Immune Cell Binding Polypeptides

Abstract

The present disclosure provides an immune cell binding polypeptide (ICBP) comprising a binding moiety capable of binding to an ILT2 and / or ILT4 polypeptide, at least one immunosuppressive polypeptide, and a scaffold polypeptide. The ICBP is useful for inducing immune tolerance. The present disclosure provides a method of inducing immune tolerance. TIFF2025511703000004.tif25169

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 328,563, filed April 7, 2022, which is incorporated by reference in its entirety herein.

[0002] Incorporation by Reference of Electronically Submitted Materials The Sequence Listing is provided herein as Sequence Listing XML "CUEB-147WO_SEQ_LIST" having a size of 188,080 bytes, created on April 5, 2023. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] preface Inflammatory diseases and disorders are conditions in which abnormal or otherwise unregulated inflammatory responses contribute to the pathogenesis or severity of disease.Examples of such diseases include autoimmune diseases.Inflammatory diseases and disorders can be characterized by the infiltration of mononuclear cells at the site of inflammation.Examples of mononuclear cells include lymphocytes (e.g., T lymphocytes) and cells of the mononuclear phagocyte system, including monocytes, macrophages, dendritic cells, and microglial cells.Therefore, pharmaceutical compositions that can induce immune tolerance can be useful in the treatment of autoimmune diseases and disorders, or other conditions involving adverse immune responses, such as metabolic disorders. Summary of the Invention

[0004] overview The present disclosure provides immune cell binding polypeptides (ICBPs) comprising a binding moiety capable of binding to an ILT2 and / or ILT4 receptor polypeptide on a cell, at least one immunosuppressant polypeptide, and a scaffold polypeptide. The ICBPs are useful for inducing immune tolerance. The present disclosure provides methods of inducing immune tolerance. [Brief description of the drawings]

[0005] [Figure 1] 1A-1B provide the amino acid sequence of a wild-type human β2M polypeptide (FIG. 1A; SEQ ID NO:1), and a β2M polypeptide having an R12C substitution (FIG. 1B; SEQ ID NO:2). [Figure 2A] 2A-2M provide the amino acid sequences of immunoglobulin Fc polypeptides (SEQ ID NOs: 3-15, respectively). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 2G] See legend to Figure 2A. [Figure 2H] See legend to Figure 2A. [Figure 2I] See legend to Figure 2A. [Figure 2J] See legend to Figure 2A. [Figure 2K] See legend to Figure 2A. [Figure 2L] See legend to Figure 2A. [Figure 2M] See legend to Figure 2A. [Diagram 3] The HLA-G consensus sequence (SEQ ID NO: 16) is provided. [Figure 4A] 4A-4F provide the amino acid sequences of the HLA-G heavy chain (SEQ ID NOs: 17-22, respectively). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 5A]5A-5B provide the amino acid sequences of the single-chain ICBP polypeptides (SEQ ID NOs:23 and 29, respectively). [Figure 5B] See legend to Figure 5A. [Figure 6A] Figures 6A-6C provide the amino acid sequences of the polypeptides of (i) construct 4601+4602 (Figure 6A) (SEQ ID NO: 30), (ii) construct 4607+4602 (Figure 6B) (SEQ ID NO: 33), and (iii) construct 4607-2+4602 (Figure 6C) (SEQ ID NO: 35). [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7A] FIG. 7 shows the binding of constructs 4601+4602 and 4607+4602 to ILT2, ILT4, and PD1. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8A] 8A-8F provide the amino acid sequences of the ILT2 polypeptide (SEQ ID NOs:36-41, respectively). [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 9A] Figures 9A-9E provide the amino acid sequence of the ILT4 polypeptide (SEQ ID NOs: 42-46, respectively). [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 9E] See legend to Figure 9A. [Figure 10A]Figures 10A-10F provide the amino acid sequences of the CD155 isoform (Figures 10A and 10B; SEQ ID NOs: 47 and 48, respectively), the CD112 isoform (Figures 10C and 10D; SEQ ID NOs: 49 and 50, respectively), the amino acid sequence of FGL1 (Figure 10E; SEQ ID NO: 51), and the amino acid sequence of the extracellular portion of LSECtin (Figure 10F; SEQ ID NO: 52). [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 10F] See legend to Figure 10A. [Figure 11A] Figures 11A-11D provide the amino acid sequences of wild-type and variant PD-L1 polypeptides (SEQ ID NOs: 34 and 53-55, respectively). [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 12A] 12A-12G provide the amino acid sequences of TGF-β polypeptides (SEQ ID NOs:56-62, respectively). [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 12E] See legend to Figure 12A. [Figure 12F] See legend to Figure 12A. [Figure 12G] See legend to Figure 12A. [Figure 13] FIG. 13A and FIG. 13B provide the amino acid sequences of IL-12α and IL-27β polypeptides (SEQ ID NOs:63 and 64, respectively). [Figure 14A] 14A-C provide the amino acid sequence of KIR2DL4 (SEQ ID NOs:65-67, respectively). [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] definition The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Furthermore, as used herein, "polypeptide" refers to a protein containing modifications such as deletions, additions, and substitutions (generally of a conservative nature as known to those of skill in the art) to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as site-directed mutagenesis, or may be accidental, such as mutations of the host producing the protein, or errors due to polymerase chain reaction (PCR) amplification or other recombinant DNA methods. Reference herein to a particular residue or residue number in a known polypeptide will be understood to refer to the amino acid at that position in the wild-type polypeptide. To the extent that the sequence of the wild-type polypeptide is modified by either the addition or deletion of one or more amino acids, one of skill in the art will understand that reference to a particular residue or residue number will be correspondingly altered to refer to the same particular amino acid in the modified polypeptide, but at the altered position number. For example, if an HLA-G polypeptide is modified by adding an amino acid to the N-terminus, reference to position 84 or a specific residue at position 84 will be understood to refer to the amino acid at position 85 on the modified polypeptide. Similarly, reference herein to a specific amino acid substitution at a specific position, for example Y84, will be understood to refer to the substitution of that amino acid for the amino acid at position 84 of the wild-type polypeptide. Thus, a Y84C substitution will be understood to be the substitution of a Cys residue for the Tyr residue present in the wild-type sequence. For example, if a wild-type polypeptide is modified to change the amino acid at position 84 from its wild-type amino acid to an alternative amino acid, the substitution of the amino acid at position 84 will be understood to refer to the substitution of the alternative amino acid.Where such polypeptides are also modified by the addition or deletion of one or more amino acids, reference to a substitution will be understood to refer to the substitution of an alternative amino acid at the altered position number. Reference to a "non-naturally occurring Cys residue" in a polypeptide, e.g., an HLA-G polypeptide, means that the polypeptide contains a Cys residue at a position where there is no Cys in the corresponding wild-type polypeptide. This can be accomplished through routine protein engineering, where a cysteine ​​is replaced with an amino acid that occurs in the wild-type sequence.

[0007] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases, or other natural, chemically or biochemically modified non-natural, or derivatized nucleotide bases.

[0008] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, which means that when the two sequences are compared and aligned, that percentage of bases or amino acids are the same and in the same relative positions. Sequence identity can be determined with BLAST, which is available on the World Wide Web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / . Unless otherwise specified, "sequence identity" as referred to herein is determined by BLAST (Basic Local Alignment Search Tool) with default settings selected.

[0009] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues with similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine, the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine, the group of amino acids with amide-containing side chains consists of asparagine and glutamine, the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan, the group of amino acids with basic side chains consists of lysine, arginine, and histidine, the group of amino acids with acidic side chains consists of glutamic acid and aspartate, and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0010] "T cells" are helper T cells (CD4 + cells), cytotoxic T cells (CD8 + These include all types of immune cells that express CD3, including T-cells, regulatory T cells (Tregs), and natural killer (NK)-T cells.

[0011] As used herein, the term "immunosuppressive polypeptide" refers to a polypeptide that specifically binds to a cognate polypeptide (e.g., a receptor or co-receptor) on an immune cell (e.g., a T cell, a dendritic cell, etc.), thereby providing a signal that inhibits an immune cell response, including, but not limited to, proliferation, activation, secretion of inflammatory cytokines, etc. As discussed herein, immunosuppressive polypeptides can include, but are not limited to, wild-type or mutant forms of wild-type polypeptides such as cytokines (e.g., IL-10, IL-35, IL-2), PD-L1, Fas-L, LAG-3 ligand, TIGIT ligand (e.g., CD155), and TGF-β.

[0012] As used herein, the term "in vivo" refers to any process or procedure that occurs inside the body.

[0013] As used herein, the term "in vitro" refers to any process or procedure that occurs outside the body.

[0014] As used herein, "heterologous" means a nucleotide or polypeptide that is not found in the native nucleic acid or protein, respectively.

[0015] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and / or ligation steps that result in a construct having structural coding or non-coding sequences distinguishable from endogenous nucleic acids present in natural systems. A DNA sequence encoding a polypeptide may be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide a synthetic nucleic acid expressible from a recombinant transcription unit contained within a cellular or cell-free transcription and translation system.

[0016] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule that includes a vector and at least one insert. Recombinant expression vectors are typically constructed for the purpose of expressing and / or propagating an insert or for the construction of other recombinant nucleotide sequences. The insert may or may not be operably linked to a promoter sequence and may or may not be operably linked to a DNA regulatory sequence.

[0017] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen), and is also referred to as the dissociation constant (K D As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution.

[0018] As used herein (e.g., with respect to binding of a first polypeptide to a second polypeptide (e.g., where the first polypeptide is an ICBP of the present disclosure and the second polypeptide is an ILT2 polypeptide)), the term "binding" refers to a non-covalent interaction between two molecules. Non-covalent binding means a direct association between two molecules, e.g., by electrostatic, hydrophobic, ionic, and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. "Affinity" refers to the strength of the non-covalent bond, and increased binding affinity is indicated by a lower K D "Specific binding" generally refers to the binding of a ligand to a moiety that is its designated binding site or receptor. "Non-specific binding" generally refers to the binding of a ligand to a moiety other than its designated binding site or receptor. As used herein, "covalently binding" or "covalent bond" means the formation of one or more covalent chemical bonds between two distinct molecules.

[0019] As used herein, the terms "treatment", "treating" and the like generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing the onset of the disease or condition in a subject who may or may not be susceptible to the disease or condition, but has not yet been diagnosed as having it, (b) suppressing the disease or one or more symptoms associated with the disease, e.g., arresting its onset, and / or (c) relieving the disease, i.e., regressing the disease. Therapeutic agents may be administered before, during, and / or after the onset of the disease or injury. Of particular interest are treatments of ongoing diseases that stabilize or reduce the undesirable clinical symptoms of the patient. Such treatments are desirably performed before complete loss of function of the affected tissue. The therapy will desirably be administered during, and in some cases after, the symptomatic stage of the disease.

[0020] The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), etc. Unless otherwise indicated, the terms "individual," "subject," "host," and "patient" refer to humans.

[0021] Unless otherwise indicated, the term "substantially" is intended to encompass both "entirely" and "largely, but not entirely." For example, an Ig Fc that "substantially does not induce ADCC" means an Ig Fc that does not induce ADCC at all or that does not induce ADCC for the most part.

[0022] As used herein, the term "about" used in connection with an amount indicates that the amount may vary by 10% of the amount stated. For example, "about 100" means an amount of 90 to 110. When about is used in the context of a range, "about" used in connection with a lower amount in a range means that the lower amount includes an amount 10% lower than the lower amount in the range, and "about" used in connection with a higher amount in a range means that the higher amount includes an amount 10% higher than the upper amount in the range. For example, about 100 to about 1000 means that the range extends from 90 to 1100.

[0023] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope will be limited only by the appended claims.

[0024] Where a range of numerical values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may be independently included in the narrower range, and are also encompassed within the disclosure, subject to any specifically excluded value in the stated range. Where an explicit range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the disclosure.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although any method and material similar or equivalent to the methods and materials described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described below.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with the content in which they are cited.

[0026] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "ICBP" includes a plurality of such ICBPs, a reference to "immunosuppressant polypeptide" includes a reference to one or more immunosuppressant polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0027] It is to be understood that certain features of the present disclosure that are described in the context of individual embodiments for clarity may also be provided in combination within a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the present disclosure are expressly embraced by the present disclosure and are disclosed herein as if all combinations were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present disclosure and are disclosed herein as if all such subcombinations were individually and expressly disclosed herein.

[0028] Publications discussed herein are provided solely for their disclosure. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publications. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0029] Detailed Description The present disclosure provides a therapeutic polypeptide (ICBP) comprising (a) a binding moiety comprising a human leukocyte antigen-G (HLA-G) molecule and a stabilizing peptide, (b) optionally at least one immunosuppressive polypeptide, and (c) a scaffold polypeptide. The HLA-G molecule comprises an HLA-G heavy chain polypeptide and a beta-2 microglobulin (β2M) polypeptide. The binding moiety of the ICBP binds to immunoglobulin-like transcript 2 (ILT2) and / or immunoglobulin-like transcript 4 (ILT4) polypeptides present on the surface of immune cells (e.g., T cells, B cells, natural killer (NK) cells, monocytes, macrophages, and dendritic cells), while the immunosuppressive polypeptide binds to a cognate polypeptide (e.g., a receptor or co-receptor) on the surface of a T cell. Thus, the ICBP of the present disclosure binds to cells expressing both ILT2 and a cognate polypeptide that binds to the immunosuppressive polypeptide, or to cells expressing both ILT4 and a cognate polypeptide that binds to the immunosuppressive polypeptide. These cells are generally inflammatory immune cells, such as T cells, that exhibit inflammatory activity. In some examples, when ICBP is administered to an individual in need thereof, it suppresses the activity of inflammatory immune cells in the individual. In some examples, when ICBP is administered to an individual in need thereof, it increases the number of regulatory T cells (Tregs) in the individual. In some examples, when ICBP is administered to an individual in need thereof, it increases the number of tolerogenic dendritic cells in the individual.

[0030] As mentioned above, ICBPs include stabilizing peptides. The stabilizing peptides bind to and stabilize HLA-G molecules. However, ICBPs function in an antigen-nonspecific manner to suppress the activity of inflammatory immune cells. As discussed below, the stabilizing peptides of the binding moiety do not contribute to the binding between the binding moiety and the ILT2 or ILT4 receptors. Because the ICBPs of the present disclosure function in an antigen-nonspecific manner, such ICBPs are useful for treating, for example, autoimmune diseases (including autoimmune diseases that may involve immune cells that are reactive against multiple self-epitopes) or other conditions involving undesirable immune responses (including metabolic disorders).

[0031] In some instances, the ICBP does not include at least one immunosuppressive polypeptide. That is, the ICBP may be "MOD-free," i.e., it may include (a) a binding moiety that includes a human leukocyte antigen-G (HLA-G) molecule and a stabilizing peptide, and (b) a scaffold polypeptide. In such cases, immune tolerance is provided substantially through the interaction of the binding moiety with its receptor on T cells (e.g., ILT2 and ILT4).

[0032] joining part As mentioned above, the binding moieties present in the ICBPs of the present disclosure include an HLA-G molecule and a stabilizing peptide. The HLA-G molecule includes an HLA-G heavy chain polypeptide and a β2M polypeptide.

[0033] HLA-G heavy chain polypeptide Figure 3 shows the consensus sequence for an HLA-G heavy chain polypeptide, with variable amino acid positions shown as consecutively numbered "X" residues, with double underlined positions at amino acid (aa) 84, 139, and 236. An HLA-G heavy chain polypeptide can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to an amino acid sequence shown in any one of Figures 4A-4F.

[0034] In some examples, the HLA-G heavy chain polypeptide present in ICBP comprises one or more amino acid substitutions relative to a reference HLA-G polypeptide (the reference HLA-G polypeptide can be a wild-type HLA-G polypeptide). In some examples, the HLA-G heavy chain polypeptide comprises one or more mutations that can prevent homodimerization, such as a C42S substitution (see, e.g., Boyson et al. (2002) Proc. Natl. Acad. Sci. USA 99:16180). In some examples, the amino acid substitution replaces an amino acid with a cysteine ​​(Cys) residue. Such a cysteine ​​residue can form a disulfide bond with a natural or non-natural cysteine ​​residue present in the HLA-G heavy chain of ICBP. As used herein, reference to a "non-natural Cys residue" in an HLA-G polypeptide means that the polypeptide comprises a Cys residue at a position where there is no Cys in the corresponding wild-type polypeptide. This can be achieved through routine protein engineering, where a cysteine ​​is replaced with an amino acid that occurs in the wild-type sequence.

[0035] The amino acid sequences of suitable HLA-G heavy chain polypeptides are provided in Figures 4A-4D, where Figure 4A provides the amino acid sequence of HLA-G*01:01 (wild type), Figure 4B provides the amino acid sequence of HLA-G*01:01 with Y84C and A236C substitutions, Figure 4C provides the amino acid sequence of HLA-G*01:01 with C42S, Y84C and A236C substitutions, Figure 4D provides the amino acid sequence of HLA-G*01:04 (wild type), Figure 4E provides the amino acid sequence of HLA-G*01:04 with Y84C and A236C substitutions, and Figure 4F provides the amino acid sequence of HLA-G*01:04 with C42S, Y84C and A236C substitutions. As discussed below, providing a Cys at residue 84 of the HLA-G heavy chain polypeptide may allow for the formation of a disulfide bond with a Cys in the linker that connects the stabilizing peptide to β2M. Providing a Cys at residue 236 of the HLA-G heavy chain polypeptide may allow for the formation of a disulfide bond that can link the HLA-G heavy chain to a Cys residue in the β2M polypeptide (e.g., at amino acid residue 12 of the β2M polypeptide). Such a disulfide bond may be an interchain disulfide bond (e.g., when ICBP is a heterodimeric polypeptide) or an intrachain disulfide bond (e.g., when ICBP is a single-chain polypeptide). In some examples, thus, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 4A, 4B, 4C, 4D, 4E, or 4F. In some examples, the HLA-G heavy chain polypeptide comprises a C42S substitution in addition to the Y84C and A236C substitutions. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 95%, or at least 98%, amino acid sequence identity to the amino acid sequence shown in Figure 4A. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 95%, or at least 98%, amino acid sequence identity to the amino acid sequence shown in Figure 4B.In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 95% or at least 98% amino acid sequence identity to the amino acid sequence shown in FIG. 4C. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 95% or at least 98% amino acid sequence identity to the amino acid sequence shown in FIG. 4D. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 95% or at least 98% amino acid sequence identity to the amino acid sequence shown in FIG. 4E. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 95% or at least 98% amino acid sequence identity to the amino acid sequence shown in FIG. 4F.

[0036] In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 99% or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4A. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 99% or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4B. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 99% or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4C. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 99% or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4D. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 99% or at least 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4E. In some examples, the HLA-G heavy chain polypeptide of ICBP may comprise an amino acid sequence having at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4F. The HLA-G heavy chain polypeptide present in ICBP typically does not comprise a signal peptide, a transmembrane domain, or a cytoplasmic tail. The HLA-G heavy chain polypeptide present in ICBP generally comprises only three extracellular domains (α1 domain, α2 domain, and α3 domain). The length of the HLA-G polypeptide may range from about 270 amino acids to about 280 amino acids (e.g., 270 amino acids (aa), 271aa, 272aa, 273aa, 274aa, 275aa, 276aa, 277aa, 278aa, 279aa, or 280aa). In some examples, the length of the HLA-G polypeptide is 275 amino acids.

[0037] β2M Polypeptide As described above, the HLA-G molecule comprises a β2-microglobulin (β2M) polypeptide. The β2M polypeptide can be a human β2M polypeptide, a non-human primate β2M polypeptide, a mouse β2M polypeptide, etc. In some examples, the β2M polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 1A (wild-type human β2M). In some examples, the β2M polypeptide present in ICBP comprises an amino acid sequence shown in FIG. 1A (wild-type human β2M). In some examples, the β2M polypeptide comprises an amino acid sequence having at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence shown in FIG. 1A. In some examples, the β2M polypeptide comprises an amino acid sequence having at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 1A.

[0038] In some examples, the β2M polypeptide present in ICBP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 1B, where amino acid 12 is Cys, i.e., the β2M comprises a non-naturally occurring Cys at position 12 as a result of an R12C substitution. In some examples, the β2M polypeptide present in ICBP comprises an amino acid sequence shown in FIG. 1B. In some examples, the β2M polypeptide comprises an amino acid sequence having at least 90%, or at least 95%, amino acid sequence identity to the amino acid sequence shown in FIG. 1B. In some examples, the β2M polypeptide comprises an amino acid sequence having at least 98%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 1B.

[0039] Stabilizing Peptides As described above, the binding moiety comprises a peptide (which may be referred to herein as a "stabilizing peptide") that binds to an HLA-G molecule. The peptide binds to a peptide bond cleft formed by the α1 and α2 domains of an HLA-G heavy chain polypeptide. The stabilizing peptide may have a length of about 5 amino acids to about 15 amino acids. For example, the peptide may have a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some examples, the stabilizing peptide has a length of 8 amino acids to 12 amino acids. In some examples, the peptide has a length of 9 amino acids to 12 amino acids. In some examples, the stabilizing peptide has a length of 9 amino acids to 11 amino acids. In some examples, the peptide has a length of 9 amino acids.

[0040] The binding of ICBP is driven not by the binding of the stabilizing polypeptide in the binding moiety, but rather by the binding of ILT2 and ILT4 to the complex formed between the α3 domain of the HLA-G heavy chain and the β2M polypeptide. Thus, the peptide presented by the binding moiety does not contribute to the binding between the binding moiety and the ILT2 or ILT4 receptor. See, for example, Attia et al., Int. J. Mol. Sci. 2020, 21(22), 8678; https: / / doi.org / 10.3390 / ijms21228678. Thus, the origin of the stabilizing peptide, e.g., autoantigen or viral antigen, is not important if the peptide functionally stabilizes the HLA-G moiety.

[0041] In some examples, the stabilizing peptide has the following amino acid sequence: X1X2PX3X4X5X6X7L, where X1 is Lys or Arg, X2 is Gly, Ile, or Leu, X3 is Ala or Pro, X4 is Ala, Arg, or Gln, X5 is Ala, Phe, or Tyr, X6 is Ile, Leu, Val, or Tyr, and X7 is Ile, Met, Thr, or Gln. In some examples, the stabilizing peptide has the following amino acid sequence: X1X2X3PX4X5X6X7L, where X1 is Lys or Arg, X2 is Gly, Ile, or Leu, X3 is Ala, Gln, or Ile, X4 is Ala, Arg, or Gln, X5 is Ala, Phe, His, or Tyr, X6 is Ile, Leu, Val, or Tyr, and X7 is Ile, Met, Thr, or Gln. See, e.g., Diehl et al. (1996) Current Biol. 6:305, and Walpole et al. (2010) J. Mol. Biol. 397:467.

[0042] The following are non-limiting examples of stabilizing peptides: RIIPRHLQL (SEQ ID NO:24), RLPKDFRIL (SEQ ID NO:68), KGPPAALTL (SEQ ID NO:69), KLPQAFYIL (SEQ ID NO:70), KLPAQFYIL (SEQ ID NO:71), RIPQGFGNLL (SEQ ID NO:72), KIPAQFYIL (SEQ ID NO:73), KGPAQFYIL (SEQ ID NO:74), RGPPQRPKL (SEQ ID NO:75), RGPPQRPKL (SEQ ID NO:75), RIHDKAVAL (SEQ ID NO:76), RAIQKKIDL (SEQ ID NO:77), RLKKSADTL (SEQ ID NO:78), KSPPPMNL (SEQ ID NO:79), KYIHSANVL (SEQ ID NO:80), KIIDSGPQL (SEQ ID NO:81), TAVISIGNQL (SEQ ID NO:82), VVPKDRVAL (SEQ ID NO:83)RSPVYLTVL, (SEQ ID NO:84)RHPKYKTEL , (SEQ ID NO:85), HVPEHAVVL (SEQ ID NO:86) MQPTHPIRL, (SEQ ID NO:87), KIAGYVTHL (SEQ ID NO:88), KGPPAALTL (SEQ ID NO:69), SYPTRIASL (SEQ ID NO:89), RLPDGRVVL (SEQ ID NO:90), MRPRKAFLL (SEQ ID NO:91), RLPKDFVDL (SEQ ID NO:92), and VLPKLYVKL (SEQ ID NO:93).

[0043] In some cases, the stabilized peptide is a self-peptide, i.e., a peptide associated with an autoantigen. For example, in some cases, the stabilized peptide presents an autoimmune disease-related epitope. Numerous epitopes associated with autoimmune disorders are known. For example, see WO2020 / 181062 (Cue Biopharma, Inc.), paragraphs

[0386] and after.

[0044] In some examples, the stabilized peptide epitope may be a peptide that presents an epitope associated with an infectious disease, such as a viral infection. See, for example, paragraphs

[0321] and following of WO2020 / 243315 (Cue Biopharma, Inc.).

[0045] ILT2 and ILT4 As discussed above, the binding moiety of ICBP of the present disclosure binds to ILT2 and / or ILT4 polypeptides. ILT2 and ILT4 polypeptides are present on the surface of immune cells. ILT2 (also known as "leukocyte immunoglobulin-like receptor B1" (LILRB1), "myeloid inhibitory receptor 7 (MIR-7), and "CD85 antigen-like family member J" (CD85J)) polypeptides are known in the art. ILT2 is expressed on the surface of dendritic cells (DCs), B cells, natural killer (NK) cells, and T cells. The amino acid sequence of ILT2 polypeptides is known in the art. Examples include those shown in Figures 8A-8F.

[0046] ILT4 (also known as "leukocyte immunoglobulin-like receptor B2" (LILRB2), "myeloid inhibitory receptor 10 (MIR-10), and "CD85 antigen-like family member D" (CD85D)) polypeptides are known in the art. ILT4 is expressed on monocytes and on tolerogenic IL10-producing dendritic cells. The amino acid sequence of the ILT4 polypeptide is known in the art. Examples include those shown in Figures 9A-9E.

[0047] KIR2DL4 The binding portion of ICBP of the present disclosure has been reported for KIR2DL4. See, e.g., Rajagopalan and Long (2012) Front. Immunol. 3:1. KIR2DL4 ("Killer cell immunoglobulin-like receptor, two Ig domains, and long cytoplasmic tail 4") is also known as CD158D, KIR-103AS, and G9P. KIR2DL4 polypeptides are expressed by natural killer cells and certain T cells. Amino acid sequences of KIR2DL4 polypeptides are known in the art. Examples include those shown in Figures 14A-14C.

[0048] Immunosuppressant Polypeptides The ICBPs of the present disclosure include one or more immunosuppressant polypeptides. In some examples, at least one of the one or more immunosuppressant polypeptides is a wild-type ("wt") immunosuppressant polypeptide. In other examples, at least one of the one or more immunosuppressant polypeptides is a variant of the wt immunosuppressant polypeptide, e.g., such a variant has between one amino acid (aa) and 20 aa differences from the wt immunosuppressant polypeptide. In some examples, the variant immunosuppressant polypeptide includes one or more conservative amino acid substitutions compared to the wt immunosuppressant polypeptide.

[0049] In some examples, the variant immunosuppressive polypeptide binds to a cognate co-receptor (co-immunosuppressive polypeptide) with a reduced affinity compared to the affinity of the binding of the corresponding wt immunosuppressive polypeptide to the cognate co-receptor. A suitable immunosuppressive polypeptide exhibiting a reduced affinity for a receptor or co-receptor may have a difference of 1 amino acid (aa) to 20 aa from the wild-type immunosuppressive polypeptide. For example, in some examples, the variant immunosuppressive polypeptide present in ICBP has an amino acid sequence that differs from the corresponding wt immunosuppressive polypeptide by 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa. As another example, in some examples, the variant immunosuppressive polypeptide present in ICBP has an amino acid sequence that differs from the corresponding wt immunosuppressive polypeptide by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, or 20 aa. As an example, in some instances, a variant immunosuppressant polypeptide present in ICBP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to a corresponding reference (e.g., wild-type) immunosuppressant polypeptide.

[0050] An ICBP may include a single immunosuppressant polypeptide. Alternatively, an ICBP may include two or more immunosuppressant polypeptides. For example, an ICBP may include two immunosuppressant polypeptides. The two immunosuppressant polypeptides may be the same or different (e.g., may have the same amino acid sequence or may have different amino acid sequences). When an ICBP includes two immunosuppressant polypeptides, in some cases, the immunosuppressant polypeptides are tandemly arranged, optionally separated by a linker (e.g., a peptide linker described below).

[0051] PD-L1 Polypeptide In some examples, the immunosuppressive polypeptide present in ICBP is a PD-L1 polypeptide. PD-L1 variants that may be suitable as immunosuppressive polypeptides are disclosed in published PCT applications WO2019 / 051091 and WO2017 / 201131. The amino acid sequences of wild-type and reduced affinity PD-L1 polypeptides are provided in Figures 11A-11D.

[0052] In some examples, the PD-L1 polypeptide of ICBP comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO:54).

[0053] In some examples, the PD-L1 polypeptide of ICBP comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 34).

[0054] In some examples, the PD-L1 polypeptide has the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO:54), and having one or more substitutions of the following amino acids: D8, T19, I36, E42, Q48, E54, I98, G101, G102, A103, D104, Y105, K106, and R107.

[0055] For example, a PD-L1 polypeptide may have the following PD-L1 extracellular domain amino acid sequence: TIFF2025511703000002.tif30153 (SEQ ID NO:53), wherein X1 is any amino acid other than Asp, X2 is any amino acid other than Thr, X3 is any amino acid other than Ile, X4 is any amino acid other than Glu, X5 is any amino acid other than Gln, X6 is any amino acid other than Glu, X7 is any amino acid other than Ile, X8 is any amino acid other than Gly, X9 is any amino acid other than Gly, and X 10 is any amino acid other than Ala, and X 11 is any amino acid other than Asp, and X 12 is any amino acid other than Tyr, and X 13 is any amino acid other than Lys, and X 14 is any amino acid other than Arg.

[0056] By way of example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPK(X)LYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO:94), where X is any amino acid other than Asp, for example, X is Ala or Arg.

[0057] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNM(X)IEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO:95), where X is any amino acid other than Thr, for example, X is Ala.

[0058] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AAL(X)VYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO:96), where X is any amino acid other than Ile, for example, X is Asp.

[0059] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEM(X) DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO:97), wherein X is any amino acid other than Glu, for example, X is Asp or Arg.

[0060] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNII(X)FVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 98), wherein X is any amino acid other than Gln, for example, X is Asp.

[0061] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG E(X)DLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO:99), wherein X is any amino acid other than Glu, for example, X is Asp or Arg.

[0062] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCM(X)SYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 100), wherein X is any amino acid other than Ile, for example, X is Asp or Arg.

[0063] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISY(X)G ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 101), wherein X is any amino acid other than Gly, for example, X is Ala, Asp, or Arg.

[0064] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYG(X) ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 102), where X is any amino acid other than Gly, for example, X is Ala, Asp, or Arg.

[0065] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence:FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG (X)DYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 103), where X is any amino acid other than Ala, for example, X is Arg or Asp.

[0066] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG A(X)YKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 104), where X is any amino acid other than Asp, for example, X is Arg or Ala.

[0067] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence:FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG AD(X)KRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 105), wherein X is any amino acid other than Tyr, for example, X is Asp, Arg, or Ala.

[0068] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADY(X)RITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 106), where X is any amino acid other than Lys, for example, X is Ala, Asp, or Arg.

[0069] As another example, in some examples, a PD-L1 polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYK(X)ITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELPLAHP PNER (SEQ ID NO: 107), where X is any amino acid other than Arg, for example, X is Ala or Asp.

[0070] For example, a PD-L1 polypeptide may have the following PD-L1 extracellular domain amino acid sequence: TIFF2025511703000003.tif29153 (SEQ ID NO:55), wherein X1 is any amino acid other than Asp, X2 is any amino acid other than Thr, X3 is any amino acid other than Ile, X4 is any amino acid other than Glu, X5 is any amino acid other than Gln, X6 is any amino acid other than Glu, X7 is any amino acid other than Ile, X8 is any amino acid other than Gly, X9 is any amino acid other than Gly, and X 10 is any amino acid other than Ala, and X 11 is any amino acid other than Asp, and X 12 is any amino acid other than Tyr, and X 13 is any amino acid other than Lys, and X 14 is any amino acid other than Arg.

[0071] FasL Polypeptide In some examples, the immunosuppressive polypeptide present in ICBP is a FasL polypeptide, e.g., an extracellular domain of a FasL polypeptide. In some examples, the FasL polypeptide of ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following FasL extracellular domain amino acid sequence: QLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L (SEQ ID NO: 108).

[0072] TIGIT Ligand As another example, in some cases, the immunosuppressive polypeptide present in ICBP is a TIGIT ligand. TIGIT ("T cell immunoreceptor with Ig and ITIM domains") is also known as "V-set and immunoglobulin domain-containing protein 9" (VSIG9) and "V-set and transmembrane domain-containing protein 3" (VSTM3). TIGIT inhibits NK cells and T cells (CD4 + T cells, CD8 + TIGIT is expressed on NK cells, including T cells, T cell markers, and regulatory T cells (Tregs). In some instances, the TIGIT ligand is CD155. CD155 (also known as "poliovirus receptor" (PVR) and "nectin-like protein 5" (Necl-5)) mediates NK cell adhesion and induces NK cell effector function. In other instances, the TIGIT ligand is CD112. CD112 (also known as "poliovirus receptor-like 2" (PVRL2), "herpes virus entry mediator B" (HVEB), and "nectin-2") is a modulator of T cell signaling.

[0073] In some cases, the CD155 polypeptide suitable for inclusion in ICBP does not include the transmembrane domain present in wild-type CD155, and instead, the CD155 polypeptide may only include the extracellular portion of the CD155 polypeptide.For example, the beta and gamma isoforms of CD155 lack the internal segment that includes the putative transmembrane domain compared to the alpha isoform.

[0074] In some examples, a CD155 polypeptide suitable for inclusion in an ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CD155 amino acid sequence shown in Figure 10A or Figure 10B.

[0075] In some examples, a CD112 polypeptide suitable for inclusion in an ICBP does not include a transmembrane domain present in wild-type CD112; instead, the CD112 polypeptide may include only the extracellular portion of the CD112 polypeptide. As a non-limiting example, in some examples, a CD112 polypeptide suitable for inclusion in an ICBP includes an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CD112 amino acid sequence shown in Figure 10C or Figure 10D.

[0076] LAG-3 ligand In some instances, the immunosuppressive polypeptide present in ICBP is a ligand of the LAG-3 ("lymphocyte activation gene 3") polypeptide. The LAG-3 polypeptide is also known as CD223. LAG-3 delivers an inhibitory signal upon binding to ligands such as fibrinogen-like protein 1 (FGL1). LAG-3 ligands include FGL1, galectin-3 (Gal-3), lymph node sinusoidal endothelial cell C-type lectin (LSECtin), and α-synuclein fibrils.

[0077] FGL1 ("hepatocyte-derived fibrinogen-related protein 1" (HFREP1), "liver fibrinogen-related protein 1" (LFIRE-1), and "hepasocin") suppresses T cell activation by acting as a ligand for LAG-3. Wang et al. (2019) Cell 176:334.

[0078] In some examples, an FGL1 polypeptide suitable for inclusion in an ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the FGL1 amino acid sequence shown in FIG. 10E.

[0079] LSECtin (also known as "C-type lectin domain family 4 member G" (CLEC4G)) can negatively regulate T cell receptor-mediated signaling. In some examples, an LSECtin polypeptide suitable for inclusion in an ICBP comprises only the extracellular portion of LSECtin (not including the transmembrane region). The extracellular portion may comprise amino acids 55-293 of LSECtin. See, e.g., Dominguez-Soto et al. (2007) Blood 109:5337. Thus, in some examples, an LSECtin polypeptide suitable for inclusion in an ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the LSECtin amino acid sequence shown in FIG. 10F.

[0080] IL-10 As another example, in some instances, the immunosuppressive polypeptide present in an ICBP is an IL-10 polypeptide. IL-10 is also known as "cytokine synthesis inhibitory factor" (CSIF) and "T cell growth inhibitory factor" (TGIF). In some examples, the IL-10 polypeptide of ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following IL-10 amino acid sequence: SP GQGTQSENSC THFPGNLPNM LRDLRDAFSR VKTFFQMKDQ LDNLLLKESL LEDFKGYLGC QALSEMIQFY LEEVMPQAEN QDPDIKAHVN SLGENLKTLR LRLRRCHRFL PCENKSKAVE QVKNAFNKLQ EKGIYKAMSE FDIFINYIEA YMTMKIRN (SEQ ID NO: 109), and has a length of about 160 amino acids to about 170 amino acids (e.g., about 166 amino acids).

[0081] IL-27 As another example, in some examples, the immunosuppressive polypeptide present in ICBP is an IL-27 polypeptide. In some examples, the IL-27 polypeptide of ICBP has the following IL-27 amino acid sequence: MGQTAGDLGW RLSLLLLPLL LVQAGVWGFP RPPGRPQLSL QELRREFTVS LHLARKLLAE VRGQAHRFAE SHLPGVNLYL LPLGEQLPDV SLTFQAWRRL SDPERLCFIS TTLQPFHALL GGLGTQGRWT NMERMQLWAM RLDLRDLQRH LRFQVLAAGF NLPEEEEEEE EEEEEERKGL LPGALGSALQ GPAQVSWPQL LSTYRLLHSL ELVLSRAVRE LLLLSKAGHS VWPLGFPTLS It comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to PQP (SEQ ID NO: 110), and has a length of about 225 amino acids to about 250 amino acids (e.g., about 243 amino acids).

[0082] IL-35 Polypeptides In some examples, the immunosuppressive polypeptide present in ICBP is IL-35 polypeptide.IL-35 is a dimeric protein that includes IL-12α chain and IL-27β chain.Therefore, in some examples, the immunosuppressive polypeptide present in ICBP includes both IL-12α polypeptide and IL-27β polypeptide.

[0083] For example, IL-12α and IL-27β polypeptides suitable for inclusion in ICBPs include amino acid sequences having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the IL-12α and IL-27β amino acid sequences shown in Figures 13A and 13B.

[0084] IL-2 Polypeptides and TGF-β Polypeptides As another example, in some cases, the immunosuppressive polypeptide present in ICBP is an IL-2 polypeptide. When IL-2 is used as the immunosuppressive polypeptide, typically it will not be a T cell activating polypeptide, but rather an IL-2 polypeptide that is a variant of IL-2 that contains one or more modifications that favor the binding of the variant IL-2 polypeptide to the IL-2 receptor alpha (IL-2Rα), which can result in an increase in regulatory T cells (Tregs). Such variants are well known in the art.

[0085] Alternatively, however, an activated form of IL-2 can be used when used in combination with a masked TGF-β polypeptide, as described in WO2021 / 081258 (Cue Biopharma, Inc.).

[0086] scaffold The ICBP comprises a scaffold polypeptide. Suitable scaffold polypeptides include antibody-based scaffold polypeptides (e.g., immunoglobulin (Ig) Fc polypeptides) and non-antibody-based scaffolds. Non-antibody based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptides, transferrin, Fc receptor polypeptides, elastin-like polypeptides (see, e.g., Hassouneh et al. (2012) Methods Enzymol. 502:215; e.g., polypeptides comprising a pentapeptide repeat unit (Val-Pro-Gly-X-Gly; SEQ ID NO:153), where X is an amino acid other than proline), albumin-binding polypeptides, silk-like polypeptides (see, e.g., Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), silk-elastin-like polypeptides (SELPs; see, e.g., Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTEN polypeptides include, for example, those disclosed in WO2009 / 023270, WO2010 / 091122, WO2007 / 103515, US2010 / 0189682, and US2009 / 0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitable albumin polypeptides include, for example, human serum albumin.

[0087] Other suitable scaffold components capable of presenting both the HLA-G binding moiety and the optional immunosuppressive polypeptide include carriers such as lipid vesicles (e.g., liposomes) or micelles, nanoparticles, PEGylated proteins (including site-specific PEGylation), fibronectin-based scaffold proteins, or artificial antigen-presenting cells such as genetically engineered red blood cells and enucleated cells (e.g., platelets).

[0088] A suitable scaffold polypeptide will in some cases be a polypeptide that extends half-life. Thus, in some cases, a suitable scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of ICBP compared to a control ICBP lacking the scaffold polypeptide. For example, in some cases, the scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of ICBP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold compared to a control ICBP lacking the scaffold polypeptide. By way of example, in some instances, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of an ICBP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold, compared to a control ICBP lacking the Fc polypeptide.

[0089] Ig Fc Polypeptide In some examples, the scaffold polypeptide present in ICBP is an Ig Fc polypeptide. In some examples, the Ig Fc polypeptide in ICBP comprises an Ig heavy chain constant region (CH2-CH3) polypeptide sequence that functions as a dimerization or multimerization sequence (see, e.g., Figures 2A-2M). In some examples, the Ig polypeptide may comprise a mutation that results in a decreased ability to induce cell lysis, e.g., via activation of complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC), thus reducing or substantially eliminating the ability of the Ig polypeptide to induce cell lysis. In some examples, the Ig Fc polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Ig Fc polypeptide shown in any one of Figures 2A-2M. Such an Ig Fc polypeptide can covalently link together the polypeptides of ICBP, e.g., by forming one or two interchain disulfide bonds. As discussed below, additional disulfide bonds can be introduced to stabilize the dimer, particularly when a pair of interspecies Ig sequences is employed, such as a knob-in-hole (KiH) polypeptide pair.

[0090] In some examples, the Fc polypeptide present in the ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to an Fc amino acid sequence shown in any one of Figures 2A-2M.

[0091] In some examples, the Fc polypeptide present in the ICBP is an IgG1 Fc polypeptide, or a variant of an IgG1 Fc polypeptide. For example, in some examples, the Fc polypeptide present in the ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in FIG. 2A. As another example, in some examples, the Fc polypeptide present in the ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Fc polypeptide shown in FIG. 2B, and the Ig Fc polypeptide comprises an Ala at position 14 and an Ala at position 15. In any of the above embodiments, the Ig Fc polypeptide can have an N77 substitution, i.e., the Ig Fc polypeptide can have an amino acid other than Asn at position 77, and in some examples, the Ig Fc polypeptide has an Ala at position 77. In some examples, the Fc polypeptide present in the ICBP comprises the amino acid sequence shown in Figure 2A. In some examples, the Fc polypeptide present in the ICBP comprises the amino acid sequence shown in Figure 2B.

[0092] In some examples, the Fc polypeptide present in the ICBP is an IgG1 Fc polypeptide or a variant of the IgG1 Fc polypeptide, the variant including a naturally occurring variant, a non-naturally occurring variant, and a combination thereof. For example, in some examples, the Fc polypeptide present in the ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in FIG. 2C, the Ig Fc polypeptide comprises a Glu at position 136 and a Met at position 138. As another example, in some examples, the Fc polypeptide present in the ICBP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in FIG. 2D, the Ig Fc polypeptide comprises an Ala at positions 14 and 15, the Fc polypeptide comprises a Glu at position 136 and a Met at position 138. In any of the above embodiments, the Ig Fc polypeptide can have an N77 substitution, i.e., the Ig Fc polypeptide can have an amino acid other than Asn at position 77, and in some examples, the Ig Fc polypeptide has Ala at position 77. In some examples, the Fc polypeptide present in the ICBP comprises the amino acid sequence shown in Figure 2C. In some examples, the Fc polypeptide present in the ICBP comprises the amino acid sequence shown in Figure 2D.

[0093] In some examples, the Fc polypeptide present in ICBP comprises the amino acid sequence shown in FIG. 2E (human IgG1 Fc with L234F, L235E, and P331S substitutions, where L234 corresponds to amino acid 14 of the amino acid sequence shown in FIG. 2A, L235 corresponds to amino acid 15 of the amino acid sequence shown in FIG. 2E, and P331 corresponds to amino acid 111 of the amino acid sequence shown in FIG. 2E). In some examples, the Fc polypeptide present in ICBP comprises the amino acid sequence shown in FIG. 2F, which comprises an N279A substitution (amino acid sequence of N77A shown in FIG. 2F). The substitution at N297 leads to the removal of carbohydrate modification, resulting in an antibody sequence with reduced complement component 1q ("C1q") binding compared to the wild-type protein, thus reducing complement-dependent cytotoxicity (CDC). In some examples, the Fc polypeptide present in ICBP comprises a substitution at K322. K322 (e.g., K322A) substitutions show a substantial reduction in FcγR binding affinity and antibody-dependent cell-mediated cytotoxicity (ADCC), while C1q binding and CDC function are substantially or completely eliminated.

[0094] In some examples, the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG2 Fc polypeptide shown in FIG. 2G, e.g., the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 99-325 of the human IgG2 Fc polypeptide shown in FIG. 2G (e.g., an Ig Fc polypeptide has a length of about 227 amino acids). In some examples, the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG3 Fc polypeptide shown in FIG. 2H, e.g., the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide shown in FIG. 2H (e.g., an Ig Fc polypeptide has a length of about 228 amino acids). In some examples, the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgM Fc polypeptide shown in FIG. 2J, e.g., the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide shown in FIG. 2J.In some examples, the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgA Fc polypeptide shown in FIG. 2K, e.g., the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 1-234 of the human IgA Fc polypeptide shown in FIG. 2K.

[0095] In some examples, the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG4 Fc polypeptide shown in Figure 2M. In some examples, the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 100-327 of the human IgG4 Fc polypeptide shown in Figure 2M (e.g., an Ig Fc polypeptide has a length of about 228 amino acids).

[0096] In some examples, the Fc polypeptide present in ICBP comprises the amino acid sequence shown in FIG. 2A (human IgG1 Fc). In some examples, the Fc polypeptide present in ICBP comprises the amino acid sequence shown in FIG. 2A (human IgG1 Fc), except for the substitution of N297 (N77 in the amino acid sequence shown in FIG. 2A) with an amino acid other than asparagine (e.g., N77A substitution). In some examples, the Fc polypeptide present in ICBP comprises the amino acid sequence shown in FIG. 2F. The substitution at N297 (N77) leads to the removal of carbohydrate modification, resulting in an antibody sequence with reduced complement component 1q ("C1q") binding compared to the wild-type protein, and thus reduced complement-dependent cytotoxicity (CDC). In some examples, the Fc polypeptide present in ICBP comprises a substitution at K322 (corresponding to amino acid 102 in the amino acid sequence shown in FIG. 2A). The K322 (e.g., K322A) substitution shows a substantial reduction in FcγR binding affinity and antibody-dependent cell-mediated cytotoxicity (ADCC), and C1q binding and CDC function are substantially or completely eliminated. In some examples, the Fc polypeptide present in ICBP comprises the amino acid sequence shown in FIG. 2A (human IgG1 Fc), except for the substitution of L234 (L14 in the amino acid sequence shown in FIG. 2A) with an amino acid other than leucine. In some examples, the Fc polypeptide present in ICBP comprises the amino acid sequence shown in FIG. 2A (human IgG1 Fc), except for the substitution of L235 (L15 in the amino acid sequence shown in FIG. 2A) with an amino acid other than leucine.

[0097] The Ig Fc heavy chain CH2 and CH3 domains (such as those shown in Figures 2A-2M) may also function as dimerization or multimerization sequences (e.g., when the ICBP comprises two or more heterodimers). If asymmetric pairing between two Ig Fc polypeptides is desired, the Ig Fc polypeptides may incorporate knob-in-hole modifications, for example, in the CH3 domain. Such knob-in-hole pairs include the T366Y and Y407T mutant pair at the CH3 domain interface of IgG1, or the corresponding residues in another Ig Fc ("T366" corresponds to amino acid 146 of the IgG1 Fc amino acid sequence shown in Figure 2A, and "Y407" corresponds to amino acid 187 of the IgG1 Fc amino acid sequence shown in Figure 2A). See Ridgway et al., Protein Engineering 9:7, 617-621 (1996), (substitutions are indicated by the EU numbering scheme of Kabat et al. (1991)). Another knob-into-hole pairing involves the formation of a knob by a T366W substitution and a hole by the triple substitutions T366S, L368A, and Y407V on a complementary Fc polypeptide ("T366" corresponds to amino acid 146 of the IgG1 Fc amino acid sequence shown in FIG. 2A, "L368" corresponds to amino acid 148 of the IgG1 Fc amino acid sequence shown in FIG. 2A, and "Y407" corresponds to amino acid 187 of the IgG1 Fc amino acid sequence shown in FIG. 2A). See Xu et al. mAbs 7:1, 231-242 (2015). For example, in some instances, a first ICBP may comprise an IgG1 Fc polypeptide including a T366Y substitution (e.g., a T146Y substitution based on the IgG1 Fc amino acid sequence shown in FIG. 3A), and a second ICBP may comprise an IgG1 Fc polypeptide including a Y407T substitution (e.g., a Y187T substitution based on the IgG1 Fc amino acid sequence shown in FIG. 2A).As another example, in some instances, a first ICBP may comprise an IgG1 Fc polypeptide comprising a T366W substitution (e.g., a T146W substitution based on the IgG1 Fc amino acid sequence shown in FIG. 2A), and a second ICBP heterodimer may comprise an IgG1 Fc polypeptide comprising a T366S substitution, a L368A substitution, and a Y407V substitution (e.g., a T146S substitution, a L148A substitution, and a Y187V substitution based on the IgG1 Fc amino acid sequence shown in FIG. 2A). The Fc polypeptides may be stabilized by the formation of disulfide bonds (e.g., hinge region disulfide bonds) between the Fc polypeptides, with or without knob-into-hole modifications.

[0098] Single strand ICBP The ICBP of the present disclosure may be a single-chain polypeptide, i.e., all of the components of the ICBP (e.g., HLA-G heavy chain polypeptide, stabilizing peptide, β2M polypeptide, immunosuppressive polypeptide, scaffolding polypeptide) are present in a single polypeptide chain.

[0099] The individual components may be arranged in various configurations. For example, in some examples, an ICBP may include, in order from N-terminus to C-terminus, (a) a stabilizing peptide, (b) a β2M polypeptide, (c) an HLA-G heavy chain polypeptide, (d) at least one immunosuppressive polypeptide, and (e) a scaffold polypeptide. A non-limiting example of an ICBP having such a configuration is shown in FIG. 5A. As another example, in some examples, an ICBP may include, in order from N-terminus to C-terminus, (a) a stabilizing peptide, (b) a β2M polypeptide, (c) an HLA-G heavy chain polypeptide, (d) a scaffold polypeptide, and (e) at least one immunosuppressive polypeptide. A non-limiting example of an ICBP having such a configuration is shown in FIG. 5B. As mentioned above, in some examples, an ICBP does not include at least one immunosuppressive polypeptide. In such a case, for example, an ICBP may include, in order from N-terminus to C-terminus, (a) a stabilizing peptide, (b) a β2M polypeptide, (c) an HLA-G heavy chain polypeptide, and (d) a scaffold polypeptide. As another example, in some instances, an ICBP may comprise, in order from N-terminus to C-terminus, (a) a stabilizing peptide, (b) a β2M polypeptide, (c) an HLA-G heavy chain polypeptide, and (d) a scaffold polypeptide.

[0100] In any of the above-described embodiments, one or more of the individual components (e.g., HLA-G heavy chain polypeptide, stabilizing polypeptide, β2M polypeptide, immunosuppressive polypeptide, scaffold polypeptide) are linked together by one or more linkers (e.g., peptide linkers described elsewhere herein). As an example, in some examples, an ICBP may comprise, in order from N-terminus to C-terminus, (a) a stabilizing peptide, (b) a linker, (c) a β2M polypeptide, (d) a linker, (e) an HLA-G heavy chain polypeptide, (f) a linker, (g) at least one immunosuppressive polypeptide, (h) a linker, and (i) a scaffold polypeptide, such as a variant IgG1 Fc polypeptide. As another example, in some cases, ICBP may include, in order from N-terminus to C-terminus, (a) a stabilizing peptide, (b) a linker, (c) a β2M polypeptide, (d) a linker, (e) an HLA-G heavy chain polypeptide, (f) a linker, (g) a scaffold polypeptide, such as a variant IgG1 Fc polypeptide, (h) a linker, and (i) at least one immunosuppressive polypeptide. Exemplary single-chain constructs are provided as Figures 5A and 5B. Each of these exemplary constructs includes an IgG1 Fc scaffold polypeptide with L14A and L15A substitutions. Upon expression, these single-chain ICBPs will spontaneously form homodimers, with disulfide bonds linking the IgG1 Fc polypeptide of one ICBP to the IgG1 Fc of the other single-chain ICBP. In some cases, the single-chain ICBP includes one or more intrachain disulfide bonds. For example, in some instances, the single-chain ICBP comprises (a) a disulfide bond between (i) a Cys at residue 84 of the HLA-G heavy chain polypeptide and (ii) a Cys in a linker linking the stabilizing peptide to the β2M polypeptide, and / or (b) a disulfide bond between a Cys at residue 236 of the HLA-G heavy chain polypeptide and a Cys residue in the β2M polypeptide (e.g., amino acid residue 12 of the β2M polypeptide).

[0101] Heterodimeric ICBP An ICBP of the present disclosure may be a heterodimeric polypeptide, i.e., the components of the ICBP (e.g., HLA-G heavy chain polypeptide, stabilizing peptide, β2M polypeptide, immunosuppressive polypeptide, scaffolding polypeptide) are present in two separate polypeptide chains. Figures 6A-6C provide non-limiting examples of polypeptide chains of such a heterodimeric ICBP.

[0102] The individual components can be arranged in various configurations. For example, in some cases, the ICBP comprises a first polypeptide comprising a stabilizing peptide and a β2M polypeptide, and a second polypeptide comprising an HLA-G heavy chain polypeptide, at least one immunosuppressive polypeptide, and a scaffold polypeptide. As another example, in some cases, the ICBP comprises a first polypeptide comprising a binding moiety (i.e., a stabilizing peptide, an HLA-G heavy chain polypeptide, and a β2M polypeptide), and a second polypeptide comprising at least one immunosuppressive polypeptide and a scaffold polypeptide.

[0103] As an example, in some examples, ICBP comprises, in order from N-terminus to C-terminus, a first polypeptide comprising (i) a stabilizing peptide, and (ii) a β2M polypeptide, and a second polypeptide comprising, in order from N-terminus to C-terminus, (i) at least one immunosuppressant polypeptide, (ii) an HLA-G heavy chain polypeptide, and (iii) a scaffold polypeptide. In some examples, the at least one immunosuppressant polypeptide is a PD-L1 polypeptide (wild-type or reduced affinity variant). In some examples, the scaffold polypeptide is an Ig Fc polypeptide. Figure 6B provides examples of such polypeptide chains, where an exemplary first polypeptide chain is designated "4602" and an exemplary second polypeptide chain is designated "4607". The amino acid sequences of the individual components of the second polypeptide chain (e.g., PD-L1 extracellular domain, HLA-G heavy chain polypeptide, and IgG1 Fc) are also provided. As another example, in some examples, an ICBP comprises, in order from N-terminus to C-terminus, a first polypeptide comprising: (i) a stabilizing peptide, and (ii) a β2M polypeptide; and a second polypeptide comprising, in order from N-terminus to C-terminus, (i) an HLA-G heavy chain polypeptide, (ii) at least one immunosuppressive polypeptide, and (iii) a scaffold polypeptide. In some examples, the at least one immunosuppressive polypeptide is a PD-L1 polypeptide (wild-type or reduced affinity variant). In some examples, the scaffold polypeptide is an Ig Fc polypeptide. Figure 6C provides examples of such polypeptide chains, where an exemplary first polypeptide chain is designated "4602" and an exemplary second polypeptide chain is designated "4607-2". The amino acid sequences of the individual components of the second polypeptide chain (e.g., the PD-L1 extracellular domain, the HLA-G heavy chain polypeptide, and the IgG1 Fc) are also provided. As another example, in some embodiments, an ICBP comprises, in order from N-terminus to C-terminus, a first polypeptide comprising (i) a stabilizing peptide, and (ii) a β2M polypeptide, and a second polypeptide comprising, in order from N-terminus to C-terminus, (i) an HLA-G heavy chain polypeptide, (ii) a scaffold polypeptide, and (iii) at least one immunosuppressive polypeptide.In some examples, the at least one immunosuppressive polypeptide is a PD-L1 polypeptide (wild-type or reduced affinity variant). In some examples, the scaffold polypeptide is an Ig Fc polypeptide.

[0104] As another example, in some examples, an ICBP comprises, in order from N-terminus to C-terminus, a first polypeptide comprising: (i) a stabilizing peptide, (ii) a β2M polypeptide, and (iii) at least one immunosuppressive polypeptide; and a second polypeptide comprising, in order from N-terminus to C-terminus, (i) an HLA-G heavy chain polypeptide, and (ii) a scaffold polypeptide. In some examples, the at least one immunosuppressive polypeptide is a PD-L1 polypeptide (wild-type or reduced affinity variant). In some examples, the scaffold polypeptide is an Ig Fc polypeptide.

[0105] As mentioned above, in some examples, the ICBP does not include an immunosuppressant polypeptide. For example, in some examples, the ICBP includes (a) a first polypeptide including (i) a stabilizing peptide and (ii) a β2M polypeptide, and (b) a second polypeptide including (i) an HLA-G heavy chain polypeptide and (ii) a scaffold polypeptide. In some examples, the scaffold polypeptide is an Ig Fc polypeptide. A non-limiting example of such an ICBP is provided in FIG. 6A, where an exemplary first polypeptide chain is designated as "4602" and an exemplary second polypeptide is designated as "4601". The amino acid sequences of the individual components of the second polypeptide chain (e.g., HLA-G heavy chain polypeptide, and IgG1 Fc) are also provided. In any of the above-mentioned embodiments, one or more of the individual components (e.g., HLA-G heavy chain polypeptide, stabilizing polypeptide, β2M polypeptide, immunosuppressant polypeptide, scaffold polypeptide) are linked together by one or more linkers (e.g., peptide linkers described elsewhere herein).

[0106] Dimeric form of ICBP As mentioned above, the components of the ICBP of the present disclosure (e.g., HLA-G heavy chain polypeptide, stabilizing peptide, β2M polypeptide, immunosuppressive polypeptide, scaffolding polypeptide) may be in a single polypeptide chain or may be present in two separate polypeptide chains forming a heterodimer. Regardless of whether the components are present in a single polypeptide chain or two separate polypeptide chains forming a heterodimer, the ICBP of the present disclosure can form a dimer. Thus, the present disclosure provides a protein comprising a dimer of two ICBPs.

[0107] In some cases, the two ICBPs are covalently linked to each other. The covalent bond of the dimer can be one or more disulfide bonds between the Ig Fc polypeptide in the first ICBP (which can be a single-chain ICBP or a heterodimeric ICBP) and the Ig Fc polypeptide in the second ICBP (which can be a single-chain ICBP or a heterodimeric ICBP). When an ICBP includes an Ig Fc polypeptide, the ICBP will typically self-assemble into a dimer by spontaneously forming a disulfide bond with the IgG1 Fc polypeptide of another ICBP. Thus, for example, the Ig Fc polypeptides in the first ICBP and the second ICBP can be linked to each other by one or more disulfide bonds. In many cases, the two ICBPs will be identical to each other in amino acid sequence and include an Ig Fc polypeptide that spontaneously forms one or more disulfide bonds, thereby forming a dimerized ICBP that is a homodimer.

[0108] Thus, the present disclosure provides a protein comprising (a) a first ICBP and (b) a second ICBP, which may optionally be identical to the first ICBP, wherein the first and second ICBPs are covalently bound to each other. The covalent bond may be a disulfide bond between an Ig Fc polypeptide in the first ICBP and an Ig Fc polypeptide in the second ICBP. In some examples, the first and second ICBPs are single-chain polypeptides. In some examples, the first and second ICBPs are heterodimeric polypeptides.

[0109] Alternatively, if it is desired to link two different ICBPs together to form a dimer, an interspecies linker sequence can be used, see, e.g., WO2021242935 (Cue Biopharma, Inc.).

[0110] Linker As described above, an ICBP can include one or more independently selected peptide linkers, i.e., linkers comprising a contiguous stretch of two or more amino acids, between one or more components of the ICBP (e.g., HLA-G heavy chain polypeptide, stabilizing peptide, β2M polypeptide, immunosuppressive polypeptide, scaffold polypeptide). For example, the linker can be between one or more of: (i) a stabilizing peptide and a β2M polypeptide, (ii) a β2M polypeptide and an HLA-G heavy chain polypeptide (in the case of a single-chain ICBP), (iii) an immunosuppressive polypeptide and an HLA-G heavy chain polypeptide, (iv) an HLA-G heavy chain polypeptide and a scaffold polypeptide (e.g., an Ig Fc polypeptide), (v) an immunosuppressive polypeptide and a scaffold polypeptide (e.g., an Ig Fc polypeptide), and (vi) a β2M polypeptide and an immunosuppressive polypeptide. The linker can be independently selected from (i) a flexible peptide linker, including a short flexible peptide linker, and (ii) a rigid peptide linker.

[0111] Suitable linkers (also referred to as "spacers") can be readily selected and can be any of several suitable lengths, such as 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. Suitable linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some examples, the linker has a length of 25 to 50 amino acids, e.g., 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids.

[0112] Flexible Peptide Linkers Exemplary flexible peptide linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 111), (GGGGS)n (SEQ ID NO: 112), and (GGGS) n (SEQ ID NO:113), where n is at least one integer and can be an integer between 1 and 10), glycine-alanine polymers, alanine-serine polymers, and other flexible peptide linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can be used; glycine has access to significantly more phi-psi space than alanine and is significantly less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:114), GGSGG (SEQ ID NO:115), GSGSG (SEQ ID NO:116), GSGGG (SEQ ID NO:117), GGGSG (SEQ ID NO:118), GSSSG (SEQ ID NO:119), and the like.

[0113] Exemplary flexible peptide linkers include, for example, (GGGGS)n (SEQ ID NO:112), also referred to as "G4S" linkers), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:112), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:112), where n is 2. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:112), where n is 3. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:112), where n is 4. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:112), where n is 7. In some examples, the linker comprises the amino acid sequence AAAGG (SEQ ID NO: 112). A linker having the amino acid sequence AAAGG (SEQ ID NO: 28) is also suitable. In the ICBP of the present disclosure, the β2M polypeptide may be linked to the HLA-G heavy chain polypeptide by a (GGGGS)n (SEQ ID NO: 112) linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., where n=3, n=4, or n=7.

[0114] As used in this disclosure, a "short flexible peptide linker" refers to a flexible peptide linker that contains fewer than 15 amino acids, i.e., 2-14 amino acids. For example, a short flexible peptide linker can contain 2-4, 2-5, or 3-6 amino acids (e.g., a GGS linker as discussed in Example 1), or 4-8, 5-10, or 10-14 amino acids. Included within this range are flexible peptide linkers that contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids.

[0115] Rigid Peptide Linkers In some instances, the peptide linker is a rigid peptide linker. As used herein, the term "rigid peptide linker" refers to a linker that comprises a contiguous stretch of two or more amino acids that effectively separates protein domains by maintaining a substantially fixed distance / spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between such domains. Rigid peptide linkers are known in the art and generally adopt a relatively well-defined conformation when in solution. Rigid peptide linkers include those that have a specific secondary and / or tertiary structure in solution, typically of sufficient length to impart a secondary or tertiary structure to the linker. Rigid peptide linkers include proline-rich peptide linkers and peptide linkers that have an inflexible helical structure, such as an α-helical structure. Rigid peptide linkers are described, for example, in Chen et al. (2013) Adv. Drug Deliv. Rev. 65:1357; and Klein et al. (2014) Protein Engineering, Design & Selection 27:325.

[0116] Examples of rigid peptide linkers include, for example, (EAAAK)n (SEQ ID NO: 120), A(EAAAK)nA (SEQ ID NO: 121), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO: 122), (Lys-Pro)n, (Glu-Pro)n, (Thr-Pro-Arg)n, and (Ala-Pro)n, where n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). Non-limiting examples of suitable rigid peptide linkers that contain EAAAK (SEQ ID NO: 123) include EAAAK (SEQ ID NO: 123), (EAAAK)2 (SEQ ID NO: 124), (EAAAK)3 (SEQ ID NO: 125), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 126), and AEAAAKEAAAKA (SEQ ID NO: 127). Non-limiting examples of suitable rigid peptide linkers that contain (AP)n include APAP (SEQ ID NO: 128, also referred to herein as "(AP)2"), APAPAPAP (SEQ ID NO: 129, also referred to herein as "(AP)4"), APAPAPAPAPAP (SEQ ID NO: 130, also referred to herein as "(AP)6"), APAPAPAPAPAPAPAP (SEQ ID NO: 131, also referred to herein as "(AP)8"), and APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 132, also referred to herein as "(AP)10"). Non-limiting examples of suitable rigid peptide linkers that contain (KP)n include KPKP (SEQ ID NO:133, also referred to herein as "(KP)2"), KPKPKPKP (SEQ ID NO:134, also referred to herein as "(KP)4"), KPKPKPKPKPKP (SEQ ID NO:135, also referred to herein as "(KP)6"), KPKPKPKPKPKPKPKP (SEQ ID NO:136, also referred to herein as "(KP)8"), and KPKPKPKPKPKPKPKPKPKP (SEQ ID NO:137, also referred to herein as "(KP)10").Non-limiting examples of suitable rigid peptide linkers comprising (EP)n include EPEP (SEQ ID NO:138, also referred to herein as "(EP)2"), EPEPEPEPEP (SEQ ID NO:139, also referred to herein as "(EP)4"), EPEPEPEPEPEPEP (SEQ ID NO:140, also referred to herein as "(EP)6"), EPEPEPEPEPEPEPEP (SEQ ID NO:141, also referred to herein as "(EP)8"), and EPEPEPEPEPEPEPEPEPEPEP (SEQ ID NO:142, also referred to herein as "(EP)10").

[0117] Cysteine-Containing Linkers In some examples, the heterodimer or single-chain ICBP comprises a linker between the stabilizing peptide and the β2M polypeptide that comprises a cysteine ​​residue that forms an interchain disulfide bond with a cysteine ​​residue in the HLA-G heavy chain polypeptide. For example, in some examples, the ICBP, or dimerized ICBP, such as a homodimer, comprises a cysteine-containing linker between the stabilizing peptide and the β2M polypeptide, and the cysteine ​​residue in the linker forms a disulfide bond with the cysteine ​​residue at amino acid 236 (e.g., formed by A236C substitution) in the HLA-G heavy chain polypeptide present in the ICBP.

[0118] In some examples, the peptide linker between the stabilizing peptide and the β2M polypeptide comprises the amino acid sequence GCGGS (SEQ ID NO: 143). In some examples, the peptide linker between the stabilizing peptide and the β2M polypeptide comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 144), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, 1, 2, or 3. In some examples, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 144), where n is 2. In some examples, the peptide linker between the stabilizing peptide and the β2M polypeptide comprises the amino acid sequence CGGGS (SEQ ID NO: 145). In some examples, the peptide linker comprises the amino acid sequence CGGGS (GGGGS) n (SEQ ID NO: 146), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, 1, 2, or 3. In some examples, the peptide linker between the stabilizing peptide and the β2M polypeptide comprises the amino acid sequence GGCGS (SEQ ID NO: 147). In some examples, the peptide linker comprises the amino acid sequence GGCGS (GGGGS) n (SEQ ID NO: 148), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, 1, 2, or 3. In some examples, the peptide linker between the stabilizing peptide and the β2M polypeptide comprises the amino acid sequence GGGCS (SEQ ID NO: 149). In some examples, the peptide linker comprises the amino acid sequence GGGCS (GGGGS) n (SEQ ID NO: 150), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, 1, 2, or 3. In some examples, the peptide linker between the stabilizing peptide and the β2M polypeptide comprises the amino acid sequence GGGGC (SEQ ID NO: 151). In some examples, the peptide linker comprises the amino acid sequence GGGGC(GGGGS)n (SEQ ID NO: 152), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0119] composition The present disclosure provides compositions, including pharmaceutical compositions, that comprise an ICBP of the present disclosure.

[0120] A wide variety of pharma- ceutically acceptable ingredients are known in the art and therefore will not be discussed in detail herein. Additionally, pharma- ceutically acceptable ingredients and compositions are fully described in various publications, including, but not limited to, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds 7 th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3 rd ed Amer. Pharmaceutical Assoc. Numerous other publications describing the preparation of biopharmaceutical compositions may be referenced.

[0121] The composition may be formulated according to various routes of administration, as described below. Generally, the ICBP of the present disclosure (e.g., a homodimer containing two ICBPs) will be an aqueous liquid and will typically be administered via intravenous infusion. In some cases, a pharmaceutical composition containing an ICBP may be mixed with saline (e.g., 0.9% NaCl) prior to intravenous administration. Thus, the present disclosure provides a sterile composition comprising (a) an ICBP (or a protein containing two ICBPs) and (b) saline (e.g., 0.9% NaCl). Alternatively, the ICBP may be administered as is, i.e., without further dilution, via intravenous infusion. Alternatively, a pharmaceutical composition containing an ICBP may be formulated to be administered by injection.

[0122] Common and pharma- ceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. As noted above, pharmaceutical compositions containing ICBP are typically administered intravenously, but may be administered by other routes, including injection.

[0123] Nucleic acids, expression vectors, and host cells The present disclosure provides compositions of nucleic acids comprising one or more nucleotide sequences encoding ICBP. In some examples, when ICBP is a heterodimeric polypeptide, each polypeptide chain of ICBP is encoded within a separate nucleic acid. In some examples, when ICBP is a heterodimeric polypeptide, each polypeptide chain of ICBP is encoded within a single nucleic acid. In some examples, a first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of ICBP, and a second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of ICBP.

[0124] The nucleotide sequence encoding the single-chain ICBP may be operably linked to a transcriptional control element such as a promoter. The nucleotide sequence encoding the first polypeptide chain of the heterodimeric ICBP may be operably linked to a transcriptional control element such as a promoter. The nucleotide sequence encoding the second polypeptide chain of the heterodimeric ICBP may be operably linked to a transcriptional control element such as a promoter. The nucleotide sequence encoding the first and second polypeptide chains of the heterodimeric ICBP may be operably linked to a transcriptional control element such as a promoter. Suitable promoters include promoters that function in eukaryotic cells. Suitable promoters include constitutive promoters and inducible promoters.

[0125] As described above, in some cases, each polypeptide chain of ICBP is encoded in a separate nucleic acid. In some cases, the nucleotide sequence encoding each polypeptide chain of ICBP is operably linked to a transcription control element, such as a promoter, such as a promoter that is functional in eukaryotic cells, where the promoter may be a constitutive promoter or an inducible promoter. In some cases, the transcription control element is a promoter that is functional in eukaryotic cells. In some cases, the nucleic acid is present in an individual expression vector.

[0126] ICBP The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding separate polypeptide chains (e.g., a first polypeptide and a second polypeptide) of ICBP. In some examples, the nucleotide sequence encoding the first polypeptide and the second polypeptide of ICBP comprises a proteolytically cleavable linker disposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some examples, the nucleotide sequence encoding the first polypeptide and the second polypeptide of ICBP comprises an internal ribosome entry site (IRES) disposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some examples, the nucleotide sequence encoding the first polypeptide and the second polypeptide of ICBP comprises a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL) disposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide.

[0127] Recombinant Expression Vectors The disclosure provides recombinant expression vectors comprising one or more of the nucleic acids described above.

[0128] Large numbers of suitable expression vectors are known to those of skill in the art, many of which are commercially available, see, e.g., published WO2020 / 243315 (Cue Biopharma, Inc.).

[0129] Compositions Comprising Nucleic Acids or Recombinant Expression Vectors The present disclosure provides compositions, e.g., pharmaceutical compositions, comprising one or more nucleic acids or one or more recombinant expression vectors, the nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding an ICBP of the present disclosure. A wide variety of pharma- ceutically acceptable excipients are well known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are fully described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds 7; th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3 rd ed Amer.Pharmaceutical Assoc.

[0130] Genetically modified host cells The present disclosure provides genetically modified host cells, where the host cells are genetically modified with a nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding an ICBP of the present disclosure.

[0131] Suitable host cells include eukaryotic cells such as yeast, insect cells, and mammalian cells. In some examples, the host cell is a cell of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), Chinese hamster ovary (CHO) cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0132] In some examples, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous β2M.

[0133] In some examples, the host cell is a mammalian cell that has been genetically modified to not synthesize endogenous HLA-G heavy chain. In some examples, the host cell is a mammalian cell that has been genetically modified to not synthesize endogenous β2M and not synthesize endogenous HLA-G heavy chain.

[0134] How to Generate ICBP The present disclosure provides a method for producing ICBP. The method generally includes culturing a host cell genetically modified with one or more nucleic acids (e.g., one or more recombinant expression vectors) comprising a nucleotide sequence encoding ICBP in a medium, the culturing being carried out under conditions that allow for the synthesis of ICBP. In some examples, the method includes isolating ICBP from the genetically modified host cell and / or the culture medium. A host cell genetically modified with one or more nucleic acids (e.g., one or more recombinant expression vectors) comprising a nucleotide sequence encoding ICBP is also referred to as an "expression host." As described above, in some examples, the individual polypeptide chains of ICBP are encoded within individual nucleic acids (e.g., one or more recombinant expression vectors). In some examples, all of the polypeptide chains of ICBP are encoded within a single nucleic acid (e.g., a single recombinant expression vector).

[0135] Isolation of ICBP from expression host cells (eg, lysates of expression host cells) and / or from the medium in which the host cells are cultured can be carried out using standard methods for protein purification.

[0136] For example, a lysate may be prepared from the expression host, and the lysate may be purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. Alternatively, if the ICBP is secreted into the medium from the expression host cell, the ICBP may be purified from the medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In some examples, the composition used will contain at least about 80% by weight, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight of the desired product (ICBP) relative to contaminants associated with the method of preparation of the product and its purification. The percentages can be based on total protein.

[0137] In some cases, for example, where the ICBP comprises an affinity tag, the ICBP can be purified using an immobilized binding partner of the affinity tag.

[0138] method The present disclosure provides methods of inducing immune tolerance in an individual, methods of reducing inflammatory immune cell activity in an individual, and methods of treating an autoimmune disease in an individual, which generally involve administering to an individual in need thereof an effective amount of an ICBP of the present disclosure.

[0139] In some instances, an "effective" amount of ICBP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the production of inflammatory cytokines (also called "pro-inflammatory cytokines") by immune cells of the individual. Inflammatory cytokines include T helper (T h In some instances, an effective amount of ICBP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the level of a proinflammatory cytokine in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or more than 50%, compared to the level of the proinflammatory cytokine in the individual prior to treatment with ICBP.

[0140] In some instances, an effective amount of ICBP is an amount that, when administered in one or more doses to an individual in need thereof, increases the number of regulatory T cells (Tregs) in the individual. Tregs are CD4 + , FOXP3 + , and CD25 +Tregs can suppress autoreactive T cells. In some examples, an effective amount of ICBP is an amount that, when administered in one or more doses to an individual in need thereof, increases the number of Tregs in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, or more than 2.5-fold compared to the number of Tregs in the individual prior to treatment with ICBP.

[0141] In some examples, an effective amount of ICBP is an amount that, when administered in one or more doses to an individual in need thereof, increases the number of tolerogenic dendritic cells (DCs) in the individual. In some examples, an effective amount of ICBP is an amount that, when administered in one or more doses to an individual in need thereof, increases the number of tolerogenic DCs in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, or more than 2.5-fold compared to the number of tolerogenic DCs in the individual prior to treatment with ICBP. Tolerogenic DCs can be identified by known characteristics, such as lower expression of MHC class II polypeptides compared to mature DCs, lower expression of CD80 / CD86 and CD40 compared to mature DCs, increased production of anti-inflammatory molecules such as IL-10 and TGF-β, and decreased production of IL-12p70 and other pro-inflammatory cytokines.

[0142] Treatment method In some examples, the methods of the disclosure induce immune tolerance in an individual, thereby treating an autoimmune disease. Thus, the disclosure provides a method of treating an autoimmune disease in an individual, the method comprising administering to the individual an effective amount of ICBP (or a protein comprising two ICBPs).

[0143] In some instances, an "effective amount" of an ICBP is an amount that, when administered in one or more doses to an individual in need thereof, ameliorates one or more symptoms associated with an autoimmune disease in the individual. In some instances, an ICBP inhibits the activation of CD4 + The number of autoreactive T cells (i.e., CD4 + T cell numbers), which in turn reduces the number of CD8 + This results in a reduction in autoreactive T cells. In some cases, ICBP inhibits CD4 + Increases the number of Tregs, which in turn increases the number of CD4 + Autoreactive T cells and / or CD8 + Reduces the number of autoreactive T cells.

[0144] Autoimmune diseases that may potentially be treated using the methods of the present disclosure include type 1 diabetes (T1D), Addison disease (autoimmune adrenalitis, Morbus Addison), alopecia areata, Addison's anemia (Bielmer's disease, Morbus Addison), and the like. Biermer), autoimmune hemolytic anemia (AIHA), cold autoimmune hemolytic anemia (AIHA) (cold hemagglutinin disease, cold autoimmune hemolytic anemia (AIHA) (cold agglutinin disease, (CHAD)), warm autoimmune hemolytic anemia (AIHA) (warm AIHA, warm autoimmune hemolytic anemia (AIHA)), autoimmune hemolytic Donath-Landsteiner anemia (paroxysmal cold hemoglobinuria), antiphospholipid syndrome (APS), atherosclerosis, autoimmune arthritis, temporal arteritis, Takayasu's arteritis (Takayasu's disease, aortic arch disease), temporal arteritis / giant cell arteritis, autoimmune chronic gastritis, autoimmune infertility, autoimmune inner ear disease (AIED), Graves' disease (Basedow's disease) (Graves' disease (Morbus Basedow), Bechterew's disease (Bechterew's disease (Morbus Behcet), ankylosing spondylitis, ankylosing spondylosis), Behcet's syndrome (Behcet's disease (Morbus Behcet)), intestinal diseases including autoimmune inflammatory bowel disease (ulcerative colitis (including Crohn's disease (Morbus Crohn)), autoimmune cardiomyopathy, idiopathic dilated cardiomyopathy (DCM), celiac disease, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIDP), chronic polyarthritis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, CREST syndrome (syndrome with calcinosis cutis, Raynaud's phenomenon, esophageal dysmotility, sklerodaktylia and telangiectasia), Crohn's disease Crohn's disease (MorbusCrohn), ulcerative colitis), dermatitis herpetiformis, autoimmune skin diseases, dermatomyositis, essential mixed cryoglobulinemia, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Goodpasture's syndrome (anti-GBM mediated glomerulonephritis), graft versus host disease, Guillain-Barré syndrome (GBM, polyradiculitis), autoimmune blood diseases, Hashimoto's thyroiditis, hemophilia, acquired hemophilia, autoimmune hepatitis, idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenic purpura, immune thrombocytopenia (Werlhof's disease, ITP), IgA nephropathy, autoimmune infertility, juvenile rheumatoid arthritis (Still's disease, Still's syndrome), Lambert-Eaton syndrome, systemic lupus erythematosus (SLE), discoid lupus erythematosus, Lyme arthritis (Lyme disease, Borrelia arthritis), Meniere's disease (Morbus Meniere), mixed connective tissue disease (MCTD), multiple sclerosis (MS, disseminated encephalomyelitis, Charcot disease), myasthenia gravis (MG), myositis, polymyositis, neuroautoimmune diseases, pemphigus vulgaris, bullous pemphigoid, polyglandular (autoimmune) syndrome (PGA syndrome, Schmidt syndrome), polymyalgia rheumatica, primary agammaglobulinemia, primary autoimmune cholangitis, progressive systemic sclerosis (PSS), rheumatoid arthritis (RA, chronic polyarthritis, rheumatic diseases of the joints, rheumatic fever), sarcoidosis (Beck's disease (Morbus Boeck), Benier-Beck-Schaumann disease), stiff man syndrome, dermatosclerosis, scleroderma, Sjögren's syndrome, autoimmune uveitis, and Wegner's disease (Wegner's disease (Morbus Wegner's granulomatosis and Wegner's granulomatosis.

[0145] In some examples, the method of the present disclosure induces immune tolerance in an individual, thereby reducing transplant rejection and / or one or more symptoms associated with transplant rejection. Thus, the present disclosure provides a method of inducing immune tolerance to transplanted cells, tissues, or organs in a transplant recipient. Transplanted organs include, for example, skin, kidney, lung, liver, pancreas, and heart. Transplanted tissues include, for example, small intestine, blood vessels, bone, and the like. Transplanted cells include, for example, pancreatic islet cells, allogeneic bone marrow cells, hematopoietic stem cells, and the like.

[0146] In some examples, the methods of the disclosure induce immune tolerance in an individual, thereby reducing one or more symptoms associated with graft-versus-host disease (GVH).

[0147] In some examples, the methods of the disclosure induce immune tolerance in an individual, thereby reducing one or more symptoms associated with metabolic diseases and disorders.

[0148] Metabolism is the chemical process the body uses to convert food into fuel that keeps the body alive. Nutrition (food) consists of proteins, carbohydrates, and fats. These substances are broken down by enzymes in the digestive system and then transported to cells where they can be used as fuel. The body either uses these substances immediately or stores them in the liver, body fat, and muscle tissue for later use. Metabolic disorders, which can be either inherited or acquired, are disorders that interfere with the body's metabolism and can alter the body's processing and distribution of macronutrients such as proteins, fats, and carbohydrates in harmful ways. Metabolic disorders can occur when abnormal chemical reactions in the body alter normal metabolic processes.

[0149] There are hundreds of inherited metabolic disorders caused by genetic defects, including familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, cystic fibrosis, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, sickle cell anemia, Tay-Sachs disease, and Wilson disease.

[0150] Acquired metabolic disorders are metabolic disorders acquired during a person's life and can result from a variety of factors, including obesity. Such disorders include, for example, type 2 diabetes (T2D) and pre-T2D, which can result from insulin resistance and / or insufficient insulin secretion, and non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), a severe form of NAFLD closely related to obesity, pre-T2D and T2D. ICBP can induce immune tolerance in an individual, thereby reducing one or more symptoms associated with metabolic diseases and disorders, such as type 2 diabetes (T2D), which can result from insulin resistance and / or insufficient insulin secretion, and non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH). ICBP can also induce immune tolerance in an individual with one or more metabolic disorders or metabolic dysregulation associated with obesity.

[0151] Dosage The appropriate dosage of ICBP (or a protein containing two ICBPs, e.g., dimerized ICBP) can be determined by the attending physician or other qualified medical personnel based on a variety of clinical factors. As is well known in the medical arts, the dosage for any one patient will depend on many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid being administered, the patient's sex, the duration and route of administration, general health, and other drugs being administered concomitantly. ICBP or dimerized ICBP may be administered in an amount of 0.1 mg / kg body weight to 20 mg / kg body weight per dose, e.g., 0.1 mg / kg body weight to 10 mg / kg body weight, e.g., 0.5 mg / kg body weight to 5 mg / kg body weight, 1 mg / kg body weight to 5 mg / kg body weight, 5 mg / kg body weight to 10 mg / kg body weight, 10 mg / kg body weight to 15 mg / kg body weight, 15 mg / kg body weight to 20 mg / kg body weight, however, doses beyond this exemplary range are envisioned, particularly taking into account the aforementioned factors. If the regimen is a continuous infusion, it may be in the range of 1 μg to 10 mg per kilogram of body weight per minute. ICBP or dimerized ICBP may be administered in an amount of about 1 mg / kg (body weight) to 50 mg / kg (body weight), for example, about 1 mg / kg (body weight) to about 5 mg / kg (body weight), about 5 mg / kg (body weight) to about 10 mg / kg (body weight), about 10 mg / kg (body weight) to about 15 mg / kg (body weight), about 15 mg / kg (body weight) to about 20 mg / kg (body weight), about 20 mg / kg (body weight) to about 25 mg / kg (body weight), about 25 mg / kg (body weight) to about 30 mg / kg (body weight), about 30 mg / kg (body weight) to about 35 mg / kg (body weight), about 35 mg / kg (body weight) to about 40 mg / kg (body weight), or about 40 mg / kg (body weight) to about 50 mg / kg (body weight). Exemplary amounts of ICBP or dimerized ICBP include 1 mg / kg body weight to 5 mg / kg body weight, 5 mg / kg body weight to 10 mg / kg body weight, about 1 mg / kg body weight to about 5 mg / kg body weight, and about 5 mg / kg body weight to about 10 mg / kg body weight.

[0152] One of skill in the art can readily estimate repetition rates of administration based on, for example, the patient's clinical condition, the patient's response to administration of ICBP, and the measured residence time and concentration of the administered agent in bodily fluids or tissues following administration. Following successful treatment, it may be desirable to subject the patient to maintenance therapy to prevent recurrence of the disease state, with the ICBP or dimerized ICBP of the present disclosure being administered at a maintenance dose ranging from about 1 mg / kg body weight to about 5 mg / kg body weight, about 5 mg / kg body weight to about 10 mg / kg body weight, about 10 mg / kg body weight to about 15 mg / kg body weight, about 15 mg / kg body weight to about 20 mg / kg body weight, or greater than 20 mg / kg body weight.

[0153] One of ordinary skill in the art will readily appreciate that dosage levels may vary depending on the particular ICBP or dimerized ICBP, the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for a given compound can be readily determined by those of ordinary skill in the art using a variety of means known to those of ordinary skill in the art.

[0154] In some cases, multiple doses of ICBP or dimerized ICBP are administered over the course of treatment.The frequency of administration of ICBP or dimerized ICBP can vary depending on any of a variety of factors, including those mentioned above.For example, in some cases, ICBP or dimerized ICBP is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, or less frequently than once a week, such as twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), every day (qd), twice a day (qid), or three times a day (tid). When ICBP or dimerized ICBP is administered intravenously, at the initiation of treatment, dosing once a week, once every two weeks, once every three weeks, or once every four weeks, or once a month may generally be used.

[0155] The duration of administration of ICBP or dimerized ICBP, e.g., the period over which ICBP or dimerized ICBP is administered, can vary depending on any of a variety of factors, e.g., the patient's clinical condition and response to ICBP. For example, ICBP or dimerized ICBP can be administered for periods ranging from about 1 day to about 1 week, about 2 weeks to about 4 weeks, 1 month to about 2 months, about 2 months to about 4 months, about 4 months to about 6 months, about 6 months to about 8 months, about 8 months to about 1 year, about 1 year to about 2 years, or about 2 years to about 4 years, or longer, including periodic administration for the patient's lifetime.

[0156] Route of administration ICBP or dimerized ICBP is administered to an individual using any available method and route suitable for drug delivery, including in vivo and in vitro methods, and systemic and local routes of administration. ICBP or dimerized ICBP of the present disclosure will typically be delivered by intravenous administration, although other conventional pharmacologic acceptable routes of administration may be used, including intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, subcutaneous, intradermal, topical application, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration.

[0157] In some examples, ICBP or dimerized ICBP is administered intravenously. In some examples, ICBP or dimerized ICBP is administered intramuscularly. In some examples, ICBP or dimerized ICBP is administered subcutaneously.

[0158] Suitable subjects for treatment Subjects suitable for treatment using the methods of the present disclosure include individuals with autoimmune disease, including individuals diagnosed with a condition that could benefit from increased immune tolerance, including, for example, an autoimmune disorder, a metabolic disorder, transplant, or GVHD, individuals who have been treated for such a condition but have not responded to the treatment, and individuals who have symptoms indicating the impending onset of one of such conditions.

[0159] For example, subjects suitable for treatment using the method of the present disclosure include individuals with T1D, including individuals who have been diagnosed with T1D or T2D, and individuals who have been treated for T1D or T2D but have not responded to the treatment.Suitable subjects may also include individuals who have been diagnosed as likely to develop T1D or T2D, or individuals who have symptoms indicating the impending onset of T1D or T2D.Suitable subjects for treatment using the method of the present disclosure include individuals with T1D or T2D, including individuals who have been diagnosed with celiac disease, and individuals who have been treated for celiac disease but have not responded to the treatment.Suitable subjects may also include individuals who have been diagnosed as likely to develop celiac disease, or individuals who have symptoms indicating the impending onset of celiac disease.

[0160] Other subjects suitable for treatment with the methods of the present disclosure include transplant recipients and individuals who are soon to become transplant recipients (e.g., individuals who are scheduled to receive a transplant within the next month or within the next week).

[0161] Other subjects suitable for treatment with the methods of the present disclosure include bone marrow transplant recipients and individuals who are soon to become bone marrow transplant recipients (e.g., individuals scheduled to receive a bone marrow transplant within the next month or within the next week).

[0162] Examples of Non-Limiting Aspects of the Disclosure Aspects including embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the disclosure are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or subsequent individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0163] Embodiment 1. An immune cell binding polypeptide (ICBP) comprising: (a) a binding moiety comprising an HLA-G molecule and a stabilizing peptide; (b) optionally at least one immunosuppressive polypeptide; (c) a scaffold polypeptide; and (d) optionally one or more independently selected linkers linking two or more of the components of the ICBP, wherein the HLA-G molecule comprises an HLA-G heavy chain polypeptide and a β2M polypeptide, the binding moiety is capable of binding to an ILT2 and / or ILT4 polypeptide on an immune cell, and the ICBP is capable of inhibiting activity of the immune cell.

[0164] Embodiment 2. The ICBP of embodiment 1, wherein the scaffold polypeptide is an immunoglobulin (Ig) Fc polypeptide.

[0165] Embodiment 3. The ICBP according to embodiment 2, wherein the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially induce cell lysis, and optionally the IgG1 Fc polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to the IgG1 Fc amino acid sequence depicted in FIG. 2A.

[0166] Embodiment 4. An ICBP according to embodiment 3, wherein the IgG1 Fc polypeptide comprises one or more amino acid substitutions selected from N77A, L14A, L15A, L14F, L15E, and P101S based on the numbering of the IgG1 Fc amino acid sequence shown in Figure 2A.

[0167] Embodiment 5. An ICBP according to embodiment 4, wherein the IgG1 Fc polypeptide comprises the L14A and L15A amino acid substitutions based on the numbering of the Ig Fc amino acid sequence shown in Figure 2A.

[0168] Embodiment 6. An ICBP according to any one of embodiments 1 to 5, wherein the β2M polypeptide and the HLA-G heavy chain polypeptide are linked by a disulfide bond linking a Cys residue in the β2M polypeptide to a Cys residue in the HLA-G heavy chain polypeptide.

[0169] Embodiment 7. The ICBP according to embodiment 6, wherein a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the HLA-G heavy chain polypeptide.

[0170] Embodiment 8. An ICBP according to any one of embodiments 1 to 7, wherein the β2-microglobulin polypeptide is linked to the stabilizing peptide by a first linker comprising a Cys, and a disulfide bond connects a Cys present in the first linker with a Cys present in the HLA-G heavy chain polypeptide.

[0171] Embodiment 9. An ICBP according to embodiment 8, wherein the first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 146) or GCGGS(GGGGS)n (SEQ ID NO: 144), in which n is an integer from 1 to 10, e.g., 2 or 3, and a disulfide bond connects a Cys in the linker with the Cys substituted for Tyr84 of the HLA-G heavy chain polypeptide.

[0172] Embodiment 10. An ICBP according to any one of embodiments 1 to 9, wherein the HLA-G heavy chain polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to an HLA-G heavy chain polypeptide depicted in any one of Figures 4A to 4F, and optionally wherein the HLA-G heavy chain polypeptide comprises a C42S amino acid substitution.

[0173] Embodiment 11. An ICBP according to any one of embodiments 1 to 10, wherein the ICBP comprises at least one immunoinhibitory polypeptide capable of binding to an immune cell and mediating inhibition of the immune cell, optionally wherein the one or more immunoinhibitory polypeptides are selected from a PD-L1 polypeptide, a FasL polypeptide, an IL-10 polypeptide, an IL-35 polypeptide, a TIGIT ligand, a Lag3 ligand, and combinations thereof.

[0174] Embodiment 12. An ICBP according to any one of embodiments 1 to 11, wherein at least one of the one or more immunoinhibitory polypeptides binds to its cognate co-receptor and exhibits reduced affinity for said cognate co-receptor compared to the affinity of a corresponding wild-type immunoinhibitory polypeptide for its cognate co-receptor polypeptide.

[0175] Embodiment 13. The ICBP according to any one of Embodiments 1 to 12, wherein at least one of the one or more immunoinhibitory polypeptides is a PD-L1 polypeptide, and optionally the immunoinhibitory polypeptide comprises a PD-L1 polypeptide extracellular domain.

[0176] Embodiment 14. The ICBP of Embodiment 13, wherein the PD-L1 polypeptide comprises an amino acid sequence that has at least 95% amino acid sequence identity to the PD-L1 amino acid sequence shown in Figure 11A or Figure 11C, and has a length of about 220 amino acids.

[0177] Embodiment 15. An ICBP according to any one of embodiments 1 to 14, which is a single polypeptide chain.

[0178] Embodiment 16. An ICBP according to any one of embodiments 1 to 14, which is a heterodimeric polypeptide.

[0179] Embodiment 17. A β2-microglobulin polypeptide is linked to a stabilizing peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO: 146) or GCGGS(GGGGS)n (SEQ ID NO: 144), where n is an integer between 1 and 10, e.g., 2 or 3; the HLA-G polypeptide comprises a Cys at residue 84 and a Cys at residue 236; the β2M polypeptide comprises a Cys at residue 12, where the Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to the Cys at amino acid residue 236 of the HLA-G polypeptide, and a disulfide bond connects the Cys in the linker with the Cys substituted for Tyr84 of the HLA-G polypeptide; the HLA-G polypeptide comprises a serine at amino acid residue 42; the at least one immunosuppressive polypeptide is a PD-L1 polypeptide; and the Ig Fc polypeptide is an IgG1 polypeptide comprising L14A and L15A substitutions. 17. The ICBP according to any one of aspects 1 to 16, which is an Fc polypeptide.

[0180] Embodiment 18. An ICBP according to any one of embodiments 1 to 10, wherein the ICBP comprises (a) a binding moiety comprising an HLA-G molecule and a stabilising peptide, and (b) a scaffold polypeptide, but not an immunosuppressive polypeptide, wherein the HLA-G molecule comprises an HLA-G heavy chain polypeptide and a β2M polypeptide, and wherein the binding moiety is capable of binding to an ILT2 and / or ILT4 polypeptide on an immune cell.

[0181] Embodiment 19. A dimer comprising two ICBPs according to any one of embodiments 1 to 18, optionally wherein the dimer is a homodimer of two ICBPs having the same amino acid sequence, and wherein the two polypeptides of the homodimer are linked to each other by one or more disulfide bonds linking a scaffold polypeptide of one polypeptide to a scaffold polypeptide of the other polypeptide.

[0182] Aspect 20. A pharmaceutical composition comprising an ICBP according to any one of aspects 1 to 18.

[0183] Embodiment 21. A pharmaceutical composition comprising the dimer according to embodiment 19.

[0184] A method for producing an ICBP or a dimer according to Aspect 19, comprising culturing in vitro a host cell genetically modified with one or more nucleic acids comprising a nucleotide sequence encoding one or more polypeptides of an ICBP according to any one of Aspects 1 to 18, wherein the genetically modified host cell produces one or more polypeptides of ICBP.

[0185] Embodiment 23. A method for inducing immune tolerance in an individual, the method comprising administering to the individual an ICBP according to any one of embodiments 1 to 18, a dimer according to embodiment 19 or a pharmaceutical composition according to embodiment 20 or embodiment 21.

[0186] Embodiment 24 The method of embodiment 23, wherein the individual has an autoimmune disease.

[0187] Embodiment 25 The method of embodiment 23, wherein the individual is a transplant recipient.

[0188] Embodiment 26 The method of embodiment 23, wherein the individual has graft-versus-host (GVH) disease.

[0189] Embodiment 27. The method of embodiment 23, wherein the individual has a metabolic disorder, such as T2D and / or non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH).

[0190] Embodiment 28 The method according to any one of embodiments 23 to 27, wherein said administering increases the number of regulatory T cells in the individual.

[0191] Embodiment 29 The method according to any one of embodiments 23 to 27, wherein said administering increases the number of tolerogenic dendritic cells in the individual.

[0192] Aspect 30 The method of any one of Aspects 23 to 27, wherein said administering reduces inflammatory immune cell activity in the individual.

[0193] Embodiment 31 The method of any one of embodiments 23 to 27, wherein said administering reduces the level of a pro-inflammatory cytokine in the individual. EXAMPLES

[0194] The following examples are presented to provide those of skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of the invention as contemplated by the inventors, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobase; bp, base pairs; nt, nucleotides; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; and the like.

[0195] Example 1 Two constructs, designated "4601+4602" and "4607+4602", were generated. Polypeptide chain 4601 comprises an HLA-G polypeptide with C42S, Y84C and A236C substitutions, an AAAGG (SEQ ID NO:28) linker, and an IgG1 Fc polypeptide with L14A and L15A substitutions. Polypeptide chain 4607 comprises a PD-L1 polypeptide, a (GGGGS)4 (SEQ ID NO:27) linker, an HLA-G polypeptide with C42S, Y84C and A236C substitutions, an AAAGG (SEQ ID NO:28) linker, and an IgG1 Fc polypeptide with L14A and L15A substitutions. Polypeptide chain 4602 comprises a histone 2A peptide (RIIPRHLQL, SEQ ID NO:24), a linker with the amino acid sequence GCGGSGGGSGGGGS (SEQ ID NO:25), and a β2M polypeptide with an R12C substitution. The amino acid sequence of the 4601+4602 polypeptide chain is provided in Figure 6A. The amino acid sequence of the 4607+4602 polypeptide chain is provided in Figure 6B.

[0196] As described above, each of these heterodimeric ICBPs will form an interchain disulfide bond (i) between a Cys at residue 84 of the HLA-G heavy chain and a Cys in the linker that connects the stabilizing peptide RIIPRHLQL (SEQ ID NO:24) to the β2M polypeptide, and (ii) between a Cys at residue 236 of the HLA-G heavy chain and a Cys residue at amino acid residue 12 of β2M. Upon expression, ICBP construct 4601+4602 will spontaneously dimerize to form a homodimer in which a disulfide bond links the IgG1 Fc polypeptide of one ICBP to the IgG1 Fc of the other ICBP. Similarly, upon expression, ICBP construct 4607+4602 will also spontaneously dimerize to form a homodimer in which a disulfide bond links the IgG1 Fc polypeptide of one ICBP to the IgG1 Fc of the other ICBP.

[0197] The binding of the 4601+4602 and 4607+4602 constructs to immobilized ILT2, ILT4 and PD1 was tested. As shown in Figure 7, both 4601+4602 and 4607+4602 bind to both ILT2 and ILT4, but only 4607+4602 (and not 4601+4602) binds to PD1.

[0198] Although the present invention has been described with reference to specific embodiments in this disclosure, those skilled in the art will recognize that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications can be made to particular conditions, materials, compositions of matter, processes, and process steps to adapt to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. An immune cell-binding polypeptide (ICBP), wherein the ICBP is (a) A binding site comprising an HLA-G molecule and a stabilizing peptide, (b) At least one immunosuppressive polypeptide selected from PD-L1 polypeptide, FasL polypeptide, IL-10 polypeptide, IL-35 polypeptide, TIGIT ligand, and Lag3 ligand, (c) Scaffolding polypeptide and Includes, The HLA-G molecule comprises an HLA-G heavy chain polypeptide and a β2M polypeptide. The binding portion can bind to ILT2 and / or ILT4 polypeptides on immune cells, The stabilizing peptide of the binding site does not contribute to the binding between the binding site and the ILT2 or ILT4 polypeptide on the immune cell, and The ICBP can suppress the activity of the immune cells. The aforementioned immune cell-binding polypeptide (ICBP).

2. The ICBP according to claim 1, wherein the scaffold polypeptide is an immunoglobulin (Ig) Fc polypeptide.

3. The ICBP according to claim 2, wherein the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially induce cell lysis, and optionally the IgG1 Fc polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity with the IgG1 Fc amino acid sequence shown in SEQ ID NO:

3.

4. The ICBP according to claim 3, wherein the IgG1Fc polypeptide comprises one or more amino acid substitutions selected from N77A, L14A, L15A, L14F, L15E, and P101S based on the numbering of the IgG1Fc amino acid sequence shown in SEQ ID NO:

3.

5. The ICBP according to claim 4, wherein the IgG1 Fc polypeptide includes L14A and L15A amino acid substitutions based on the numbering of the Ig Fc amino acid sequence shown in SEQ ID NO:

3.

6. The ICBP according to claim 2, wherein the β2M polypeptide and the HLA-G heavy chain polypeptide are linked by a disulfide bond that connects a Cys residue in the β2M polypeptide to a Cys residue in the HLA-G heavy chain polypeptide.

7. The ICBP according to claim 6, wherein the Cys at amino acid residue 12 of the β2M polypeptide is disulfide-bonded to the Cys at amino acid residue 236 of the HLA-G heavy chain polypeptide.

8. The ICBP according to claim 5, wherein the β2 microglobulin polypeptide is bound to the stabilizing peptide by a first linker containing Cys, and the disulfide bond links the Cys present in the first linker to the Cys present in the HLA-G heavy chain polypeptide.

9. The ICBP according to claim 8, wherein the first linker comprises the sequence CGGGS(GGGGGS)n (SEQ ID NO: 146) or GCGGS(GGGGGS)n (SEQ ID NO: 144), where n is an integer from 1 to 10, and the disulfide bond links the Cys in the linker to the Cys substituted for Tyr84 of the HLA-G heavy chain polypeptide.

10. The ICBP according to claim 5, wherein the HLA-G heavy chain polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity with respect to the HLA-G heavy chain polypeptide shown in any one of SEQ ID NOs: 17 to 22.

11. The ICBP according to claim 10, wherein the HLA-G heavy chain polypeptide comprises a C42S amino acid substitution.

12. The ICBP according to claim 10, wherein at least one of the one or more immunosuppressive polypeptides binds to its homogeneous coreceptor and exhibits a reduced affinity for the homogeneous coreceptor compared to the affinity of the corresponding wild-type immunosuppressive polypeptide to the homogeneous coreceptor polypeptide.

13. The ICBP according to claim 5, wherein at least one of the one or more immunosuppressive polypeptides is a PD-L1 polypeptide, and optionally the immunosuppressive polypeptide comprises the extracellular domain of the PD-L1 polypeptide.

14. The ICBP according to claim 13, wherein the immunosuppressive polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity with respect to the PD-L1 amino acid sequence shown in SEQ ID NO: 34 or 54, and having a length of about 220 amino acids.

15. The ICBP according to claim 5, which is a single polypeptide chain.

16. The ICBP according to claim 5, which is a heterodimeric polypeptide.

17. The β2-microglobulin polypeptide is linked to the stabilizing peptide by a first linker containing the sequence CGGGS(GGGGGS)n (SEQ ID NO: 146) or GCGGS(GGGGGS)n (SEQ ID NO: 144), where n is an integer from 1 to 10. The HLA-G polypeptide contains Cys at residue 84 and Cys at residue 236. The β2M polypeptide contains Cys at residue 12, The Cys at amino acid residue 12 of the β2M polypeptide is disulfide-bonded to the Cys at amino acid residue 236 of the HLA-G polypeptide. The disulfide bond connects the Cys in the linker to the Cys substituted for Tyr84 in the HLA-G polypeptide. The HLA-G polypeptide contains serine at amino acid residue 42, The at least one immunosuppressive polypeptide is a PD-L1 polypeptide, The IgFc polypeptide is an IgG1Fc polypeptide containing L14A and L15A substitutions. The ICBP according to claim 5.

18. A homodimer comprising two ICBPs as described in claim 17, wherein the two polypeptides are linked to each other by one or more disulfide bonds that connect the scaffold polypeptide of one polypeptide to the scaffold polypeptide of the other polypeptide.

19. A pharmaceutical composition comprising the ICBP according to claim 18 for inducing immune tolerance in an individual.

20. A pharmaceutical composition comprising the homodimer described in claim 18.

Images