De novo designed binding agents to target human EpCAM and PDL1 receptors

Abstract

Disclosed herein are polypeptides that bind to the epithelial cell adhesion molecule (EpCAM) and programmed death ligand I (PDL1) receptor, nucleic acids encoding them, and methods of their use in treating or limiting the development of cancer, autoimmune disease, or inflammation. The invention also provides nucleic acids encoding the polypeptides of the disclosure: expression vectors comprising the nucleic acids of the disclosure operably linked to a suitable regulatory control element, host cells comprising the polypeptides, fusion proteins, nucleic acids or expression vectors of any embodiment of the disclosure, and methods of using the same. And pharmaceutical compositions comprising a polypeptide, a fusion protein, a nucleic acid, an expression vector or a host cell of any embodiment; and a pharmaceutically acceptable carrier.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 336,629, filed on April 29, 2022, which is incorporated herein by reference in its entirety.

[0003] Federal Funding Statement

[0004] This invention was made with government support under Grant Nos. HR0011835403 from the Defense Advanced Research Projects Agency, R01CA240339-01 from the National Cancer Institute, T32GM008268 from the National Institute of General Medical Sciences, and 5U19AG065156-02 from the National Institute on Aging. The government has certain rights in this invention.

[0005] Reference to an electronic sequence listing

[0006] A computer readable form of the sequence listing is submitted electronically with this application and is hereby incorporated by reference in its entirety. The sequence listing is contained in an XML file created on April 19, 2023, with the file name "22-0544-WO.xml" and a size of 87,939 bytes. Background Art

[0007] Human epithelial cell adhesion molecule (EpCAM) and programmed death ligand 1 (PDL1) receptors are implicated in a variety of diseases such as cancer, autoimmunity, and inflammation. Therefore, there is a need for therapeutics that target EpCAM and PDL1. While antibodies that target these receptors exist, our designed proteins have unique properties compared to antibodies, such as rapid pharmacokinetics and enhanced tissue / tumor penetration due to their small size, which will alter the toxicity / efficacy profile of therapeutics made from our binders compared to antibodies. Summary of the invention

[0008] In one aspect, the disclosure provides a polypeptide that binds to a human epithelial cell adhesion molecule (EpCAM) receptor, wherein the polypeptide comprises 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.

[0009] In another aspect, the disclosure provides a polypeptide that binds to a human EpCAM receptor, wherein the polypeptide comprises 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 the interface residues present in an amino acid sequence selected from SEQ ID NO: 19-21. 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 NO: 19-21.

[0010] In another aspect, the disclosure provides a polypeptide that binds to a human programmed death ligand 1 (PDL1) receptor, wherein the polypeptide comprises 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 the 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 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.

[0011] In another embodiment, the present disclosure provides a fusion protein comprising 2, 3, 4 or more polypeptides according to any embodiment of the present disclosure, wherein the polypeptides may be directly linked or may be linked via an amino acid linker.

[0012] The present disclosure further provides nucleic acids encoding polypeptides of the present disclosure; expression vectors comprising nucleic acids of the present disclosure operably linked to appropriate regulatory control elements, host cells comprising polypeptides, fusion proteins, nucleic acids or expression vectors of any embodiment of the present disclosure, and pharmaceutical compositions comprising polypeptides, fusion proteins, nucleic acids, expression vectors or host cells of any embodiment; and pharmaceutically acceptable carriers.

[0013] The present disclosure also provides methods for treating cancer, autoimmune diseases or inflammation or limiting the development of cancer, autoimmune diseases 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 embodiment of the present invention to treat the disease or limit the development of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The binder design models from left to right are: EpcamBind1_cb4, EpcamBind2_cb10, and PDL1Bind_cb6.

[0015] Figure 2A is the octet data for EpcamBind1_cb4. EpcamBind1 shows binding and dissociation curves with an estimated Kd of 5.6 nM based on kinetic fits of binding and dissociation. The concentration range was from 100 nM (top curve) to 1.56 nM (bottom curve) with two-fold dilution.

[0016] Figure 2B is the octet data for EpcamBind2_cb10. EpcamBind2 shows binding and dissociation curves with an estimated Kd of 500 nM based on kinetic fits of binding and dissociation. The concentration range is from 1000 nM (top curve) to 15.6 nM (bottom curve) with a 2-fold dilution.

[0017] Figure 2C is the octet data for PDL1Bind_cb6. Based on steady-state analysis, the estimated Kd for PDL1Bind_cb6 is 2 nM.

[0018] Figure 3is a cell binding assay. Binding of the control antibody is shown at the top and binding of the designed binder is shown at the bottom. EpCAMBind1_cterm-cys-AF594 specifically binds to K562 cells engineered to express high levels of EpCAM, with no significant non-specific binding to EpCAM knockout K562 cells. Cell binding activity was observed at 50nM and 500nM. PDL1Bind1_E39C-AF594 specifically binds to PDL1+SKOV3 cells at 500nM and 5uM, with some minor non-specific binding to PDL1- cells at 5uM, comparable to that of the control antibody.

[0019] Figure 4 is the octet data for the gs-linked dimer of EpCAMBind1_cterm_cys. The figure shows the binding and dissociation curves, and the estimated Kd of the binder is less than 10pM based on the kinetic fit of binding and dissociation. The concentrations from the top curve to the bottom curve are 20nM, 10nM, 5nM, 2.5nM, 1nM and 0.5nM, respectively.

[0020] Figure 5 The designed sequences and secondary structure diagrams of EpcamBind1_cb4 (SEQ ID NO: 5), EpcamBind2_cb10 (SEQ ID NO: 20) and PDL1Bind_cb6 (SEQ ID NO: 23) are shown. Alpha helices are shown as boxes, loops are shown as lines, and interface residues are marked with asterisks. DETAILED DESCRIPTION

[0021] All cited references are incorporated herein by reference in their entirety. In this application, unless otherwise indicated, the techniques used can be found in several well-known references, for example: "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" (MP Deutshcer 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 (Animal Cell Culture: A Manual of Basic Technique), 2nd ed. (RI Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols (Gene Transfer and Expression Protocols), pp. 109-128, EJ Murray, ed., Humana Press Inc., Clifton, NJ), and Ambion 1998 Catalog (Ambion, Austin, TX).

[0022] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0023] The amino acid residues used in this article 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).

[0024] 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 may be deleted).

[0025] Unless the context clearly dictates otherwise, all embodiments of any aspect of the disclosure can be used in combination.

[0026] Unless the context clearly requires otherwise, throughout the specification and claims, the words “including”, “comprising” and the like are to be interpreted in an inclusive sense and not in an exclusive or exhaustive sense; that is, in the sense of “including but not limited to”. Words using the singular or plural number also include the plural and singular, respectively. In addition, when used in this application, the words “herein,” “above,” and “below,” and words of similar meaning, when used, shall refer to this application as a whole and not to any particular portion of this application.

[0027] In a first aspect, the present disclosure provides a polypeptide that binds to a human epithelial cell adhesion molecule (EpCAM) receptor, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:1.

[0028] As disclosed in the embodiments, the polypeptides of this aspect bind to the human EpCAM receptor. As disclosed in the following embodiments, 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 the permissible substitutions at each position, reflecting the permissive amino acids shown in Table 1. In some embodiments, these polypeptides bind to the extracellular portion of the human EpCAM receptor.

[0029] Table 1 shows the amino acid sequence of SEQ ID NO: 1 SEQ ID NO: 1 / Table 1

[0030]

[0031]

[0032]

[0033] Table 1 also shows the positions of interface residues in SEQ ID NO: 1, located at 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).

[0034] The residue numbers of the interface residues are listed in SEQ ID NO: 1 / Table 1. The residue numbers of SEQ ID NO: 4-18 in Table 2 are the same as those listed in SEQ ID NO: 1 / Table 1, except for the following:

[0035] 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 (ie, numbering shifted by +2); and

[0036] SEQ ID NO: 18 (EpCAM1_dimer): This embodiment includes two copies of the EpCAM receptor binder. The first copy follows the residue numbering in SEQ ID NO: 1 / Table 1. The second copy of the sequence (ie: residue 1 of the second copy) begins after a lowercase "ggsggs" (SEQ ID NO: 31) linker sequence.

[0037] For context, the bold 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.

[0038] Table 2

[0039]

[0040]

[0041] 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 NO: 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 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 in SEQ ID NO:6 or 18) are identical to the reference polypeptide sequence.

[0042] 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. The residues present in the α-helix in Table 2 are underlined for ease of understanding. In another embodiment, the residues present in the α-helix ("α-helix residues") are identical to the α-helix residues of the reference sequence.

[0043] In another aspect, the present disclosure provides a polypeptide that binds to human EpCAM receptor, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.

[0044] 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 the permissible substitutions at each position, reflecting the permissive amino acids shown in Table 3. In some embodiments, these polypeptides bind to the extracellular portion of the human EpCAM receptor.

[0045] SEQ ID NO:2 / Table 3

[0046]

[0047]

[0048]

[0049] Table 3 also shows the positions of interface residues in SEQ ID NO: 2, located at 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 are shown in Table 2).

[0050] The residue numbers of the interface residues are listed in SEQ ID NO:2 / Table 3, and the residue numbers of SEQ ID NO: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 is shifted by +2).

[0051] Table 4

[0052]

[0053]

[0054] 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: 19-21. 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 of SEQ ID NO: 20.

[0055] 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 ("α-helix residues") are identical to the α-helix residues of the reference sequence.

[0056] In another aspect, the present disclosure provides a polypeptide that binds to human PDL1 receptor, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:3.

[0057] 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 the allowed substitutions at each position, reflecting the allowed amino acids shown in Table 5. In some embodiments, these polypeptides bind to the extracellular portion of the human PDL1 receptor.

[0058] SEQ ID NO:3 / Table 5

[0059]

[0060]

[0061]

[0062] Table 5 also shows the positions of interface residues in SEQ ID NO:3, located at 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).

[0063] The residue numbers of the interface residues are listed in SEQ ID NO:3 / Table 5, and the residue numbers of SEQ ID NO:22-30 in Table 6 are the same as listed in SEQ ID NO:3 / Table 5, except for the following:

[0064] 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

[0065] SEQ ID NO:30 (PDL1_dimer mer): This embodiment includes two copies of the PDL1 receptor binding agent. The first copy follows the residue numbering in SEQ ID NO:3 / Table 5. The second copy of the sequence (ie: residue 1 of the second copy) begins after the lowercase "ggsggs" (SEQ ID NO:31) linker sequence.

[0066] Table 6

[0067]

[0068] 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.

[0069] 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 ("α-helix residues") are identical to the reference sequence α-helix residues.

[0070] In one embodiment of any of the polypeptides of the present disclosure, the polypeptide binds its target with nanomolar affinity as measured by octet. In an octet experiment, the target protein is immobilized on an octet sensor and the kinetics of binding of the binder to the target are measured over a range of binder concentrations to determine the binder affinity.

[0071] In one embodiment of any polypeptide disclosed herein, 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 disclosed above, and the percentage identity requirement does not include any additional residues added at either end. The additional residues may be any residue that is considered suitable for the intended use. In some embodiments, the additional amino acid residues include a functional domain. In various non-limiting embodiments, 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 parts), 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 connected by amino acid linkers, including but not limited to GS-rich linkers), scaffold connections, etc.

[0072] As disclosed in the following examples, multimerization (e.g., dimerization) significantly increases the binding affinity of a polypeptide to its target. Therefore, in another embodiment of any polypeptide disclosed herein, a fusion protein is provided, which comprises 2, 3, 4 or more polypeptides according to any embodiment or combination of embodiments herein, wherein the polypeptide can be directly connected, or can be connected by an amino acid linker. In one embodiment, the fusion protein comprises a dimer of any polypeptide disclosed herein, which is directly connected, or connected by an amino acid linker.

[0073] On the other hand, the present disclosure provides nucleic acids encoding polypeptides and fusion proteins of the present disclosure, including isolated nucleic acids. Isolated nucleic acid sequences may include RNA or DNA. Such isolated nucleic acid sequences may include additional sequences for promoting expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be clear to those skilled in the art which nucleic acid sequences will encode the polypeptides of the present invention.

[0074] In another aspect, the disclosure provides expression vectors, which include nucleic acids of any aspect of the present invention operably connected to suitable control sequences. "Expression vectors" include vectors that operably connect nucleic acid coding regions or genes to any control sequences that can affect the expression of gene products. "Control sequences" operably connected to the nucleic acid sequences of the present invention are nucleic acid sequences that can affect the expression of nucleic acid molecules. Control sequences do not have to be adjacent to the nucleic acid sequences, as long as they play a role in directing their expression. Therefore, for example, there may be untranslated but transcribed intermediate sequences between the promoter sequence and the nucleic acid sequence, and the promoter sequence may still be considered to be "operably connected" 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 include, but are not limited to, plasmids and virus-based expression vectors. The control sequences used to drive the expression of the disclosed nucleic acid sequences in mammalian systems 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 variety of inducible promoters, including but not limited to tetracycline, ecdysone, steroid response promoters). The expression vector must be replicable in the host organism, as an episome or by integration into the host chromosomal DNA. In various embodiments, the expression vector may include 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 may be administered in the method of the present disclosure to express the polypeptide in vivo to obtain a therapeutic benefit.

[0075] In another aspect, the present disclosure provides a host cell comprising a polypeptide, fusion protein, nucleic acid and / or expression vector of any embodiment disclosed herein, wherein the host cell can be prokaryotic or eukaryotic. Cells can be temporarily or stably transformed to integrate the expression vector of the present invention using techniques including, but not limited to, bacterial transformation, calcium phosphate coprecipitation, electroporation, or liposome-mediated, DEAE dextran-mediated, polycation-mediated, or viral-mediated transfection. (See, e.g., Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, Second Edition (RI Freshney. 1987. Liss, Inc. New York, New York)). A method for producing a polypeptide or fusion protein according to the present invention is an additional part of the present invention. The method comprises the following steps: (a) culturing a host according to this aspect of the present invention under conditions conducive to polypeptide expression, and (b) optionally recovering the expressed polypeptide.

[0076] In another aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide, fusion protein, nucleic acid, expression vector or host cell of any embodiment or combination of embodiments herein and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure can be used, for example, in the methods of the present disclosure described herein. The pharmaceutical composition may also include (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tension modifier; (e) a stabilizer; (f) a preservative and / or (g) a buffer.

[0077] 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 freeze-drying protective agent, such as sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative, such as benzalkonium chloride, benzethonium chloride, chlorhexidine, phenol, meta-cresol, benzyl alcohol, methyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a filler, such as glycine. In other embodiments, the pharmaceutical composition includes a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitol monolaurate, sorbitol monopalmitate, sorbitol monostearate, sorbitol monooleate, sorbitol trilaurate, sorbitol tristearate, sorbitol trioleate, or a combination thereof. The pharmaceutical composition may also include a tension regulator, such as a compound that makes the formulation substantially isotonic with human blood or isotonic with human blood. Exemplary tension regulators include sucrose, sorbitol, glycine, methionine, mannitol, glucose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition also includes a stabilizer, such as a molecule that substantially prevents or reduces the chemical and / or physical instability of the target protein in lyophilized or liquid form when combined with the target protein. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0078] The polypeptide, fusion protein, nucleic acid, expression vector or cell of any embodiment or combination of embodiments herein can be the only active agent in the pharmaceutical composition, or the composition can also contain one or more other active agents suitable for the intended use.

[0079] In another aspect, the present disclosure provides a method for treating cancer, autoimmune disease or inflammation or limiting the development 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 any one of the preceding claims for treating or limiting the development of the disease. By way of non-limiting example, PDL1 binders can act as checkpoint inhibitors that can activate the immune system to attack cancer because the sites to which they bind to antibody checkpoint inhibitors are the same as the sites on PDL1.

[0080] As used herein, "treat" or "treating" means achieving one or more of the following: (a) reducing the severity of a condition; (b) limiting or preventing the development of symptoms characteristic of the condition being treated; (c) inhibiting the worsening of symptoms characteristic of the condition being treated; (d) limiting or preventing the recurrence of the disease in a patient who previously had the condition; and (e) limiting or preventing the recurrence of symptoms in a patient who previously had symptoms of the condition.

[0081] The subject can be any subject suffering from 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.

[0082] Example

[0083] We developed mini-protein binders that can target either the human epithelial cell adhesion molecule (EpCAM) receptor or the human programmed death ligand 1 (PDL1) receptor. These receptors are important targets for many human diseases such as, but not limited to, cancer, autoimmunity, and inflammation. The de novo designed proteins bind to their targets with low to mid-nanomolar affinities as determined by octets. In addition, site-saturation mutagenesis (SSM) was performed on the binders displayed on yeast to reveal the functional tolerance of all possible single point mutations. SEC showed that the designs are well-behaved monodisperse proteins and cell binding assays showed that the designs can target human cells expressing their respective target receptors.

[0084] Introduction and Results

[0085] Human EpCAM and PDL1 receptors are implicated in a range of diseases including cancer, autoimmunity, and inflammation. To generate binders that could target these receptors and have clinical applications, we followed published methods involving rifdock and Rosetta. TM The sequence design approach designed de novo small proteins. The designs were first screened by yeast surface display to bind to their respective target proteins, and the best binders (EpcamBind1, EpcamBind2 and Pdl1Bind) were further optimized by site saturation mutagenesis (SSM) and subsequent combinatorial libraries based on the best single point mutations. This resulted in the generation of optimized EpcamBind1_cb4, EpcamBind2_cb10, which bind to the extracellular part of the human EpCAM receptor; and Pdl1Bind_cb6, which bind to the extracellular part of human PDL1 (see sequence listing and Figure 1 ).

[0086] According to Octet data, these proteins bind their targets with low to mid nanomolar affinities (see FIG. 2A to FIG. 2C). We then identified positions in the design where surface cysteine ​​residues could be introduced based on the SSM data to allow for the binding of small molecules of interest to the protein, which could potentially facilitate cell labeling and drug targeting applications. We showed that these variants, labeled with AlexaFluor 594 (AF594), were able to specifically bind to cells expressing their respective targets (see Figure 3 ), and we could significantly increase the binding affinity of EpCAMBind1 to low picomolar levels by creating gs-linked fusion dimers (see Figure 4 ). We further showed that the introduction of AF594 did not affect the binding or stability (circular dichroism) of the proteins, with the exception of PDL1_L10C, which folded poorly both with and without the tag. The remaining designs with and without bound AF594 performed well, remaining folded at 60-70°C and fully refolding upon cooling to room temperature. Figure 5 The sequences, secondary structure information, and interface locations of EpcamBind1_cb4, EpcamBind2_cb10, and PDL1Bind_cb6 are shown in FIG.

[0087] EpCAM binders and PDL1 binders have been conjugated to the cytotoxin MMAE via site-specific cysteines and demonstrated potent in vitro toxicity against cancer cell lines expressing their respective target proteins (data not shown).

Claims

1. A polypeptide that binds to a human epithelial cell adhesion molecule (EpCAM) receptor, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein 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.

3. A 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 an amino acid sequence selected from SEQ ID NOs: 4-18.

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 an amino acid sequence selected from SEQ ID NOs: 4-18.

5. The polypeptide of claim 4, wherein the interface residues are identical to the interface residues of the reference sequence.

6. The polypeptide according to any one of claims 1 to 5, wherein 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.

7. The polypeptide of claim 6, wherein the α-helical residues are identical to the α-helical residues of the reference sequence.

8. A polypeptide that binds to human EpCAM receptor, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

2.

9. The polypeptide of claim 8, wherein 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 sequence selected from SEQ ID NOs: 19-21.

10. A 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 an amino acid sequence selected from SEQ ID NOs: 19-21.

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 an amino acid sequence selected from SEQ ID NOs: 19-21.

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

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

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

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

3.

16. The polypeptide of claim 15, wherein 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 interface residues present in an amino acid sequence selected from SEQ ID NOs: 22-30.

17. A 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 an amino acid sequence selected from SEQ ID NOs: 22-30.

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 an amino acid sequence selected from SEQ ID NOs: 22-30.

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

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

21. The polypeptide of claim 20, wherein the α-helical residues are identical to the α-helical 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 2, 3, 4 or more polypeptides according to any one of claims 1 to 22, wherein the polypeptides can be directly linked or can be linked through an amino acid linker.

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

25. An expression vector comprising the nucleic acid of claim 24 operably linked to suitable regulatory control elements.

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

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

28. Use of the polypeptide, fusion protein, nucleic acid, expression vector, host cell or pharmaceutical composition of any one of the preceding claims for treating a condition selected from cancer, autoimmune disease and inflammation.

29. A method for treating or limiting the progression of cancer, autoimmune disease or inflammation comprising: An effective amount of the polypeptide, fusion protein, nucleic acid, expression vector, host cell or pharmaceutical composition of any one of the preceding claims is administered to a subject in need thereof to treat a disorder or limit the development of a disorder.

Images