Variants of human programmed cell death protein (pd-1)

Abstract

The disclosure provides amino acid sequence variants of programmed cell death protein 1 (PD-1) that can bind to either or both of its ligands, PDL-1 or PDL-2. The disclosure relates to ligand-binding fragments of the PD-1 variant sequences, conjugates, and related compositions and formulations. The disclosure also relates to methods for using the sequences, e.g., PD-1 variants and ligand-binding fragments thereof, in the treatment of checkpoint pathway-related diseases, disorders, and conditions including cell proliferative diseases such as cancer, as well as diseases, disorders, and conditions associated with immune deficiency, immune insufficiency, and / or subjects who are immune compromised.

Description

VARIANTS OF HUMAN PROGRAMMED CELL DEATH PROTEIN (PD-1)INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0001] This application contains a sequence listing that has been electronically filed in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on July 24, 2024, is named 67391WO01_SEQUENCE.xml and is 67,926 bytes in size.RELATED APPLICATIONS

[0002] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 578,712, filed August 25, 2023, the content of which is hereby incorporated by reference in its entirety into this disclosure.BACKGROUND

[0003] Programmed cell death protein 1 (PD-1 or CD279), is a membrane protein found on the surface of T cells, B cells and macrophages that functions as an immune checkpoint that down- regulates the immune system and prevents auto-immune diseases. In humans, PD-1 has 288 amino acids and is encoded by the PDCD1 gene. The protein includes an extracellular Ig like V-type domain followed by a single-pass transmembrane domain and an intracellular domain. When bound to its two known ligands, PDL-1 and PDL-2, PD-1 suppresses T-cell activation and subsequently negatively regulates immune response. Tumor cells are known to express PDL-1 and evade immune surveillance through the same immune checkpoint mechanism. As such, PD- 1 / PDL-l binding inhibitors, such as anti-PD-1 antibodies and anti-PDL-1 antibodies, have been pursued as potential cancer therapies, and in some cases have been shown to restore immune response against tumor cells. A similar inhibitory effect on T cell function against tumors has also been reported for PDL-2.

[0004] Another strategy for blocking PD-1 ligand interaction is to induce ligand binding to a recombinant PD- 1 sequence (e.g., a PD- 1 decoy, such as a fragment of PD- 1 or a fusion protein ofa PD- 1 sequence and another functional sequence). Nevertheless, this strategy may be limited by the relatively low intrinsic binding affinity of PD-1 to PDL-1 (KD of 8.2 qM) as compared to a typical anti-PD- 1 or anti-PDL- 1 antibody that finds use in clinical applications (typically having a KD of single digit nanomolar or below). Thus, to achieve maximum efficacy in immunotherapy, a PD-1 decoy with higher binding affinity to PDL-1 is desirable.

[0005] PD-1 protein engineering is generally known. For example, using yeast display technology, Maute et al. identified human PD-1 variants with improved PDL-1 binding affinities of 110 pM. Using a small rationally-designed mammalian cell library, Liu et al. obtained a PD-1 mutant with a reported PDL-1 binding EC50 value much lower (0.031 qg / mL) than that of wild type PD-1 (2.571 pg / mL). Using phage display panning Li et al. discovered a panel of PD-1 mutants with high affinity (KD of 0.687 nM). Lazar-Molnar et al. found that substitution of alanine with leucine at position 132 induced higher hydrophobic interaction strength which purportedly contributed to an approximate 40-fold increase in affinity with PDL-1 and 30-fold increase with PDL-2.

[0006] Nevertheless, alternative PD-1 sequence variants and strategies for producing the same are desirable. The disclosure provides novel PD-1 sequence variants that provide for improved functionality and therapeutic efficacy.SUMMARY

[0007] In overview, the disclosure provides a structural motif comprising PD-1 protein variant sequences that can exhibit improved binding characteristics (e.g., increased binding affinity) for PD-1 ligands such as, for example, PDL-1 and / or PDL-2, improved solubility characteristics, and / or improved bioavailability as compared to the wild type PD-1 protein. The disclosure also provides methods for generating, screening, and identifying such PD- 1 protein variant sequences.

[0008] In embodiments of this aspect, the PD-1 protein variant sequence comprises an amino acid sequence having a general structure:[PD-1 N-terminal sequence] -XaaiXaa Xaa; XatuXaa [PD-1 C-terminal sequence], wherein the protein variant sequence maintains or improves binding affinity to at least one PD-1 ligand (e.g., PDL-1 and / or PDL-2) when compared to native PD-1, and wherein XaaiXaaiXaaa Xaa4Xaa5 comprise five variable amino acid residues that comprise at least one amino acid residuethatis non-native to aPD-1 loop-1 sequence. In embodiments, both the PD- 1 N-terminal sequence and the PD- 1 C-tcrminal sequence comprise at least 80% homology to a wild type PD- 1 sequence (i.e., N-terminal and C-terminal sequences from a wild type PD-1, respectively). In embodiments, the PD-1 N-terminal sequence and the PD-1 C-terminal sequence can be identified by the regions that are N-terminal and C-terminal to the sequence of the loop 1 region of a native PD-1 sequence (e.g., amino acids 70-74 of native PD-1, or amino acids 38-42 of a PD-1 extracellular Ig like V type domain). In some further embodiments the N-terminal sequence comprises a sequence having at least 80% homology to amino acids 1-37 of SEQ ID NO.57. In some embodiments the C- terminal sequence comprises a sequence having at least 80% homology to amino acids 43-108 of SEQ ID NO.57.

[0009] In another aspect, the disclosure relates to a protein comprising the amino acid sequence: NPPTFSPALL VVTEGDNATF TCSFSNTSES FVLNWYR XaaiXaa2Xaa3Xaa4Xaa5QTDKLAAFPE DRSQPGQD Xaa6RFRVTQLPNG RDFHMSVVRA RRNDSGTYLC GAISLAPK Xaa7QIKESLRAEL RVTERRAE (SEQ ID NO: 57); whereinXaai comprises M, V, I, L, or E;Xaa2comprises S, L, H, Y, L, G, D, T, or H;Xaa3comprises P, E, S, T, D, K, Q, R, V, or A;Xaa4 comprises S, T, G, N, D, F, R, H, R, or E;Xaas comprises N, or G;Xaa6 comprises an amino acid other than cysteine; and Xaa7comprises a non-polar amino acid residue.

[0010] In some embodiments, Xaae comprises a nucleophilic amino acid residue that is not cysteine. In some embodiments, Xaae comprises alanine, glycine, threonine or serine. In some embodiments, Xaae comprises alanine, glycine, or serine. In some embodiments Xaae comprises serine.

[0011] In some other embodiments, Xaa7comprises glycine, alanine, valine, leucine, isoleucine or methionine. In some further embodiments, Xaa7comprises valine, leucine, or isoleucine. In yet further embodiments, Xaa7comprises leucine.

[0012] In yet further embodiments, the disclosure provides a protein comprising an amino acid sequence as described by: (SEQ ID NO.27), (SEQ ID NO.28), (SEQ ID NO.29), (SEQ IDNO.30), (SEQ ID N0.31 ), (SEQ ID NO.32), (SEQ ID NO.33), (SEQ ID NO.34), (SEQ IDNO.35), (SEQ ID NO.36), (SEQ ID NO.37), (SEQ ID NO.38), (SEQ ID NO.39), (SEQ IDNO.40), (SEQ ID N0.41), (SEQ ID NO.42), (SEQ ID NO.43), (SEQ ID NO.44), (SEQ IDNO.45), (SEQ ID NO.46), (SEQ ID NO.47), (SEQ ID NO.48), (SEQ ID NO.49), (SEQ IDNO.50), (SEQ ID N0.51), or (SEQ ID NO.52).

[0013] In some further embodiments of the above-describe aspects and embodiments, the C- terminus of the protein can be covalently fused to a human Fc amino acid sequence.

[0014] In another aspect, the disclosure provides polynucleotide sequences that encode the protein sequences described above. In some further embodiments, the polynucleotide sequences comprise a nucleotide sequence as described by (SEQ ID NO.l), (SEQ ID NO.2), (SEQ ID NO.3), (SEQ ID NO.4), (SEQ ID NO.5), (SEQ ID NO.6), (SEQ ID NO.7), (SEQ ID NO.8), (SEQ ID NO.9), (SEQ ID NO.10), (SEQ ID NO.11), (SEQ ID NO.12), (SEQ ID NO.13), (SEQ ID NO.14), (SEQ ID NO.15), (SEQ ID NO.16), (SEQ ID NO.17), (SEQ ID NO.18), (SEQ ID NO.19), (SEQ ID NO.20), (SEQ ID NO.21), (SEQ ID NO.22), (SEQ ID NO.23), (SEQ ID NO.24), (SEQ ID NO.25), or (SEQ ID NO.26).

[0015] In another aspect, the disclosure provides pharmaceutical composition comprising the protein according to any one of the above aspects and embodiments, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0016] In another aspect, the disclosure provides a method of treating cancer comprising administering to a human patient in need thereof an effective amount of the protein according to any one of the above aspects and embodiments, or the pharmaceutical composition of the above aspects and embodiments.

[0017] In another aspect, the disclosure provides a method of treating an immunodeficiency comprising administering to a human patient in need thereof an effective amount of the protein according to any one of the above aspects and embodiments, or the pharmaceutical composition of the above aspects and embodiments.

[0018] Other aspects and embodiments in accordance with the disclosure will become apparent in light of the following detailed description and Figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 depicts a sequence alignment of exemplary PD-1 mutant sequences identifying mutations within amino acid residues 65-79, within the targeted loop region.

[0020] FIG. 2 illustrates a schematic diagram of PD-1 mutant / Fc fusion proteins, where "-S-S-" depicts cysteine disulfide bonds.

[0021] FIG. 3 illustrates PD-1 mutant / Fc binding activity to PDL-1 as measured by ELISA assay.

[0022] FIG. 4 illustrates PD-1 mutant / Fc binding activity to PDL-2 as measured by ELISA assay.

[0023] FIG. 5 illustrates ED50 values for illustrative PD-1 mutant / Fcs as measured by ELISA assay.

[0024] FIG. 6 illustrates cell surface binding activity for illustrative PD-1 mutant / Fcs as measured by flow cytometry and relative to anti-PDL-1 antibody durvalumab.

[0025] FIG. 7 illustrates a PD-l / PDL-1 blockade assay for illustrative PD-1 mutant / Fcs relative to control anti-PDL-1 antibody durvalumab and anti-PD-1 antibody Nivolumab.

[0026] FIGS. 8A-8B illustrates in vivo tumor suppression activity of illustrative PD-1 mutant / Fcs relative to controls (vehicle / PBS and positive control Pembrolizumab). FIG. 8A rate of tumor volume increase over time. FIG. 8B illustrates change in body weight over time.DETAILED DESCRIPTION

[0027] I. Introduction

[0028] The disclosure generally provides a series of PD-1 protein variants that exhibit improved characteristics relative to native PD-1 and a number of other known PD-1 protein mutant sequences. As described herein, the PD-1 protein variants leverage structural information that lead the inventors to target a loop region near the PDL-1 binding interface that adopts a different conformation, targeting it as a site of interest for sequence variation that can modulate PD-l / PDL- 1 interaction. The disclosure provides a rationally-designed series of PD-1 variants that are produced from a targeted phage library coding the PD-1 extracellular Ig like V type domain (amino acids 33-150 of native PD-1) with selected point mutations (e.g., C93S, A132L) and mutations introduced in the loop 1 region (amino acids 70-74 of native PD-1). The variants are panned and screened against a PD-L1 extra cellular domain protein in order to identify candidatevariant sequences. As illustrated by the disclosure and embodiments herein, 24 PD-1 mutant sequences have been identified and exhibit improved binding affinities to PDL-1 and PDL-2 when compared to wild type PD-1. Additional in vitro studies demonstrate the PD-1 variant (or mutant) sequences in accordance with the disclosure function to block the PD-l / PDL-1 and / or the PD- l / PDL-2 interaction. An in vivo study in a xenograft tumor model demonstrate that the PD-1 variants in accordance with the disclosure can elicit a tumor suppression effect in a subject.

[0029] II. Definitions and Terminology

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0031] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0032] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0033] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0034] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0035] By " consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory,but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0036] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three -element set {(x), (y), (x, y) } . In other words, "x and / or y " means "one or both of x and y " . As another example, "x, y , and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z) } . In other words, "x, y and / or z" means "one or more of x, y and z".

[0037] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0038] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0039] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as usedin connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0040] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0041] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0042] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0043] When used herein, "C93S" with reference to an amino acid sequence refers to a PD-1 variant protein comprising a PD-1 extracellular Ig like V type domain (AAs 33-150) having a C93S point mutation.

[0044] When used herein, "C93S / A132L" with reference to an amino acid sequence refers to a PD-1 variant comprising a PD-1 extracellular’ Ig like V type domain (AAs 33-150) having a C93S and a A132L point mutation.

[0045] As used herein, the term "PDMx" where x is an integer (e.g., x=l-24) refers to a PD-1 variant protein comprising a PD-1 extracellular Ig like V type domain (AAs 33-150) having pointmutations C93S and A132L and comprising one or more mutations in the PD-1 protein loop 1 region (AAs 70-74) in accordance with the aspects and example embodiments of the disclosure.

[0046] As used herein, the term "PDMx / Fc" where x is an integer (e.g., x = 1-24), refers to a "PDMx" variant protein sequence further comprising a fusion to an IgG 1 Fc fragment sequence.

[0047] As used herein, "PD-1" (or programmed cell death 1, PDCD1, CD279, PD-1, PD1, SLEB2, hPD-1, hPD-1, hSLEl, and the like), refers to any PD-1 amino acid sequence. In certain embodiments PD-1 refers to an amino acid sequence disclosed as NCBI Reference Sequence: NP_005009.2 (SEQ ID NO.54).

[0048] As used herein, "PD-L1" (or programmed cell death 1 ligand 1, B7-H1, CD274, PDCD1LG1, PDCD1L1, PDCD1LG1, PDL1, hPD-Ll, and the like), refers to any PD-1 ligand 1 amino acid sequence. In certain embodiments, PDL-1 refers to an amino acid sequence disclosed as NCBI Reference Sequences: NP_054862.1 (isoform a), NP_001254635.1 (isoform b), or NP_001300958.1 (isoform c).PD-L1 isoform a: 290 aa, NP_054862.1:MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWE MEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMI SYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVL SGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNE RTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO.58).PD-L1 isoform b: 176 aa, NP_001254635.1:MRIFAVFIFMTYWHLLNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQ VLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPP NERTHL VILGAILLCLGV ALTFIFRLRKGRMMD VKKCGIQDTNS KKQSDTHLEET (SEQ ID NO.59).PD-L1 isoform c: 245 aa, NP_001300958.1:MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPGNILNVSIKIC LTLSPST (SEQ ID NO.60).

[0049] As used herein, "PD-L2" (or programmed cell death 1 ligand 2, B7-DC, CD273, PDCD1LG2, Btdc, PDCD1L2, PDL2, bA574F11.2 and the like) , refers to any PD-1 ligand 2 amino acid sequence. In certain embodiments, PDL-2 refers to an amino acid sequence disclosed as NCBI Reference Sequence: NP_079515.2.PD-L2 NP_079515.2:MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQK VENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK ASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVL RLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIAL RKQLCQKLYSSKDTTKRPVTTTKREVNSAI (SEQ ID NO.61).

[0050] The term "about" means up to a 10% variance of the value such term would be given depending on the number of significant figures. For example, "about 200" encompasses from 180 to 220 and "about 1" encompasses from 0.9 to 1.1.

[0051] In some aspects relating to sequences comprising amino acids (e.g., enzymes) and nucleotides (e.g., polynucleotides / genes), the disclosure provides sequence variants of the sequences disclosed herein. With regard to amino acid sequences, variants (e.g., substitution, deletion, addition) can be considered as "similar" to the original polypeptide or to have a certain "percent identity" or "percent homology" to the original polypeptide, and include those polypeptides wherein one or more amino acid residues of a polypeptide are removed and replaced with alternative residues, deleted from the reference polypeptide sequence, and / or inserted into the reference polypeptide sequence. In embodiments, the substitutions in amino acid sequences are conservative in nature, however, the disclosure embraces substitutions that are also nonconservative, as long as one or more functional characteristics exhibited by the reference polypeptide are maintained or improved.

[0052] For amino acid sequences, sequence identity and / or similarity can be determined by using standard techniques known in the art such as, for example, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithmof Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, using the default settings, or by inspection. Various alignment parameters can be set according to known methods (e.g., "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.). Additional useful algorithms include PILEUP, which can align multiple sequences from a group of related sequences using progressive, pairwise alignments; BLAST, including gapped BLAST, WU-BLAST-2 (see, e.g., Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; Altschul et al., 1996, Methods in Enzymology 266:460-480; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787).

[0053] Generally, the amino acid homology, similarity, or identity between sequences are at least 80%, including at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and from 99% to almost 100% identity. Similarly, the "percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequences described herein refers to the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotides disclosed herein, in the gene or coding sequence of the related polypeptide. Specific methods can include the default parameters of algorithms such as, for example, BLASTN (WU-BLAST-2).

[0054] The invention is defined in the claims. However, below is a non-exhaustive listing of nonlimiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0055] III. PD-1 protein variants

[0056] In one aspect, the disclosure provides PD-1 protein variant sequences that can exhibit increased binding affinity for PD-1 ligands (e.g., PDL-1 and / or PDL-2), improved solubility characteristics, and / or improved bioavailability as compared to the wild type PD-1 protein.

[0057] In embodiments of this aspect, the protein variant sequence comprises an amino acid sequence having a general structure:[PD-1 N-terminal sequence] -XaaiXaa2Xaa3Xaa4Xaas- [PD-1 C-terminal sequence], wherein the protein variant sequence maintains or improves binding affinity to at least one PD-1 ligand (e.g., PDL-1 and / or PDL-2) when compared to native PD-1. In embodiments, both the PD- 1 N-terminal sequence and the PD-1 C-terminal sequence comprise at least 80% homology to a wild type PD-1 sequence (i.e., N-terminal and C-terminal sequences from a wild type PD-1, respectively). In embodiments, the PD-1 N-terminal sequence and the PD-1 C-terminal sequence can be identified by the regions that are N-terminal and C-terminal to the sequence of the loop 1 region of a native PD-1 sequence (e.g., amino acids 70-74 of native PD-1, or amino acids 38-42 of a PD-1 extracellular Ig like V type domain). In some further embodiments the N-terminal sequence comprises a sequence having at least 80% homology to amino acids 1-37 of SEQ ID NO.57. In some embodiments the C-terminal sequence comprises a sequence having at least 80% homology to amino acids 43-108 of SEQ ID NO.57. In some embodiments the N-terminal sequence and / or the C-terminal sequence comprises a sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity or sequence homology to the corresponding amino acid residues of SEQ ID NO.57.

[0058] In some embodiments of this aspect, the disclosure provides a protein comprising the amino acid sequence:NPPTFSPALL VVTEGDNATF TCSFSNTSES FVLNWYR XaaiXaa2Xaa3Xaa4Xaa5QTDKLAAFPE DRSQPGQD Xaa6RFRVTQLPNG RDFHMSVVRA RRNDSGTYLC GAISLAPK Xaa7QIKESLRAEL RVTERRAE (SEQ ID NO: 57); wherein, Xaai comprises M, V, I, L, or E; Xaa2comprises S, L, H, Y, L, G, D, T, or H; Xaa3comprises P, E, S, T, D, K, Q, R, V, or A; Xaa4 comprises S, T, G, N, D, F, R, H, R, or E; Xaas comprises N, or G; Xaae comprises an amino acid that is not cysteine; and Xaa7comprises a nonpolar amino acid residue. In some further embodiments the disclosure provides a protein comprising SEQ ID NO. 57 fused to an Fc amino acid sequence.

[0059] In some embodiments, the protein comprises an amino acid sequence according to SEQ ID NO. 57, wherein Xaae comprises a nucleophilic amino acid that is not cysteine.

[0060] In some embodiments the protein comprises an amino acid sequence according to SEQ ID NO. 57, wherein Xaae comprises threonine, alanine, glycine, or serine.

[0061] In some embodiments, the protein comprises an amino acid sequence according to SEQ ID NO. 57, wherein Xaae comprises serine.

[0062] In some embodiments the protein comprises an amino acid sequence according to SEQ ID NO. 57, wherein Xaa? comprises glycine, alanine, valine, leucine, isoleucine or methionine.

[0063] In some embodiments the protein comprises an amino acid sequence according to SEQ ID NO. 57, wherein Xaa? comprises valine, leucine, or isoleucine.

[0064] In some embodiments the protein comprises an amino acid sequence according to SEQ ID NO. 57, wherein Xaa? comprises leucine.

[0065] As mentioned above, the variants sequences in accordance with the disclosure include variants of human PD-1, with certain amino acid positions within the loop 1 amino acid sequence of PD-1 mutated, as illustrated in SEQ ID NO. 57. Some non-limiting PD-1 variant protein sequences comprise any of SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, or SEQ ID NO. 52.

[0066] The PD- 1 valiant proteins may be produced by any method known in the art. For example, in some embodiments, the PD-1 variant proteins can be synthesized by recombinant organisms engineered using methods well known in the art, or alternatively, by chemical synthesis. Accordingly, other aspects and embodiments of the disclosure relate to polynucleotides encoding the variants of human PD-1 protein. Other embodiments relate to a vector comprising one or more polynucleotides, as well as a host cell comprising the vector and / or the polynucleotide sequence(s). Other embodiments relate to processes for producing a PD- 1 variant protein by culturing host cells carrying said vector containing DNA encoding said protein, expressing the protein from the host cells and recovering the protein from the culture media.

[0067] The polynucleotides that encode for a PD-1 variant protein may comprise any one or more of the following features: the coding sequence for the human PD-1 protein variant, the coding sequence for the variant and additional coding sequence such as a functional protein, or a signal or secretory sequence or a pro-domain sequence; the coding sequence for the human PD- 1 proteinvariant and non-coding sequence, such as introns or non-coding sequence 5' and / or 3' of the coding sequence for the variant. Thus, the term “polynucleotide encoding a PD-1 variant” encompasses a polynucleotide that may include not only the coding sequence for the PD- 1 protein variant but also a polynucleotide which includes additional coding and / or non-coding sequence. It is known in the art that a polynucleotide sequence that is optimized for a specific host cell / expression system can readily be obtained from the amino acid sequence of the desired protein. Examples of polynucleotides in accordance with the disclosure are described in Table 1 (i.e., SEQ ID NO.s 1- 26) which are DNA sequences that encode the human PD-1 protein variants as shown in Table 2 (i.e., SEQ ID NO.s: 27-52). In some further embodiments, the polynucleotide may comprise a sequence encoding a PD-1 variant protein fused to an Fc sequence.

[0068] The polynucleotides are typically expressed in a host cell after the sequences have been operably linked to an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., tetracycline, neomycin, and / or dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences. The vectors containing the polynucleotide sequences of interest (e.g., the variants of PD-1 protein and expression control sequences) are transferred into a host cell by well-known methods, which vary depending on the type of cellular host.

[0069] The PD- 1 protein variants may readily be produced in mammalian cells such as CHO, NSO, HEK293 or COS cells, in bacterial cells such as E. coli or Pseudomonas or in fungal or yeast cells. In embodiments the host cell is a mammalian cell, such as a CHO cell. The host cells are cultured using techniques well known in the art.

[0070] In embodiments, the disclosure relates to a vector containing and expressing in a host a PD-1 variant gene. The PD-1 gene encoding the PD-1 variant protein may originate from a mammal. In further embodiments, the expression vector may comprise a polynucleotide that encodes a PD-1 variant protein that is operatively linked to a promoter and optionally to an enhancer.

[0071] In some embodiments, the invention relates to a host cell harboring a vector comprising a polynucleotide encoding a PD-1 variant protein. In some further embodiments, the host cell harbors a vector that comprises a polynucleotide that encodes for the expression of PD-1 variant protein having the amino acid sequence of any one of SEQ ID NO.s 27-52, or SEQ ID NO. 57. Insome further embodiments the vector may comprise a polynucleotide encoding a PD-1 variant protein fused to an Fc sequence. In some further embodiments the host cell may be a mammalian host cell.

[0072] Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Protein Purification: Principals, High Resolution Methods, and Applications, 2nd Edition, Wiley-VCH Inc. (Germany, 1998).

[0073] IV. Method of Use

[0074] In another aspect the disclosure relates to methods of treating cancer comprising administering to a patient in need thereof an effective amount of a PD-1 protein variant in accordance with the above aspects and embodiments. In some embodiments the method comprises the administration in further combination (e.g., simultaneous, separate, or sequential combination) with an effective amount of one or more cancer therapies (e.g., chemotherapeutic agents, surgical resection, immune-oncology agents, or ionizing radiation, and the like).

[0075] In one aspect, the disclosure provides a method of treating cancer in a patient comprising administering a PD-1 protein variant of the disclosure to the patient. The methods can treat a variety of cancers and, in particular, those cancers that are characterized by production of one or both of PDL-1 and / or PDL-2. Some non-limiting examples of such cancers include cancers that are responsive to an immunotherapy (e.g., cancers that involve a checkpoint pathway) such as, for example, lung cancer, liver cancer, brain cancer, bladder cancer, breast cancer, cervical cancer, skin cancer, melanoma, sarcoma, head and neck cancer, esophageal cancer, stomach cancer, renal cancer, leukemia, lymphoma, myeloma, glioblastoma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, uterine (endometrial) cancer, or colorectal cancer. In some embodiments, the method comprises administering the PD-1 protein variant of the disclosure to the patient prior to the administration of one or more other therapies such as, for example, chemotherapeutic agent(s) and / or ionizing radiation.

[0076] In another aspect, the disclosure provides a method for treating an immunocompromised patient comprising administering a PD-1 protein variant of the disclosure to the patient. In embodiments, the method comprises an immunotherapy.

[0077] As used herein "treat", "treating", or "treatment" refers to administering a PD-1 protein variant, or compositions thereof, as described herein to a subject in order to eliminate or reduce the clinical signs of a condition, disease, or disorder; arrest, inhibit, or slow the progression of acondition, disease, or disorder in a subject; and / or decrease the number, frequency, or severity of the clinical symptoms and / or recurrence of a condition, disease, or disorder in a subject who currently has or who previously had a condition, disease, or disorder (e.g., a cancer and / or a recurrence of cancer). In particular, the term "treatment of a disease" (e.g., disease associated with cell proliferation, cancer, etc.) includes stopping, ameliorating, reducing, shortening the duration, slowing down, or inhibiting progression or worsening, or delaying the onset of a disease or the symptoms thereof in a subject who has the disease.

[0078] In some embodiments, the disclosure provides methods for preventing disease (i.e., preventing a condition, disease, or disorder, preventing the onset of a condition, disease, or disorder or the clinical symptoms of disease) in a subject. In some further embodiments of the methods relating to preventing disease, the subject may be at risk of one or more PD-1, PDL-1, and / or PDL-2 associated conditions, diseases, or disorders. By "being at risk" or having an “increased risk” a subject is identified as having a higher than normal chance of developing a condition, disease, or disorder and / or clinical symptoms associated therewith, compared to the general population. In some embodiments, a subject who has had, or who currently has, a condition, disease, or disorder, or who is of a certain age is a subject who has an increased risk for developing a condition, disease, or disorder. In some aspects, a subject exhibiting increased PDL- 1 and / or PDL-2 expression or activity can also be considered as having an increased risk for developing a condition, disease, or disorder. In some embodiments, the subject may also be immunocompromised. As used herein “immunocompromised” refers to a subject having a weakened immune system or a reduced ability to fight infections or other diseases, due to a genetic disorder or disease, an infection, an environmental disorder or disease, or other environmental factors.

[0079] The term "immunotherapy" relates to a treatment involving a specific immune reaction. In the context of the disclosure, terms such as "protect", "prevent", "prophylactic", "preventive", or "protective" relate to the prevention of the occurrence and / or the propagation of a condition, disease, or disorder and clinical symptoms in an individual and, in some embodiments, to minimizing the chance that a subject will develop further clinical symptoms or disease progression. For example, a person at risk for a condition, disease, or disorder associated with one or more of PD-1, PDL-1, and / or PDL-2, as described herein, would be a candidate for preventative therapy to prevent such a condition, disease, or disorder or associated clinical symptom(s).

[0080] A prophylactic administration of an immunotherapy, for example, a prophylactic administration of an antibody or a composition comprising the antibody or fragment thereof as disclosed herein, can in certain embodiments protect the recipient from a condition, disease, or disorder associated with one or more of PD-1, PDL-1, and / or PDL-2, or reduce the risk of the recipient from developing such a condition, disease, or disorder. In some alternative embodiments, a prophylactic administration may reduce chances of developing increased severity of a condition, disease, or disorder associated with one or more of PD-1, PDL-1, and / or PDL-2 in the recipient.

[0081] A therapeutic administration of an immunotherapy, for example, a therapeutic administration of a PD-1 variant protein or a composition thereof as disclosed herein, may lead to the inhibition of the progress of the condition, disease, or disorder and / or clinical symptoms associated with the condition, disease, or disorder. Such methods may include embodiments that comprise the reduction or inhibition of the expression of one or more of PD- 1 , PDL- 1 , and / or PDL- 2, the activity of one or more of PD-1, PDL-1, and / or PDL-2, and / or the total amount of cells associated with one or more of PD-1, PDL-1, and / or PDL-2 expression which preferably leads to elimination of the condition, disease, or disorder and the associated clinical symptoms.

[0082] In certain aspects, the disclosure provides methods of treatment comprising a PD-1 variant protein administered in combination or in conjunction with additional cancer therapies. In particular embodiments the methods comprise a combination of a PD-1 variant protein and a therapy used to treat various stages of a cancer.

[0083] The term "administering" refers to an act to transfer a pharmaceutical composition of the present invention into the body of a mammal, preferably a human, in need thereof.

[0084] Administration may be via any route known to be effective by the physician of ordinary skill. Parenteral administration is one form of administration commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump. The parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Administration at the time of surgery or through the use of x-ray imaging (fluoroscopy) are additional forms.

[0085] Pharmaceutically acceptable excipient refers to a pharmaceutically acceptable formulation carrier, solution, or additive to enhance the formulation characteristics. Such excipients must becompatible with the other ingredients of the formulation and not deleterious to the recipient thereof and arc well known to the skilled artisan, sec, e.g., Remingtons Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, 1995.

[0086] A stable formulation is one in which the protein remains soluble for an extended period of time under the conditions of storage.

[0087] Potency or specific activity is a measurement of the relative activity of variants of human PD-1 protein including those in accordance with the example embodiments of the disclosure, and may be measured in any of the various assays described herein (e.g., in the Examples) or by any other method known in the art. Generally, the relative activity is compared to a control such as, for example, a wild type PD- 1 sequence to yield a relative potency for the human PD-1 protein variant.

[0088] The terms "subject", "patient", or "individual", are used interchangeably herein, and all of them refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals (i.e., sheep, etc.), mammalian sport animals (i.e., horses), and mammalian pets (i.e., dogs or cats). In some embodiments, the term refers to humans. In a certain embodiment, the subject, for example a human, is further characterized as having a disease, disorder, or condition that would benefit from treatment with a PD-1 variant protein in accordance with the disclosure.

[0089] The phrase "therapeutically effective amount" refers to the amount of an active agent necessary to impart a therapeutic benefit to a patient.

[0090] A therapeutically effective amount is the amount of an active agent necessary to impart a therapeutic benefit to a patient. For example, a therapeutically effective amount administered to a human patient in need of treatment for cancer or an immunodeficiency comprises an amount which induces, ameliorates, or otherwise causes an improvement in the clinical and / or pathological symptoms, disease progression, or physiological conditions associated with the disease or condition. Furthermore, a therapeutically effective amount of a human PD- 1 protein variant of the disclosure comprises an amount administered to a subject (e.g., human patient) in need of treatment for cancer is an amount which in mammals, preferably, humans, reduces the number of cancer cells; reduces the tumor size; inhibits (i.e., slow to some extent or stop) cancer cell infiltration into peripheral tissues organs; inhibit (i.e., slow to some extent or stop) tumor metastasis; inhibits, to some extent, tumor growth; and / or relieves to some extent one or more of the symptoms associated with the cancer. An effective amount of a human PD-1 protein variant of the invention may be administered in a single dose or in multiple doses. Furthermore, an effective amount of a humanPD- 1 protein variant of the invention may be administered in multiple doses of amounts that would be less than an effective amount if not administered more than once.

[0091] As is known in the medical ails, dosages for any one subject depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, gender, time and route of administration, general health, and other drugs being administered concurrently. Dose may further vary depending on the type and severity of the disease. A typical dose of a PD-1 protein variant described herein can be, for example, in the range of about 10 mg to about 1000 mg; preferably, about 50 mg to about 500 mg; more preferably, about 200 mg to about 500 mg; even more preferably, about 200 mg to about 400 mg, even more preferably, about 200 mg to about 300 mg; even more preferably, about 225 mg to about 275 mg; even more preferably, about 250 mg to about 275 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. A daily parenteral dosage regimen can be from about 250 pg / kg to about 10 mg / kg. Progress may be monitored by periodic assessment, and the dose adjusted accordingly.

[0092] In some embodiments, a single dose of a PD-1 protein variant as described herein may be administered intravenously for treating a cancer in an adult patient. A typical single dose for intravenous administration can be, for example, in the range of about 10 mg to about 1000 mg; preferably, about 10 mg to about 500 mg; more preferably, about 10 mg to about 500 mg; more preferably, about 10 mg to about 400 mg; more preferably, about 10 mg to about 350 mg; more preferably, about 10 mg to about 300 mg; even more preferably, about 10 mg to about 275 mg; even more preferably, about 10 mg to about 250 mg; even more preferably, about 10 mg to about 200 mg; even more preferably, about 10 mg to about 175 mg; even more preferably, about 10 mg to about 150 mg; or most preferably, about 10 mg to about 125 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Alternatively, a typical single dose for intravenous administration of a PD-1 valiant protein in accordance with the disclosure can be, for example, from about 0.2 mg / kg to about 15 mg / kg body weight; more preferably, about 0.2 mg / kg to about 10 mg / kg; even more preferably, about 0.2 mg / kg to about 7.5 mg / kg; even more preferably, about 0.2 mg / kg to about 5 mg / kg; even more preferably, about 0.2 mg / kg to about 4 mg / kg; even more preferably, about 0.2 mg / kg to about 3 mg / kg; even more preferably about 0.2 mg / kg to about 2.5 mg / kg; or most preferably, about 0.2 mg / kg to about 2 mg / kg. Such doses can be administered intravenously once every week, onceevery two weeks, once every three weeks, or once every month, for example. Progress may be monitored by periodic assessment, and the dose adjusted accordingly.

[0093] These suggested amounts of a PD-1 variant protein are subject to therapeutic discretion. One factor in selecting an appropriate dose and scheduling is the result obtained. Other factors for consideration in this context include the particular disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the protein, the particular type of form of the protein (e.g., PD-1 protein variant or a longer PD-1 protein variant, or fusion thereof) the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0094] The presently described technology and its advantages will be better understood by reference to the following examples. These examples arc provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.EXAMPLESExample 1: Generating high affinity PD-1 variant sequences

[0095] Random mutations within the PD-1 loop 1 region (amino acid residues 70-74) were introduced by site-directed mutagenesis PCR using NNS oligonucleotides. Two additional mutations were introduced in the PD-1 coding region, namely a C93S mutation (to provide protein stability) and a A132L mutation (to improve binding affinity). PCR products were ligated to phage p3 coat protein coding sequence and the resulting phagemids were transformed into bacteria TG- 1 (Lucigen, cat# 60502). The mutational phage library was created by introduction of M13KO7 helper phage (ThermoFisher, cat# 18311019) to package and release phage particles carrying PD- 1 mutants on the surface. The phage particles were precipitated from the culture supernatant using PEG and sodium chloride (4% and 0.5M final concentration, respectively).

[0096] The PD-1 mutational library was screened for high affinity binders using M-280 streptavidin magnetic Dynabeads (ThermoFisher, 11205D) and biotin-PDL-1 protein (SinoBiological, cat# 10084-H08H-B) as bait for 3 rounds, at biotin-PDL-1 concentrations of 200nM, 40 nM, and 20 nM, respectively. After the final round, phage ELISA was performed for positive clone selection. A total of 24 clones were picked and sequenced based on ELISA results. All leads from the mutational library contained the C93S and A132L mutations (see Tables 1 and 2).Table 1. Nucleic acid sequences coding for PD-1 mutants.Table 2. Amino acid sequences of PD-1 mutantsExample 2: Binding Analysis of Fusion Proteins

[0097] Example 2A: Generating Fusion Proteins.

[0098] The 24 variant sequences along with the C93S and C93S / A132L mutants were converted to IgGl Fc fusion proteins in order to characterize binding properties. A schematic of the fusion protein structure is depicted in FIG. 2. The IgGl Fc DNA and amino acid sequences used in the fusions are shown in Table 3. In brief overview, PD-1 mutant coding sequences were each cloned into a mammalian expression vector pJL263 upstream of the Fc coding sequence. The resulting vectors were transfected into HEK293 cells using polyethylenimine (PEI), and the cultures were maintained for six days for protein expression. Each culture was harvested by centrifugation and the supernatant was loaded onto a HiScreen MabSelect SuRe column(Cytiva). The column was washed with PBS and protein was eluted with 20 mM sodium citrate, 25 mM sodium phosphate at pH 3.2. The eluted protein was buffer-exchanged to PBS (pH 6.5) and sterile filtered for 2-8°C storage.

[0099] Example 2B: PDL-1 and PDL-2 binding

[0100] ELISA assays were performed to evaluate the binding of the PD-l / Fc fusion proteins to PDL-1 and PDL-2. Briefly, 96-well plates were coated with 50 pL of 1 pg / mL PDL-1 or PDL-2 in each well. PD-l / Fc fusion sample proteins were added at three different concentrations (0.17 pg / mL, 0.085 pg / mL and 0.0425 pg / mL). Results were read on a Molecular Device SpectraMax M3 plate reader following the standard ELISA protocol.

[0101] Results indicated that certain fusions (e.g., PDM3 / Fc, PDM8 / Fc, PDM9 / Fc, and PDM15 / Fc) had improved binding to PDL-1 as compared to C93S / A132L / Fc. In line with expectation, the C93S / Fc fusion exhibited poor binding activity to PDL-1 in the ELISA assay (FIG. 3). A number of the fusion constructs (e.g., PDM3 / Fc, PDM5 / Fc, PDM6 / Fc, PDM7 / Fc, PDM8 / Fc, PDM9 / Fc, PDMH / Fc, PDM12 / Fc, PDM13 / Fc, PDM14 / Fc, PDM15 / Fc, PDM16 / Fc, PDM18 / Fc, PDM21 / Fc, PDM22 / Fc, and PDM24 / Fc had improved binding to PDL-2 relative to the C93S / Fc and C93S / AL32L / Fc fusions. (FIG. 4).Table 3. IgGl Fc sequences

[0102] Example 2C: PDL-1 binding EC50

[0103] As an illustrative embodiment of the class of PD-1 variants that fall within the scope of the disclosure, the PDM15 / Fc and C93S / A132L / Fc sequences were characterized to determine PDL-1 EC50 values (by ELISA). Briefly, 96-well plates were coated with 50 pL of 1 pg / mL PDL-1 in each well. PDM15 / Fc or C93S / A132L / Fc protein was added at the following 12 concentrations: 1.00E-01, 1.25E-02, 1.56E-03, 1.95E-04, 2.44E-05, 3.05E-06, 3.81E-07, 4.77E- 08, 5.96E-09, 7.45E-10, 9.31E-11, and 1.16E-11 pg / mL. Results were read on a Molecular Device SpectraMax M3 plate reader following the standard ELISA protocol. The EC50s for PDM15 / Fc and C93S / A132L / Fc were determined to be 70 nM and 121 nM respectively (FIG. 5).

[0104] Example 2D: Cell surface binding

[0105] A representative variant fusion sequence, PDM15 / Fc, was selected to test cell surface binding to NC1-H1975 cells relative to Durvalumab (anti-PDL-1 antibody) and the C93S / A132L / Fc mutant fusion. The proteins were incubated at various concentrations (5.00E+00, 2.50E+00, 5.00E-01, 1.00E-01, 2.00E-02, 4.00E-03, 8.00E-04, 1.60E-04, 3.20E-05, 6.40E-06, 1.28E-06, 2.56E-07 pg / mL) with NCLH1975 cells. After washing and probing with a fluorescence-labeled secondary antibody, the cell surface binding was analyzed on ThermoFisher Attune flow cytometer. The PDM15 / Fc variant was determined to have a binding EC50 of 0.0253 nM, comparable to Durvalumab (0.0288 nM) and slightly better than C93S / A132L / Fc (0.0302 nM).

[0106] Example 2E: PD-l / PDL-1 blockade assay

[0107] A PD-l / PDL-1 blockade bioassay was performed using a commercial kit from Promega (Cat# J 1250) following the manufacturer's protocol. Anti-PD-1 antibody (Nivolumab) and anti- PDL-1 antibody (Durvalumab) served as controls. Representative variant fusion sequence PDM15 / Fc exhibited a better EC50 than C93S / A132L / Fc, while C93S / Fc showed poor efficacy in this assay (Table 4 and FIG. 7).Table 4. EC50s in PD-l / PDL-1 blockade bioassayExample 3: In vivo tumor suppression

[0108] The tumor suppression effect of representative variant fusion sequence PDM15 / Fc was evaluated using a C57BL / 6 xenograft mouse model with MC38 cells. PDM15 / Fc was dosed at two levels, 3 mg / kg and 15 mg / kg, along with vehicle (PBS) and a positive control (Pembrolizumab, 3 mg / kg), through IV injection, 7x Q.O.D. Body weight and tumor volume were measured on day 0, 2, 5, 7, 9, 12, 14, 16, and 21. Results showed that PDM15 / Fc had comparable tumor suppression effect to the positive control (Pembrolizumab, see FIGs. 8A and 8B).

[0109] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0110] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

ClaimsWe claim:

1. A protein comprising the amino acid sequence: NPPTFSPALL VVTEGDNATF TCSFSNTSES FVLNWYR XaaiXaa2Xaa3Xaa4Xaa5QTDKLAAFPE DRSQPGQD-Xaa6- RFRVTQLPNG RDFHMSVVRA RRNDSGTYLC GAISLAPK-Xaa7-QIKESLRAEL RVTERRAE (SEQ ID NO: 57); whereinXaai comprises M, V, I, L, or E;Xaa2comprises S, L, H, Y, L, G, D, T, or H;Xaa3comprises P, E, S, T, D, K, Q, R, V, or A;Xaa4 comprises S, T, G, N, D, F, R, H, R, or E;Xaa7comprises N, or G;Xaae comprises an amino acid residue that is not cysteine; and Xaa7comprises a non-polar amino acid residue; or a sequence having about 80% sequence identity to SEQ ID NO.

572. The protein of claim 1, wherein Xaa6 comprises a nucleophilic amino acid that is not cysteine.

3. The protein of claim 1 or claim 2, wherein Xaae comprises threonine, alanine, glycine, or serine.

4. The protein of any one of claims 1 to 3, wherein Xaae comprises serine.

5. The protein of any one of claims 1 to 4, wherein Xaa7comprises glycine, alanine, valine, leucine, isoleucine or methionine.

6. The protein of any one of claims 1 to 5, wherein Xaa7comprises valine, leucine, or isoleucine.

7. The protein of any one of claims 1 to 6, wherein Xaa7comprises leucine.

8. The protein of any one of claims 1 to 7, wherein the protein comprises an amino acid sequence selected from the group consisting SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ IDNO.30, SEQ ID N0.31 , SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID N0.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO .47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, and SEQ ID NO.52.

9. The protein according to claim 8, wherein the C-terminus of the protein is covalently fused to a human Fc amino acid sequence.

10. A pharmaceutical composition comprising the protein according to any one of claims 1 to 9, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

11. A method of treating cancer comprising administering to a human patient in need thereof an effective amount of the protein of any one of claims 1 to 9, or the pharmaceutical composition of claim 10.

12. A method of treating an immunodeficiency comprising administering to a human patient in need thereof an effective amount of the protein of any one of claims 1 to 9, or the pharmaceutical composition of claim 10.

13. The method according to either one of claims 11 or 12 further comprising administering to the patient in need thereof an effective amount of a second therapy.

14. A PD-1 protein variant sequence that comprises improved binding affinity for a PD-1 ligand compared to a wild type PD-1 protein, wherein the PD-1 protein variant sequence comprises the structure:[PD-1 N-terminal sequence]-XaaiXaa2Xaa3 Xaa4Xaas-[PD-l C-terminal sequence], whereinXaaiXaaoXaaa Xaa4Xaas comprise five variable amino acid residues that comprise at least one amino acid residue that is non-native to a PD-1 loop-1 sequence; the PD-1 N-terminal sequence and the PD-1 C-terminal sequence comprise at least 80% homology to a wild type PD-1 sequence.

15. The PD-1 protein variant sequence of claim 14, whereinXaai comprises M, V, I, L, or E;Xaa? comprises S, L, H, Y, L, G, D, T, or H;Xaaa comprises P, E, S, T, D, K, Q, R, V, or A;Xaa4 comprises S, T, G, N, D, F, R, H, R, or E;Xaas comprises N, or G;Xaa6 comprises an amino acid residue that is not cysteine; andXaa? comprises a non-polar amino acid residue.

16. The PD-1 protein variant sequence of claim 14 or claim 15, wherein the N-terminal sequence comprises an amino acid sequence that is N-terminal to the loop 1 region of a native PD-1 sequence; and the C-terminal sequence comprises an amino acid sequence that is C-terminal to the loop 1 region of a native PD- 1 sequence; or one or both of the N-terminal sequence and / or the C-terminal sequence comprises an amino acid sequence that is at least 80% homologous to a native N-terminal sequence and / or a C-terminal PD-1 sequence.

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