] Table 1 also shows the interface residue positions of SEQ ID NO: 1, residues 21, 22, 24, 25, 28, 29, 32, 33, 36, 42, 45, 46, 49, 50, 52, 53, 54, and 57. In one embodiment, interface residues 21, 22, 24, 25, 28, 29, 32, 33, 36, 42, 45, 46, 49, 50, 52, 53, 54, and 57 are selected from the corresponding interface residues present in any one of SEQ ID NOs: 4-18 (sequences shown in Table 2).

[0023] The residue numbers of the interface residues are listed in SEQ ID NO:1 / Table 1, and the residue numbers of SEQ ID NOs:4-18 in Table 2 are the same as those listed in SEQ ID NO:1 / Table 1, except for the following: SEQ ID NO:6 (EpCAM1_nterm-cys): This embodiment has two additional amino acid residues at the N-terminus relative to SEQ ID NO:1. * i.e., the numbering has shifted by +2); and SEQ ID NO:18 (EpCAM1_dimer): This embodiment contains two copies of an EpCAM receptor binding agent. The first copy follows the residue numbering of SEQ ID NO:1 / Table 1. The second copy of the sequence (i.e., residue 1 of the second copy) begins after the lowercase "ggsggs" (SEQ ID NO:31) linker sequence.

[0024] For context, the bold font residues in SEQ ID NO:5 are interface residues 21, 22, 24, 25, 28, 29, 32, 33, 36, 42, 45, 46, 49, 50, 52, 53, 54, and 57.

[0025] [Table 2]

[0026] In one embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 4-18. In another embodiment, the polypeptide comprises an amino acid sequence that is 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:5 (EpcamBind1_cb4). In one such embodiment, the interface residues in the polypeptide are identical to the reference polypeptide sequence. For example, residues 21, 22, 24, 25, 28, 29, 32, 33, 36, 42, 45, 46, 49, 50, 52, 53, 54, and 57 of the polypeptide (or the corresponding residues of SEQ ID NO:6 or 18) are identical to the reference polypeptide sequence.

[0027] In another embodiment, residues 1-16 relative to the reference sequence (or the corresponding residues in SEQ ID NO: 6 or 18) form a first α-helix, residues 20-36 relative to the reference sequence (or the corresponding residues in SEQ ID NO: 6 or 18) form a second α-helix, and residues 40-57 relative to the reference sequence (or the corresponding residues in SEQ ID NO: 6 or 18) form a third α-helix. For context, residues present in the α-helices are underlined in Table 2. In a further embodiment, the residues present in the α-helices ("α-helix residues") are identical to the α-helix residues of the reference sequence.

[0028] In another aspect, the disclosure provides a polypeptide that binds to a human EpCAM receptor, the polypeptide comprising the amino acid sequence of SEQ ID NO:2.

[0029] As disclosed in the Examples below, the inventors designed polypeptides according to SEQ ID NO:2 (exemplary such polypeptides are listed in Table 4) and performed site saturation mutagenesis studies to determine permissible substitutions at each position that reflect the permissible amino acids shown in Table 3. In some embodiments, these polypeptides bind to the extracellular portion of the human EpCAM receptor.

[0030] [Table 3] TIFF2025516169000006.tif138132

[0031] Table 3 also shows the interface residue positions of SEQ ID NO:2, residues 1, 4, 5, 7, 8, 9, 11, 12, 15, 16, 19, 20, 43, 44, 45, 47, 48, 50, 51, 52, 54, and 55. In one embodiment, interface residues 1, 4, 5, 7, 8, 9, 11, 12, 15, 16, 19, 20, 43, 44, 45, 47, 48, 50, 51, 52, 54, and 55 are selected from the corresponding interface residues present in any one of SEQ ID NOs:19-21 (sequences shown in Table 2).

[0032] Residue numbers for the interface residues are listed in SEQ ID NO:2 / Table 3, and residue numbers for SEQ ID NOs:19-21 in Table 4 are the same as those listed in SEQ ID NO:2 / Table 3, except for SEQ ID NO:21 (EpcamBind2_cb10_cys). This embodiment has two additional amino acid residues at the N-terminus relative to SEQ ID NO:2. * i.e. the numbering has shifted by +2).

[0033] [Table 4]

[0034] In another 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 an amino acid sequence selected from SEQ ID NOs: 19-21. In another 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 an amino acid sequence of SEQ ID NO:20.

[0035] In another embodiment, residues 1-20 relative to the reference sequence form a first α-helix, residues 24-41 relative to the reference sequence form a second α-helix, and residues 44-57 relative to the reference sequence form a third α-helix. In a further embodiment, the residues present in the α-helix (the "α-helical residues") are identical to the α-helical residues of the reference sequence.

[0036] In a further aspect, the disclosure provides a polypeptide that binds to the human PDL1 receptor, the polypeptide comprising the amino acid sequence of SEQ ID NO:3.

[0037] As disclosed in the Examples below, the inventors designed polypeptides according to SEQ ID NO:3 (exemplary such polypeptides are listed in Table 6) and performed site saturation mutagenesis studies to determine permissible substitutions at each position that reflect the permissible amino acids shown in Table 5. In some embodiments, these polypeptides bind to the extracellular portion of the human PDL1 receptor.

[0038] [Table 5] TIFF2025516169000009.tif130132

[0039] Table 5 also shows the interface residue positions of SEQ ID NO:3, residues 1, 4, 5, 8, 11, 12, 15, 16, 19, 42, 43, 44, 47, 48, 51, 52, 54, 55, and 56. In one embodiment, interface residues 1, 4, 5, 8, 11, 12, 15, 16, 19, 42, 43, 44, 47, 48, 51, 52, 54, 55, and 56 are selected from the corresponding interface residues present in any one of SEQ ID NOs:22-30 (sequences shown in Table 6).

[0040] The residue numbers of the interface residues are listed in SEQ ID NO:3 / Table 5, and the residue numbers of SEQ ID NOs:22-30 in Table 6 are the same as those listed in SEQ ID NO:3 / Table 5, except for the following: SEQ ID NO:24 (PDL1_nterm-cys): This embodiment has two additional amino acid residues at the N-terminus relative to SEQ ID NO:3; and SEQ ID NO:30 (PDL1_dimer mer): This embodiment contains two copies of a PDL1 receptor binding agent. The first copy follows the residue numbering of SEQ ID NO:3 / Table 5. The second copy of the sequence (i.e., residue 1 of the second copy) starts after the lowercase "ggsggs" (SEQ ID NO:31) linker sequence.

[0041] [Table 6]

[0042] In another embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 22-30. In one embodiment, interface residues 1, 4, 5, 8, 11, 12, 15, 16, 19, 42, 43, 44, 47, 48, 51, 52, 54, 55, and 56 are selected from the corresponding interface residues present in any one of SEQ ID NOs: 22-30.

[0043] In another embodiment, residues 1-20 relative to the reference sequence form a first α-helix, residues 24-41 relative to the reference sequence form a second α-helix, and residues 44-55 relative to the reference sequence form a third α-helix. In a further embodiment, the residues present in the α-helix (the "α-helical residues") are identical to the α-helical residues of the reference sequence.

[0044] In one embodiment of any of the polypeptides of the present disclosure, the polypeptides bind to their targets with nanomolar affinity as measured by Octet. In an Octet experiment, a target protein is immobilized on an Octet sensor and the kinetics of binding of a binder to the target is measured over a range of binder concentrations to determine the affinity of the binder.

[0045] In one embodiment of any of the polypeptides of the present disclosure, the polypeptide may include additional amino acid residues at the N-terminus and / or C-terminus. In these embodiments, the residue numbering is based on the reference sequence as disclosed above, and the percent identity requirement does not include any additional residues added to either end. The additional residues may be any residues deemed appropriate for the intended use. In some embodiments, the additional amino acid residues include a functional domain. In various non-limiting embodiments, the additional residues / functional domains may include, but are not limited to, detectable residues / domains (i.e.: GFP, etc.), expression tags (i.e.: N-terminal methionine or other residues), cysteine ​​residues (e.g., to facilitate covalent attachment to other moieties), functional domains (i.e., cell or tissue targeting moieties; therapeutic peptide domains, diagnostic peptide domains, cytotoxins, etc.), additional copies of EpCAM or PDL1 receptor binding agents (i.e., multimers such as dimers, trimers, etc., optionally linked by amino acid linkers, including but not limited to GS-rich linkers), scaffolding, etc.

[0046] As disclosed in the examples below, multimerization (such as dimerization) significantly increases the binding affinity of polypeptides to their targets. Therefore, in another embodiment of any of the polypeptides of the present disclosure, a fusion protein is provided that comprises two, three, four or more polypeptides according to any of the embodiments or combinations of embodiments herein, where the polypeptides may be directly linked or linked via an amino acid linker. In one embodiment, the fusion protein comprises a dimer of any of the polypeptides disclosed herein, linked directly or via an amino acid linker.

[0047] In a further aspect, the present disclosure provides nucleic acids, including isolated nucleic acids, encoding the polypeptides and fusion proteins of the present disclosure. The isolated nucleic acid sequences may include RNA or DNA. Such isolated nucleic acid sequences may include additional sequences useful for facilitating expression and / or purification of the encoded proteins, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings of the present specification, it will be clear to one skilled in the art what nucleic acid sequences encode the polypeptides of the present invention.

[0048] In another aspect, the disclosure provides an expression vector comprising the nucleic acid of any aspect of the invention operably linked to a suitable regulatory sequence. An "expression vector" includes a vector that operably links a nucleic acid coding region or gene to any regulatory sequence capable of effecting expression of the gene product. A "regulatory sequence" operably linked to a nucleic acid sequence of the invention is a nucleic acid sequence capable of effecting expression of a nucleic acid molecule. A regulatory sequence need not be contiguous with a nucleic acid sequence, so long as it functions to direct its expression. Thus, for example, an intervening untranslated but transcribed sequence can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such regulatory sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors include, but are not limited to, plasmids and viral-based expression vectors. Regulatory sequences used to drive expression of the disclosed nucleic acid sequences in mammalian systems can 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 within the host organism, either as an episome or by integration into the host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, a virus-based vector (including but not limited to a retroviral vector or an oncolytic virus), or any other suitable expression vector. In some embodiments, the expression vector can be administered in the methods of the present disclosure to express the polypeptide in vivo for therapeutic benefit.

[0049] In a further aspect, the present disclosure provides a host cell comprising the polypeptide, fusion protein, nucleic acid, and / or expression vector of any embodiment disclosed herein, which may be either a prokaryotic or eukaryotic cell. The cell may be transiently or stably engineered to incorporate the expression vector of the present invention using techniques including but not limited to bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-mediated, DEAE-dextran-mediated, polycation-mediated, or viral-mediated transfection (see, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition (RI Freshney. 1987. Liss, Inc. New York, NY)). A method for producing the polypeptide or fusion protein according to the present invention is an additional part of the present invention. The method comprises the steps of (a) culturing a host under conditions conducive to expression of a polypeptide in accordance with this aspect of the invention, and (b) optionally recovering the expressed polypeptide.

[0050] In another aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide, a fusion protein, a nucleic acid, an expression vector, or a host cell of any embodiment or combination of embodiments herein and a pharma- ceutical acceptable carrier. The pharmaceutical composition of the present disclosure can be used, for example, in the disclosed methods described herein. The pharmaceutical composition may further comprise (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 buffering agent.

[0051] 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 cryoprotectant, such as sucrose, sorbitol or trehalose. In some embodiments, the pharmaceutical composition includes a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, 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, such as glycine. In yet other embodiments, the pharmaceutical composition comprises a surfactant, such as polysorbate-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 trioleate, or a combination thereof. The pharmaceutical composition may also comprise a tonicity adjuster, such as a compound that renders the formulation substantially isotonic or isosmotic with human blood. Exemplary tonicity adjusters include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises a stabilizer, e.g., a molecule that, when combined with the 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.

[0052] The polypeptide, fusion protein, nucleic acid, expression vector, or cell of any embodiment or combination of embodiments herein may be the only active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for the intended use.

[0053] In another aspect, the present disclosure provides a method for treating or limiting the onset of cancer, autoimmune disease, or inflammation, comprising administering to a subject in need thereof an effective amount of a polypeptide, fusion protein, nucleic acid, expression vector, host cell, or pharmaceutical composition of the preceding claims to treat or limit the onset of the disorder.As a non-limiting example, PDL1-binding agents bind to the same site as PDL1 as antibody checkpoint inhibitors, and therefore can act as checkpoint inhibitors that can activate the immune system to attack cancer.

[0054] As used herein, "treat" or "treating" means achieving one or more of the following: (a) lessening the severity of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disorder(s) being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing the recurrence of the disorder(s) in patients who have previously suffered from the disorder(s); and (e) limiting or preventing the recurrence of symptoms in patients previously exhibiting symptoms of the disorder(s).

[0055] The subject can be any subject with the relevant disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cows, etc. In one embodiment, the subject is a human subject.

[0056] Working Example The inventors have developed miniprotein binders capable of targeting either the human epithelial cell adhesion molecule (EpCAM) receptor or the human programmed cell death ligand 1 (PDL1) receptor. These receptors are important targets for many human diseases, including but not limited to cancer, autoimmunity, and inflammation. The novel designed proteins bind to their targets with low to moderate nanomolar affinity as measured by octet. Furthermore, site saturation mutagenesis (SSM) of the binders displayed on yeast was performed to reveal the functional tolerance of all possible single point mutations. SEC shows that the designs are well-functioning monodisperse proteins, and cell binding assays show that the designs are capable of targeting human cells expressing the respective target receptors.

[0057] Introduction and Results Human EpCAM and PDL1 receptors are involved in various diseases such as cancer, autoimmunity and inflammation. To generate binders that can target these receptors and may be clinically applicable, we designed novel miniproteins following published protocols with rifdock and Rosetta™ sequence design. We first screened the designs for binding to their respective target proteins by yeast surface display, and the best binders (EpcamBind1, EpcamBind2, and Pdl1Bind) were further optimized by site-saturation mutagenesis (SSM) followed by combinatorial libraries based on the best single-point mutations. This generated the optimized EpcamBind1_cb4, EpcamBind2_cb10, which bind to the extracellular portion of the human EpCAM receptor; and Pdl1Bind_cb6, which binds to the extracellular portion of the human PDL1 (see sequence listing and Figure 1).

[0058] These proteins bind their targets with low to moderate nanomolar affinity based on the octet data (see Figure 2A-C). We then introduced surface cysteine ​​residues based on the SSM data to identify positions in the design where small molecules of interest could be conjugated to the protein that may facilitate cell labeling and drug targeting applications. We showed that these mutants, labeled with Alexa Fluor 594 (AF594), can specifically bind cells expressing their respective targets (see Figure 3) and that through the creation of gs-linked fusion dimers, the binding affinity of EpCAMBind1 can be significantly increased to low picomolar (see Figure 4). Furthermore, we show that the introduction of AF594 does not affect the binding or stability of the proteins by cyclic dichroism, with the exception of PDL1_L10C, which appears to be poorly folded with or without labeling. The remaining designs, with or without conjugated AF594, perform well, remaining folded up to 60-70 °C and fully refolding upon cooling to room temperature. Figure 5 shows the sequences, secondary structure information, and interface positions of EpcamBind1_cb4, EpcamBind2_cb10, and PDL1Bind_cb6.

[0059] EpCAM- and PDL1-binding agents were conjugated to the cytotoxin MMAE via site-specific cysteines and showed potent in vitro toxicity against cancer cell lines expressing the respective target proteins (data not shown).

Claims

**Claim 1** A polypeptide that binds to the human epithelial cell adhesion molecule (EpCAM) receptor, the polypeptide comprising the amino acid sequence of SEQ ID NO:

1. **Claim 2** The polypeptide according to claim 1, wherein the interface residues 21, 22, 24, 25, 28, 29, 32, 33, 36, 42, 45, 46, 49, 50, 52, 53, 54, and 57 are selected from the corresponding interface residues present in any one of SEQ ID NOs: 4-18. **Claim 3** The polypeptide according to claim 1 or 2, comprising an amino acid sequence that is 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 SEQ ID NOs: 4-18. **Claim 4** A polypeptide comprising an amino acid sequence that is 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 SEQ ID NOs: 4-18. **Claim 5** The polypeptide according to claim 4, wherein the interface residues are identical to the interface residues of the reference sequence. **Claim 6** The polypeptide according to any one of claims 1-5, wherein residues 1-16 (or the corresponding residues of SEQ ID NO: 6 or 18) relative to the reference sequence form a first α-helix, residues 20-36 (or the corresponding residues of SEQ ID NO: 6 or 18) relative to the reference sequence form a second α-helix, and residues 40-57 (or the corresponding residues of SEQ ID NO: 6 or 18) relative to the reference sequence form a third α-helix. **Claim 7** The polypeptide according to claim 6, wherein the α-helix residues are identical to the α-helix residues of the reference sequence. **Claim 8** A polypeptide that binds to the human EpCAM receptor, the polypeptide comprising the amino acid sequence of SEQ ID NO:

2. **Claim 9** The polypeptide according to claim 8, wherein the interface residues 1, 4, 5, 7, 8, 9, 11, 12, 15, 16, 19, 20, 43, 44, 45, 47, 48, 50, 51, 52, 54, and 55 are selected from the interface residues present in the amino acid sequences selected from SEQ ID NOs: 19-21. **Claim 10** The polypeptide according to claim 8 or 9, comprising an amino acid sequence that is 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 SEQ ID NOs: 19 to 21.

11. A polypeptide comprising an amino acid sequence that is 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 SEQ ID NOs: 19 to 21.

12. The polypeptide according to claim 11, wherein the interfacial residues are identical to the interfacial residues of the reference sequence.

13. The polypeptide according to any one of claims 8 to 12, wherein residues 1 to 20 form a first α-helix, residues 24 to 41 form a second α-helix, and residues 44 to 57 form a third α-helix.

14. The polypeptide according to claim 13, wherein the α-helix residues are identical to the α-helix residues of the reference sequence.

15. A polypeptide that binds to the human programmed cell death ligand 1 (PDL1) receptor, comprising the amino acid sequence of SEQ ID NO:

3.

16. The polypeptide according to claim 15, wherein the interfacial residues 1, 4, 5, 8, 11, 12, 15, 16, 19, 42, 43, 44, 47, 48, 51, 52, 54, 55, and 56 are selected from the interfacial residues present in the amino acid sequences selected from SEQ ID NOs: 22 to 30.

17. The polypeptide according to claim 15 or 16, comprising an amino acid sequence that is 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 SEQ ID NOs: 22 to 30.

18. A polypeptide comprising an amino acid sequence that is 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 SEQ ID NOs: 22 to 30.

19. The polypeptide according to claim 18, wherein the interfacial residues are identical to the interfacial residues of the reference sequence.

20. The polypeptide according to any one of claims 15 to 19, wherein residues 1 to 20 form a first α - helix, residues 24 to 41 form a second α - helix, and residues 44 to 55 form a third α - helix.

21. The polypeptide according to claim 20, wherein the α - helix residues are identical to the α - helix residues of the reference sequence.

22. The polypeptide according to any one of claims 1 to 21, comprising additional amino acid residues at the N - terminus and / or C - terminus.

23. A fusion protein comprising two, three, four, or more polypeptides according to any one of claims 1 to 22, wherein the polypeptides may be directly linked or linked via an amino acid linker.

24. A nucleic acid encoding the polypeptide of the fusion protein according to any one of claims 1 to 23.

25. An expression vector comprising the nucleic acid according to claim 24, operably linked to appropriate regulatory elements.

26. A host cell comprising the polypeptide, fusion protein, nucleic acid, or expression vector according to any one of claims 1 to 25.

27. A pharmaceutical composition comprising: (a) the polypeptide, fusion protein, nucleic acid, expression vector, or host cell according to any one of claims 1 to 26; and (b) a pharmaceutically acceptable carrier A pharmaceutical composition.

28. Use of the polypeptide, fusion protein, nucleic acid, expression vector, host cell, or pharmaceutical composition according to any one of claims 1 to 27 for the treatment of a disorder selected from cancer, autoimmune diseases, and inflammation.

29. A method for treating or restricting the onset of cancer, autoimmune disease, or inflammation, the method comprising administering to a subject in need thereof an effective amount of the polypeptide, fusion protein, nucleic acid, expression vector, host cell, or pharmaceutical composition according to any one of claims 1 to 28.

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