PD-L1 variant with improved affinity for PD-1

Abstract

The present invention relates to PD-L1 polypeptides comprising at least a first amino acid sequence that is at least 70% identical to SEQ ID NO: 8 and a second sequence that is at least 70% identical to SEQ ID NO: 10, wherein the polypeptide carries amino acid substitutions at least at amino acid positions Y56 and P76, which amino acid positions are based on the murine PD-L1 amino acid sequence (SEQ ID NO: 6). The present invention also relates to polynucleotides encoding the above PD-L1 polypeptides, and host cells, methods, and uses related thereto.

Description

[Technical Field]

[0001] The present invention relates to a PD-L1 polypeptide comprising at least a first amino acid sequence that is at least 70% identical to SEQ ID NO: 8, and a second sequence that is at least 70% identical to SEQ ID NO: 10, wherein the polypeptide carries amino acid substitutions at least at amino acid positions Y56 and P76, where the amino acid positions are based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6). The present invention also relates to a polynucleotide encoding the above PD-L1 polypeptide, as well as related host cells, methods, and uses. [Background technology]

[0002] Sepsis is a life-threatening illness that can occur when the entire body reacts to infection. Despite extensive research, sepsis remains the third leading cause of death in intensive care units. Pathophysiologically, sepsis progresses through an initial hyper-inflammatory phase, which in turn leads to the development of a hypo-inflammatory phase that occurs in part in parallel (Vincent, et al. (2013) Lancet 381:774-775).

[0003] Recent therapeutic approaches have primarily focused on treating the hyperinflammatory response by limiting the release of inflammatory mediators, blocking their function, or removing them from circulation. One of the most promising candidates, whose inhibition has been shown to significantly improve sepsis survival in rodent models, was TNFα. Using neutralizing antibodies, this approach was adapted for humans but failed to improve sepsis survival (Reinhart et al. (2001) Crit Care Med 29:765-769). By blocking immune stimulation, hyperinflammation is limited, and the majority of patients survive this period; however, by blocking the pro-inflammatory immune response, the host's ability to fight and control primary and secondary infections is reduced, so this therapeutic approach ultimately failed to significantly improve sepsis survival, although it did induce or promote a low-inflammatory phase. This immunosuppression often leads to multiple organ failure syndrome (MODS) and patient death (Otto et al. (2011) Crit Care 15:R183). In a different approach, an Fc-fusion protein of the extracellular portion of the human PD-L1 polypeptide has been offered for the treatment and prevention of organ failure during sepsis (International Publication No. 2017 / 029389A1).

[0004] Therapeutic approaches to rescue patients with immunoparalysis have also been applied. Considering the inactivation of monocytes, GM-CSF therapy restored monocyte function during sepsis (Meisel et al. (2009) Am J Respir Crit Care Med 180:640-648). Nevertheless, due to the multiple causes of sepsis, various pre-existing complications, or the patient's genetic predispositions, appropriate patient-specific treatment remains difficult to achieve (Hotchkiss and Opal (2010) N Engl J Med 363:87-89).

[0005] Generally, disease severity is quite advanced by the time sepsis is diagnosed in a patient, with liver damage, a relatively late event in the progression of sepsis, already occurring. During sepsis, organ failure, often followed by multiple organ failure syndrome (MODS), frequently leads to patient death. Therefore, understanding the mechanisms leading to organ damage is essential to improving existing treatment options or proposing novel therapeutic approaches.

[0006] The process that leads to organ damage in sepsis is the undesirable autoimmune activation of cytotoxic T cells (CTLs) that initiate an attack against host cells. Cytotoxic T cell activation is a tightly regulated process requiring the simultaneous occurrence of multiple signaling events. In a healthy state, binding events at immune synapses, such as the T cell receptor (TCR) binding to major histocompatibility complex 1 (MHC-1) or programmed cell death-1 (PD-1) binding to PD-1 ligand 1 (PD-L1) on epithelial cells, regulate CTL activation. However, during infection that triggers an inflammatory response by CTLs, epithelial cells undergo several types of changes. For example, the presentation of non-self peptides to the MHC-I protein on infected cells activates CTLs, which leads to the rapid destruction of infected tissue. Another example of CTL activation is the absence of PD-L1 on the surface of host cells, which is triggered among other factors by the presence of bacterial toxins in their environment. Through recognition of Toll-like receptors, these bacterial toxins induce the expression of phagocytic NADPH oxidase (NOX2), which leads to an increase in reactive oxygen species (ROS) levels in the cytoplasm and the removal of PD-L1 from the cell surface (von Knethen et al. (2019), Theranostics 9(7):2003). While both of these processes are essential for eliminating infected cells during normal infection, PD-L1 deficiency in particular becomes problematic during massive infections of large organs or bacterial infections of large portions of body tissue. Subsequently, the organ is attacked by the patient's own immune response without infection, which leads to permanent damage or, in the worst case, organ failure and death. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2017 / 029389A1 [Non-patent literature]

[0008] [Non-Patent Document 1] Vincent, et al. (2013) Lancet 381:774-775 [Non-Patent Document 2] Reinhart et al. (2001) Crit Care Med 29:765-769 [Non-Patent Document 3] Otto et al. (2011) Crit Care 15:R183 [Non-Patent Document 4] Meisel et al. (2009) Am J Respir Crit Care Med 180:640-648 [Non-Patent Document 5] Hotchkiss and Opal (2010) N Engl J Med 363:87-89 [Non-Patent Document 6] von Knethen et al. (2019), Theranostics 9(7):2003 [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, there is a strong need for improved means and methods for modulating PD-1 signaling, particularly on the surface of CTLs, for example, to treat and / or prevent organ failure, especially during sepsis.

[0010] The underlying technical problem of the present invention can be seen as providing means and methods to address the aforementioned needs. The technical problem is solved by embodiments characterized by the claims and embodiments described below herein. [Means for solving the problem]

[0011] Therefore, the present invention relates to a PD-L1 polypeptide comprising at least a first amino acid sequence that is at least 70% identical to SEQ ID NO: 8, and a second sequence that is at least 70% identical to SEQ ID NO: 10, wherein, (I) The polypeptide carries an amino acid substitution at at least one of the following positions: V54, Y56, Q63, Q66, V68, A69, P76, I115, A121, D122, Y123, K124, and R125; if the polypeptide contains an amino acid substitution at position Y56, C113, or I115, the polypeptide further carries at least one of the above substitutions; where the amino acid positions are based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6); (II) The polypeptide carries amino acid substitutions at least at amino acid positions Y56 and P76, where the amino acid positions are based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6); and / or (III) The polypeptide carries at least one amino acid substitution at at least one of the following amino acid positions in the first amino acid sequence: V54, Y56, Q63, Q66, V68, A69, P76; and at least one amino acid substitution at at least one of the following amino acid positions in the second amino acid sequence: I115, A121, D122, Y123, K124, and R125, where the amino acid positions are based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6).

[0012] In general, terms used herein are given their usual and customary meanings to those skilled in the art, and are not limited to special or exclusive meanings unless otherwise indicated. Where used below, the terms “have,” “comprise,” or “include,” or any grammatical variation thereof, are used in a non-exclusive manner. That is, these terms can mean both a situation in which no further features exist in the entity described in this context, in addition to the features introduced by these terms, and a situation in which one or more further features exist. For example, the expressions “A has B,” “A includes B,” and “A is listed as B” can mean both a situation in which no other elements exist in A besides B (i.e., A consists solely and exclusively of B), and a situation in which one or more further elements exist in entity A besides B, such as elements C, elements C and D, or other further elements. Furthermore, as will be understood by those skilled in the art, the expressions “comprising a” and “comprising an” preferably mean “comprising one or more,” that is, equivalent to “comprising at least one.”

[0013] Furthermore, where used below, the terms “preferably,” “more preferably,” “most preferably,” “particularly,” “more specifically,” “specifically,” “more specifically,” or similar terms are used in conjunction with optional features without limiting further possibilities. That is, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. The present invention can be carried out by using alternative features, as those skilled in the art will recognize. Similarly, features introduced by “in one embodiment” or similar expressions are intended to be optional features without any limitation on further embodiments of the present invention, without any limitation on the scope of the present invention, and without any limitation on the possibility of combining the features thus introduced with other optional or non-optional features of the present invention.

[0014] As used herein, the term "standard conditions" relates to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e., preferably, a temperature of 25 °C and an absolute pressure of 100 kPa, when not otherwise specified; also preferably, standard conditions include a pH of 7. Further, when not otherwise indicated, the term "about" relates to the indicated value with the normal technical accuracy acceptable in the relevant field, preferably, the indicated value ±20%, more preferably ±10%, most preferably ±5%. Further, the term "essentially" indicates that there is no deviation having an impact on the indicated result or use, i.e., the possible deviation does not cause the indicated result to deviate by more than ±20%, more preferably ±10%, most preferably ±5%. That is, "consisting essentially of" means excluding other components except for substances present as impurities, inevitable substances present as a result of the process used to provide the components, and components added for purposes other than achieving the technical effects of the present invention, while including the specified components. For example, a composition defined using the phrase "consisting essentially of" includes any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of a set of components will contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of unspecified components.

[0015] The degree of identity (e.g., expressed as "% identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences across a comparison window, where the sequence fragments in the comparison window may contain additions or deletions (e.g., gaps or overhangs) when compared to the sequence being compared for optimal alignment. The percentage is preferably calculated by determining the number of positions in both sequences where identical residues exist across the entire length of the polynucleotide or polypeptide, thereby obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), by computer implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Assuming that two sequences have been identified for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment and, therefore, the degree of identity. Preferably, default values ​​of 5.00 for gap weights and 0.30 for gap weight lengths are used. In the context of biological sequences as referred to herein, the term “essentially identical” refers to a percentage identity value of at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99%. As will be understood, the term “essentially identical” includes 100% identity.The above applies mutatis mutandis to the term "essentially complementary."

[0016] The term “fragment” of a biological macromolecule, preferably a polynucleotide or polypeptide, is used herein in a broad sense relating to any sub-part, preferably sub-domain, of each biological macromolecule, including the sequence, structure, and / or function shown. That is, the term includes sub-parts produced by the actual fragmentation of a biological macromolecule, but also includes, in an abstract form, sub-parts derived from each biological macromolecule, for example, in silico. In other words, as used herein, Fc or Fab fragments, such as single-chain antibodies, bispecific antibodies, and nanobodies, can also be referred to as immunoglobulin fragments.

[0017] Unless specifically indicated otherwise herein, the compounds identified, particularly the PD-L1 polypeptides, can be included in a larger structure and can be covalently or non-covalently linked, for example, to carrier molecules, retardants, and other excipients. In particular, the polypeptides identified can be included in fusion polypeptides containing additional peptides that can function, for example, as tags for purification and / or detection, can function as linkers, or can extend the in vivo half-life of the compound. The term "detectable tag" means an extension of amino acids added or introduced to a fusion polypeptide; preferably, the tag is added to the C-terminus or N-terminus of the fusion polypeptide of the present invention. Such an extension of amino acids preferably enables the detection of the fusion polypeptide by an antibody that specifically recognizes the tag; or preferably enables the formation of a functional conformation, such as a chelator; or preferably enables visualization, for example, in the case of a fluorescent tag. Preferred detectable tags are Myc tag, FLAG tag, 6-His tag, HA tag, GST tag, or fluorescent protein tags, such as GFP tag. All of these tags are well known in the art. Other additional peptides preferably included in the fusion polypeptide can contain additional amino acids or other modifications that can function as mediators of secretion, mediators of blood-brain barrier passage, cell-penetrating peptides, and / or immunostimulants. Further polypeptides or peptides to which the polypeptide can be fused are signal and / or transport sequences, such as the IL-2 signal sequence, linker sequences, such as the GSRS (SEQ ID NO: 25) peptide linker.

[0018] As used herein, the term "polypeptide" refers to a molecule consisting of several, typically at least 20, amino acids covalently linked to one another by peptide bonds. Molecules consisting of fewer than 20 amino acids covalently linked by peptide bonds are generally considered to be "peptides." Preferably, polypeptides contain 50 to 1000, more preferably 75 to 1000, even more preferably 100 to 500, and most preferably 110 to 400 amino acids. Preferably, polypeptides are included in fusion polypeptides and / or polypeptide complexes.

[0019] As used in this description, “fusion polypeptide” means a polypeptide composed of at least two polypeptides or peptides that are included in a continuous chain of peptide bonds. That is, a fusion polypeptide is expressible in vivo from a single expression construct, more preferably from a single open reading frame. Preferably, the fusion protein comprises at least the PD-L1 polypeptide and at least one further peptide or polypeptide, preferably a polypeptide that extends the in vivo half-life of the fusion polypeptide in which it is included; preferred polypeptides are described elsewhere in this specification. However, a fusion polypeptide may include two, three, four, five or more additional polypeptides or peptide moieties, such as signal sequences, linkers, hinge regions, and / or polypeptides that extend the in vivo half-life. A fusion polypeptide can be expressed in vivo from a polynucleotide encoding the fusion polypeptide, which can be synthesized, for example, chemically or by recombinant DNA technology and expressed in a suitable expression system. The expressed fusion polypeptide can then be purified from the expression system.

[0020] The term “polypeptide complex,” as used herein, refers to any compound comprising at least two polypeptides and / or peptides that are not linked via peptide bonds. That is, the polypeptides and / or peptides in a fusion polypeptide can be linked covalently, particularly via disulfide bonds, or non-covalently, particularly via ionic bonds, hydrogen bonds, and / or van der Waals forces, for example, by affinity bonds. Various affinity-binding systems, including ligand and receptor moieties, are known to those skilled in the art, enabling the construction of affinity pairs of polypeptides containing different peptide or polypeptide moieties reversibly linked to each other without further complications. Typical examples of such affinity-binding systems are antibody / antigen, streptavidin / biotin, avidin / biotin, and others well known in the art. Preferably, the polypeptide complex comprises at least a PD-L1 polypeptide and at least one further peptide or polypeptide, preferably a polypeptide that extends the in vivo half-life of the fusion polypeptide in which it is contained; preferred polypeptides are described elsewhere herein.

[0021] The term “PD-L1 polypeptide,” as used herein, refers to a polypeptide comprising at least a first amino acid sequence that is at least 70% identical to SEQ ID NO: 8, and a second sequence that is at least 70% identical to SEQ ID NO: 10; further, the PD-L1 polypeptide comprises at least one of the amino acid substitutions or a combination of amino acid substitutions compared to the sequence of SEQ ID NO: 6 shown above. As those skilled in the art will understand, the term “substitution” in the context of amino acid sequences refers to the substitution of amino acids with non-identical amino acids. As those skilled in the art will understand, the underlying structures of the PD-L1 polypeptides referenced herein are preferably derived from known structures of PD-L1 proteins, preferably human PD-L1 protein (Genbank registration number NP_054862.1, SEQ ID NO: 26) and / or mouse PD-L1 protein (Genbank registration number ADK70950.1, SEQ ID NO: 6, preferably encoded by the sequence of SEQ ID NO: 3). Thus, all indications of amino acid positions within the PD-L1 polypeptide are provided with respect to the amino acid sequence of mouse PD-L1 (SEQ ID NO: 6) identified above. As those skilled in the art will also understand, the corresponding positions in the PD-L1 polypeptide having more or fewer amino acids are preferably determined by aligning the PD-L1 polypeptide having SEQ ID NO: 6 as specified above herein. The PD-L1 polypeptide has biological activity to bind to PD-1, preferably human PD-1 (Genbank registration number NP_005009.2) and / or mouse PD-1 (Genbank registration number NP_032824.1). Preferably, the PD-L1 polypeptide has biological activity to bind to a recombinant fragment of mouse PD-1 containing amino acids 31-150 of the complete protein. Preferably, the Kd of binding between the PD-L1 polypeptide and PD-1, preferably the recombinant fragment of mouse PD-L1, is up to 50 nM, more preferably up to 20 nM, even more preferably up to 10 nM, even more preferably up to 7.5 nM, even more preferably up to 5 nM, and most preferably up to 2.5 nM.As disclosed in more detail herein by the Examples, for example, the presence of further polypeptides in the fusion polypeptide may affect the affinity of the PD-L1 polypeptide; that is, the above Kd value is preferably determined with respect to amino acids 18-132 of wild-type PD-L1, preferably the peptide corresponding to SEQ ID NO: 4. Preferably, the PD-L1 polypeptide further has activity to inhibit cytotoxic T cells induced by sepsis; also preferably, the PD-L1 polypeptide further has activity to induce long-term tolerance in cytotoxic T cells in the subject to activation caused by sepsis. In preferred embodiments, the PD-L1 polypeptide has activity to reduce, and more preferably inhibit, IFN-γ and / or TNF-α secretion by leukocytes, preferably T cells. In preferred embodiments, the PD-L1 polypeptide is active in T cells, preferably CD4. + and / or CD8 + It has the activity to reduce, and more preferably inhibit, IFN-γ secretion by T cells and / or TNF-α secretion by macrophages. In a further preferred embodiment, the PD-L1 polypeptide is a PD-1 agonist, preferably a PD-1 agonist having increased affinity for PD-1 compared to wild-type PD-L1.

[0022] The PD-L1 polypeptide comprises at least the first and second amino acid sequences identified above. Preferably, the first amino acid sequence is at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO: 8, and / or the second amino acid sequence is at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO: 10. Most preferably, the PD-L1 polypeptide comprises at least the first amino acid sequence selected from SEQ ID NO: 7 and SEQ ID NO: 8, and the second sequence selected from SEQ ID NO: 9 and SEQ ID NO: 10, comprising one or more amino acid substitutions identified herein. As will be understood, the above first and second amino acid sequences are derivable from human and / or mouse PD-L1 proteins. In other words, the PD-L1 polypeptide preferably includes a further sequence derivable from the PD-L1 protein, in particular a sequence that is at least 70% identical to the sequence linking the first and second amino acid sequences in human and / or mouse PD-L1 protein, i.e., an amino acid sequence that is at least 70% identical to amino acids 77-112 of human PD-L1 protein. However, it is also conceivable that the first and second amino acid sequences may be linked via a sequence that is not derivable from the PD-L1 protein, e.g., a suitable linker peptide. Nevertheless, preferably, the PD-L1 polypeptide includes, and preferably consists of, an amino acid sequence that is at least 70% identical to amino acids 18-132 of human PD-L1, i.e., the amino acid sequence of SEQ ID NO: 5. Preferably, the PD-L1 polypeptide includes, and preferably consists of, an amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 5. More preferably, the PD-L1 polypeptide includes, and preferably consists of, the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, including substitutions as specified elsewhere herein.As will be understood, the expression “the PD-L1 polypeptide comprises amino acid sequence X, comprising substitutions as specified elsewhere herein” refers to the fact that the PD-L1 polypeptide comprises amino acid sequence X in essence, but comprising the substitutions indicated, i.e., preferably the PD-L1 polypeptide comprises amino acid sequence X except that the amino acid at the indicated position is replaced by a substitutional amino acid.

[0023] The PD-L1 polypeptides identified herein carry an amino acid substitution at at least one of the following positions: V54, Y56, Q63, Q66, V68, A69, P76, I115, A121, D122, Y123, K124, and R125, wherein if the polypeptide includes an amino acid substitution at position Y56, C113, or I115, the polypeptide further carries at least one of the above substitutions selected from the list consisting of V54, Y56, Q63, Q66, V68, A69, P76, I115, A121, D122, Y123, K124, and R125. More preferably, if the polypeptide includes an amino acid substitution at position Y56, Q63, A69, P76, C113, or I115, the polypeptide further carries at least one of the above substitutions. More preferably, the PD-L1 polypeptide comprises at least one amino acid substitution at position Y56 and / or P76, more preferably at positions Y56 and P76. Also preferably, the PD-L1 polypeptide carries at least one further substitution at at least one of the amino acid positions V54, Q66, V68, A69 and / or I115. More preferably, the PD-L1 polypeptide carries at least one amino acid substitution at at least one of the following amino acid positions in the first amino acid sequence: V54, Y56, Q63, Q66, V68, A69, P76; and at least one amino acid substitution at at least one of the following amino acid positions in the second amino acid sequence: I115, A121, D122, Y123, K124, and R125. Preferably, the Q63 substitution is not the Q63N substitution, the A69 substitution is not the A69H substitution, the P76 substitution is not the P76V substitution, and / or the I115 substitution is not the I115M substitution. More preferably, the V54 substitution is the V54L substitution, the Y56 substitution is the Y56G, Y56A, Y56D, or Y56S substitution, the Q63 substitution is the Q63H substitution, the Q66 substitution is the Q66R substitution, the V68 substitution is the V68E substitution, the A69 substitution is the A69T or A69S substitution, the P76 substitution is the P76F or P76H substitution, and / or the I115 substitution is the I115L substitution.Preferably, the PD-L1 polypeptide carries at least one further substitution at amino acid position C113. Also preferably, the PD-L1 polypeptide carries at least one further substitution at at least one of the amino acid positions V54, Q66, V68, A69 and / or I115.

[0024] Preferably, the PD-L1 polypeptide comprises substitutions (i) Y56S, P76F, and I115L; (ii) V54L, Y56D, Q66R, V68E, A69S, and P76H; (iii) Y56G, Q63H, P76F, and I15L; (iv) Y56A, Q63H, A69T, and P76F; or (v) Y56A, Q63H, and P76H. Most preferably, the PD-L1 polypeptide comprises substitutions Y56S, P76F, and I115L; or V54L, Y56D, Q66R, V68E, A69S, and P76H. In other words, preferably the PD-L1 polypeptide comprises, and preferably consists of, one of the amino acid sequences shown in SEQ ID NOs: 16 to 20; preferably the PD-L1 polypeptide comprises, and preferably consists of, the amino acid sequence shown in SEQ ID NOs: 16 or 17.

[0025] Preferably, the PD-L1 polypeptide is included in a fusion polypeptide and / or polypeptide complex, as also specified above herein. Preferably, the PD-L1 polypeptide is included in a fusion polypeptide further comprising a tag, linker, and / or polypeptide for purification and / or detection, and / or a polypeptide for extending the in vivo half-life of the compound. Polypeptides for extending the in vivo half-life of a compound are known in the art and include, in particular, antibody fragments, e.g., Fc fragments of immunoglobulins, and ovalbumin or fragments thereof. Also preferably, the PD-L1 polypeptide is included in a polypeptide complex; for example, the tag, linker, and / or polypeptide for extending the in vivo half-life of the compound can be linked to the PD-L1 polypeptide via a disulfide bond or via affinity bonds, as specified elsewhere herein. The fusion polypeptide can also form a multimer, e.g., a dimer, in which case the PD-L1 polypeptide can be included in the fusion polypeptide and polypeptide complex, such as in a dimeric Fc fusion polypeptide. In other words, preferably, the PD-L1 polypeptide comprises, and preferably consists of, the amino acid sequence shown in SEQ ID NO: 21 or 22, which is preferably encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23 or 24.

[0026] The term "crystallizable (Fc) portion of immunoglobulin fragments" refers to the C portion of an antibody. H 2 and C HThis refers to an antibody fragment containing three domains and obtainable by proteolytic cleavage of the antibody, for example, using papain. Various immunoglobulins from various species are known in the art. These immunoglobulins include IgA, IgD, IgE, IgG, IgM, IgW, or IgY. Of the immunoglobulins, preferred according to the present invention are, however, those found in mammals, particularly humans, i.e., IgA, IgD, IgE, IgG, and IgM. The Fc portion of the antibody determines the class effect. Since only the constant domain of the heavy chain forms the Fc portion of the antibody, the class of the heavy chain determines the class effect. Possible classes of heavy chains in an antibody include α, γ, δ, ε, and μ. These heavy chain classes define the isotype. Different isotypes of antibodies have different class effects due to their respective Fc portions. Such Fc-mediated class effects include those that affect effector cells or effector molecules, such as opsonization, aggregation, hemolysis, complement activation, and mast cell degranulation. H 2 and C H The amino acid sequences for the three domains are well known in the art for different antibody isotypes and can be provided to those skilled in the art without further difficulty. The Fc portion, as referred to in the present invention, can preferably be post-translationally modified and more preferably glycosylated. Preferably, the immunoglobulin according to the present invention is IgG, and more preferably human IgG. The amino acid sequence encoding human IgG, and the nucleic acid sequence encoding it, are well known in the art. Furthermore, it is also well known which amino acids correspond to the Fc portion in the amino acid sequence.

[0027] Preferably, the fusion polypeptide or polypeptide complex includes a third portion for targeting, in particular a polypeptide capable of specifically binding to cytotoxic T cells. More preferably, the polypeptide capable of specifically binding to cytotoxic T cells is selected from the group consisting of polypeptides comprising a portion of an MHC-I complex capable of binding to CD8, a portion of CD80 capable of binding to CD28, a polypeptide which is an antibody or fragment thereof capable of specifically binding to CD8, a polypeptide which is an antibody or fragment thereof capable of specifically binding to CD28, and the CD2-binding portion of lymphocyte function-associated antigen-3 (LFA-3). How such portions can be derived from their respective proteins is well known to those skilled in the art.

[0028] Advantageously, the studies underlying the present invention have found that the described PD-L1 derivatives have increased affinity for the ligand PD-1. Furthermore, it has been found that significantly smaller molecules can be used. That is, therapeutic effects, particularly in the treatment of sepsis-associated organ failure, can be achieved at lower concentrations, thereby reducing the required dose. In other words, the PD-L1 variants preferably replace lost signals from host cells and thereby prevent autoimmune activation of CTLs. The PD-L1 variants preferably retain mutations that enhance affinity for PD-1 but maintain the same binding mode as PD-L1wt. As a result, the genetically engineered PD-L1 variants preferably have the potential to function as drugs that can be administered to prevent organ / tissue damage in patients caused by sepsis.

[0029] The definitions set forth above shall apply mutatis mutandis to the following. The additional definitions and descriptions set forth below shall also apply mutatis mutandis to all embodiments described herein.

[0030] The present invention also relates to a polynucleotide encoding a PD-L1 polypeptide according to the present invention.

[0031] The term “polynucleotide,” as used herein, means a single-stranded or double-stranded DNA or RNA molecule. All naturally occurring or artificial derivatives of such molecular species, including genomic DNA, cDNA, hnRNA, mRNA, and fragments thereof, are encompassed by this term. Polynucleotides may preferably be linear or cyclic molecules. Furthermore, in addition to the nucleic acid sequence encoding the PD-L1 polypeptide described above, polynucleotides according to the present invention may include additional sequences required for proper transcription and / or translation, such as 5'- or 3'-UTR sequences, or sequences required for splicing or RNA stability. Preferred polynucleotides encoding the PD-L1 polypeptide according to the present invention are also described elsewhere herein. Preferably, a polynucleotide comprises, and preferably consists of, a sequence at least 60% identical to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; preferably, a polynucleotide comprises, and preferably consists of, any one of SEQ ID NOs: 11-15, more preferably the sequence shown in SEQ ID NO: 11 or 12.

[0032] Preferably, the polynucleotide is included in an expression construct that enables the expression of the polynucleotide in the subject. The term “expression construct,” as used herein, means heterologous polynucleotides including the nucleic acids required for the expression of the aforementioned polynucleotide encoding the PD-L1 polypeptide and the polynucleotide encoding the fusion polypeptide. Typically, such additional nucleic acids, preferably heterologous to the polynucleotide encoding the PD-L1 polypeptide, may be transcription termination sequences such as promoter sequences, enhancer sequences, and / or terminators. Furthermore, the expression construct may also include additional nucleic acids required to introduce the expression construct into a host. For example, if expression in host cells is desired, the expression construct may include additional nucleic acids required for transformation or transfection and for the proliferation of the introduced expression construct in the host cells.

[0033] The present invention also relates to vectors comprising the polynucleotides of the present invention.

[0034] Vectors as used herein preferably include phages, plasmids, viruses, or retroviral vectors, as well as artificial chromosomes such as bacterial or yeast artificial chromosomes. Vectors containing polynucleotides encoding the PD-L1 polypeptide preferably further include selectable markers for host replication and / or selection. Vectors can be incorporated into host cells by various techniques well known in the art. For example, plasmid vectors can be introduced in precipitates such as calcium phosphate precipitate or rubidium chloride precipitate, or in complexes containing charged lipids or in carbon-based clusters such as fullerenes. Alternatively, plasmid vectors can be introduced by heat shock or electroporation techniques. If the vector is to be a virus, it can be packaged in vitro using a suitable packaging cell line prior to application to host cells. Retroviral vectors may be replication-capable or replication-deficient. In the latter case, viral replication will generally occur only in complementary hosts / cells. Furthermore, the polynucleotides are typically functionally linked to expression regulatory sequences that enable expression in prokaryotic or eukaryotic host cells or isolated fractions thereof in the vector. Polynucleotide expression involves the transcription of polynucleotides into translatable mRNA. Regulatory elements that ensure expression in host cells are well known in the art and are described above by example. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the lac, trp, or tac promoters in Escherichia coli, while examples of regulatory elements that allow expression in eukaryotic host cells include the AOX1 or GAL1 promoter in yeast, or the CMV, SV40, or RSV promoters (Roussarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells. Other expression systems envisioned by the present invention, such as those based on polyhedrin promoters, would allow expression in insect cells. Furthermore, inducible expression regulatory sequences can be used in vectors encompassed by the present invention.Such inducible vectors may include tet or lac operator sequences or sequences that are inducible by heat shock or other environmental factors. Suitable expression regulatory sequences are well known in the art. Apart from elements that are responsive to transcription initiation, such regulatory elements may also include transcription termination signals downstream of polynucleotides, such as SV40-polyA sites or tk-polyA sites. In this context, suitable expression vectors are well known in the art, such as Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (Invitrogen), or baculovirus-derived vectors. Preferably, the vectors are expression vectors and gene transfer or targeting vectors. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomaviruses can be used for the delivery of expression constructs according to the present invention to targeted cell populations, for example, in gene therapy approaches. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).

[0035] Furthermore, it is envisioned that the expression construct be introduced into the host genome. In such cases, the expression construct may also include nucleic acids that enable either heterogeneous or homogeneous integration of the expression construct. That is, the expression construct referred to herein may also be a targeted construct that enables random or site-specific integration of the targeted construct into genomic DNA. Such a targeted construct preferably includes DNA of sufficient length for either homogeneous or heterogeneous recombination adjacent to an expression cassette having a polynucleotide encoding the PD-L1 polypeptide. Furthermore, the expression construct may also be introduced using an integration system such as Cre / LoxP or CRISPR / CAS. In such cases, the expression construct may include further nucleic acids that enable the use of such an integration system. Appropriate modifications / additions depend on the envisioned integration system and are well known to those skilled in the art.

[0036] The present invention also relates to PD-L1 polypeptides or polynucleotides according to the present invention for use as pharmaceuticals. The present invention further relates to PD-L1 polypeptides or polynucleotides according to the present invention for use in the treatment and / or prevention of organ failure in subjects suffering from sepsis, for use in the treatment of immunodeficiency, preferably lupus erythematosus, and / or for use in immuno-oncological treatment.

[0037] The term "treating," as used herein, means improving or curing a disease or at least one symptom associated with it. That is, if improvement or cure of a disease or at least one symptom associated with it is observed, the treatment will be considered effective. It will be understood that the treatment may not be effective in all subjects. However, according to the present invention, it is assumed that the treatment is preferably effective in at least a statistically significant portion of the subjects being treated. How to determine the statistically significant portion of subjects that can be effectively treated is well known to those skilled in the art. Whether a portion is statistically significant can be determined without further difficulty by those skilled in the art using various well-known statistical evaluation tools, such as confidence interval calculation, p-value calculation, Student's t-test, and Mann-Whitney test. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. The preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-values ​​are preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probabilities assumed by the present invention allow the findings of an effective treatment to be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in a given cohort or population.

[0038] The term “preventing,” as used herein, means avoiding the onset of a disease or at least one symptom associated with it, or preventing the worsening of a disease or at least one symptom. Prevention, as used herein, can typically be achieved during the period in which the drug is administered. However, if drug administration is discontinued, prevention may persist for a specific prophylactic time window after drug administration, rather than indefinitely. Typically, a prophylactic time window according to the present invention may be at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days. However, the prophylactic time window may also depend on the dosage of the drug and the mode of administration or type of formulation. For example, a relatively long prophylactic time window can usually be achieved when a high dose is administered. The same is true when a sustained-release formulation of the drug is administered, or when the drug is administered via a route that does not result in immediate metabolism of the drug in the subject. In such cases, the prophylactic time window can be extended to several weeks, months, or even years. It will be understood that prevention may not be effective in all subjects. However, according to the present invention, prevention is preferably assumed to be effective in at least a statistically significant portion of the subjects. How to determine the statistically significant portion of subjects that can be effectively prevented is well known to those skilled in the art. Whether a portion is statistically significant can be determined without further difficulty by those skilled in the art using the various well known statistical evaluation tools described above.

[0039] Since the PD-L1 polypeptide according to the present invention will be used for medical treatment, it will preferably be formulated as a pharmaceutical. Pharmaceutical, in the sense of the present invention, preferably means a pharmaceutical composition containing an expression construct encoding the PD-L1 polypeptide according to the present invention as a biologically active PD-L1 polypeptide or a pharmaceutically active compound, and one or more other components such as one or more pharmaceutically acceptable carriers. The pharmaceutically active compound may exist in liquid or lyophilized form. For example, the pharmaceutically active compound may exist with glycerol and / or a protein stabilizer (e.g., human serum albumin). The pharmaceutical is typically administered systemically, and preferably intravenously or intramuscularly. However, depending on the nature of the formulation and the desired therapeutic use, the pharmaceutical may similarly be administered by other routes. The pharmaceutically active compound is the active ingredient or drug of the pharmaceutical and is preferably administered in a conventional dosage form, prepared by combining the drug with a standard pharmaceutical carrier according to conventional procedures. These procedures may include mixing, granulating, and compressing or dissolving the components into the desired preparation, where appropriate. It will be understood that the form and properties of a pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient being combined, the route of administration, and other well-known variables. The carrier must be acceptable in the sense that it is compatible with the other components of the formulation and is not harmful to its recipient. Pharmaceutical carriers used may be solids, gels, or liquids. Examples of solid carriers include lactose, clay, sucrose, talc, gelatin, agar, pectin, acacia gum, magnesium stearate, and stearic acid. Examples of liquid carriers include phosphate-buffered saline solutions, syrups, oils, water, emulsions, and various types of wetting agents. Similarly, carriers or diluents may include time-delaying materials well-known in the art, such as glycerol monostearate or glycerol distearate, either alone or in combination with wax.Suitable carriers include those mentioned above and others well known in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. Diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hanks' solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers. The pharmaceuticals referenced herein are preferably administered at least once, for example, as a bolus. However, the pharmaceuticals may be administered two or more times, preferably at least twice, for example, permanently or periodically after a predetermined time window.

[0040] The therapeutically effective dose refers to the amount of the PD-L1 polypeptide or expression construct encoding the PD-L1 polypeptide used in the drug that prevents, improves, or cures the symptoms associated with the disease or condition referenced herein. The therapeutic effect and toxicity of a drug can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. The administration regimen will be determined by the attending physician and based on clinical factors. As is well known in the medical field, the dose for any one patient depends on numerous factors, including the patient's size, age, the specific formulation of the drug being administered, sex, time and route of administration, overall health status, and other drugs being administered concurrently. Progress can be monitored through periodic evaluations. Recommended doses will be indicated in the prescriber or user instructions to allow for dose adjustments based on the recipient being considered.

[0041] In further embodiments of the present invention, a pharmaceutical product according to the present invention may include a drug in addition to the above-mentioned compound added during formulation. Preferably, the pharmaceutically active compound according to the present invention is subject to application together with at least one further drug, that is, it can be formulated as a pharmaceutical product together with these other drugs. More preferably, the at least one further drug is selected from the group consisting of antibiotics, vasopressors, steroids, anticoagulants, antithrombotic agents, inflammatory cytokines, and DAMP inhibitors. Furthermore, the formulation of the pharmaceutical composition is preferably carried out under GMP standardization conditions, etc., to ensure the quality, pharmaceutically safe, and efficacy of the pharmaceutical product.

[0042] The term "organ failure," as used herein, means a dysfunction of any organ that affects the physiologically expected function of the organ to such an extent that normal homeostasis cannot be maintained and cannot be endogenously compensated for. Organ failure can be acute or chronic. The symptoms associated with organ failure depend on the affected organ and are usually revealed by the pathophysiology of the subject, which can be determined, for example, by clinical or biochemical parameters. The symptoms of organ failure are also well known in the art and are described in medical textbooks. Preferably, as referred herein, organ failure is multiple organ failure. Multiple organ failure is characterized by the failure of two or more organs simultaneously or consecutively within a short period of time. In many cases, it can be observed as a result of a severe infection or inflammatory response, such as systemic inflammatory response syndrome (SIRS) or sepsis. Typical organs that fail during SIRS or sepsis are the lungs, kidneys, heart and / or systemic circulatory system, the digestive system, particularly the liver, and the nervous system. Preferably, the multiple organ failure as referred to herein is caused by autoreactive cytotoxic cells, and more preferably, CD8 cytotoxic T cell-dependent multiple organ failure. Also preferably, the organ failure as referred to herein is hepatic failure, in particular hepatic failure in septic subjects.

[0043] The term “subject” as used herein means any type of animal, including, for example, mammals, birds, fish, or reptiles. However, typically, animals are mammals such as those used as pets (including dogs, cats, horses, or rodents), laboratory animals (e.g., rats, mice, or apes), or livestock animals (e.g., pigs, cattle, goats, or sheep). More preferably, mammals are primates, and most preferably humans. Subjects according to the present invention are preferably known or suspected to have sepsis, or are expected to develop sepsis; that is, subjects preferably exhibit at least one pathological change, such as clinically apparent symptoms or changes in physiological or molecular parameters typically associated with sepsis. Preferably, subjects are known or suspected to have an immunodeficiency, preferably lupus erythematosus. Also preferably, subjects are known or suspected to have an immuno-oncological need.

[0044] The term "sepsis," as used herein, refers to an inflammatory response affecting the entire organism. Typical symptoms associated with sepsis are well known in the art and are described in standard medical textbooks. These include significantly altered body temperature (low or high), tachypnea, tachycardia, hypotension due to decreased peripheral vascular resistance, confusion, and edema. Biochemical parameters such as coagulation disorders or metabolic acidosis are also typical signs of sepsis. Preferably, sepsis is caused by a severe infection by bacteria, viruses, parasites, or fungi. Furthermore, there are cofounding factors that influence the development or outcome of sepsis, such as diabetes or cancer. Preferably, sepsis as referred to herein is characterized by the presence of two or more of the following symptoms in response to infection: abnormal body temperature (preferably less than 36°C or greater than 38°C), abnormal heart rate (preferably greater than 90 beats / min), abnormal respiratory rate (preferably greater than 20 breaths / min), or blood gas composition (preferably less than 4.3 kPa of CO2), and abnormal white blood cell count (preferably 4 × 10⁶). 9 / L less than or 12 x 10 9 (Histological presence of neutrophils with a hypertrichosis or band neutrophils).

[0045] It will be understood by those skilled in the art that the term “immunodeficiency” relates to any disorder involving the type of cells, tissues, and / or organs of a subject in which an immune response occurs. Immune disorders are preferably caused or exacerbated by an alloimmune response; that is, an immune disorder preferably involves an immune response of the host immune system against exogenous tissue or organ, in particular organ transplant rejection and / or an immune response of the exogenous immune system against host tissue or organ, in particular graft-versus-host disease. Preferably, an immune disorder is caused or exacerbated by an autoimmune response, i.e., an immune disorder; that is, an immune disorder is preferably a disorder in which T cells, preferably CD8 T cells, lyse the subject’s own cells and / or cause an inflammatory response in the absence of exogenous stimuli. Preferably, an autoimmune disorder is lupus erythematosus.

[0046] The term “immuno-oncological therapy” is also understood by those skilled in the art. This term preferably relates to the treatment of cancer by altering the immune response of a subject. Such alteration may be induction, enhancement, or suppression of the immune response.

[0047] The present invention also relates to a host cell comprising a PD-L1 polypeptide according to the present invention, a polynucleotide according to the present invention, and / or a vector according to the present invention.

[0048] As used herein, the term “host cell” refers to any cell capable of receiving, and preferably maintaining and / or expressing, the PD-L1 polypeptide, polynucleotide and / or vector of the present invention. More preferably, the host cell is capable of expressing the PD-L1 polypeptide, as specified herein, encoded on the polynucleotide and / or vector. Preferably, the cell is a bacterial cell, more preferably a cell of a common research bacterial strain known in the art, most preferably an Escherichia strain, particularly an Escherichia coli strain. Also preferably, the host cell is a eukaryotic cell, preferably a yeast cell, for example, a strain of baker's yeast, or an animal cell. More preferably, the host cell is an insect cell or mammalian cell, preferably derived from a mammalian subject as specified herein, particularly a mouse or rat cell. Most preferably, the host cell is a human cell. However, it is also conceivable that the host cell is a plant cell.

[0049] The present invention also relates to a non-human transgenic organism comprising a host cell, a PD-L1 polypeptide according to the present invention, a polynucleotide according to the present invention, and / or a vector according to the present invention.

[0050] The term "non-human transgenic organism," when used, refers to any multicellular organism, excluding humans, particularly plants and animals. Preferably, the non-human transgenic organism is a subject, preferably a mammalian subject as specified above, but not a human subject. Also preferably, the subject is a plant, preferably a monocotyledonous or dicotyledonous plant. The present invention also involves the following steps: (i) the step of expressing a polynucleotide according to the present invention in a host cell; and (ii) Steps to obtain PD-L1 polypeptide The present invention also relates to a method for producing a PD-L1 polypeptide according to any one of the claims, including the above.

[0051] The present invention further relates to a PD-L1 polypeptide obtainable by the above method.

[0052] A method for producing PD-L1 polypeptide is preferably an in vitro method. Preferably, the method is carried out under GMP and / or GLP conditions. Furthermore, the method may include further steps, for example, a step of providing host cells containing a suitable expression construct prior to step (i), and / or one or more purification steps for purifying the PD-L1 polypeptide. Furthermore, one or more steps may be assisted or performed by an automated apparatus.

[0053] The present invention also provides a method for treating and / or preventing organ failure in a subject, in particular a method for treating organ failure in a subject suffering from sepsis, the method comprising the steps of (a) administering a therapeutically effective amount of PD-L1 polypeptide to the subject or (b) administering a therapeutically effective amount of polynucleotide encoding the PD-L1 polypeptide.

[0054] Typical embodiments of the present invention relating to the types of organ failure, subjects to be treated, sepsis, and fusion polypeptides or polynucleotides are described above and apply to methods of the present invention for treating and / or preventing organ failure in a subject. Preferably, the method includes identifying the subject to be treated by determining the presence of sepsis prior to the administration of the fusion polypeptide or the polynucleotide encoding it.

[0055] Preferably, the method includes the steps of monitoring the subject for signs of organ failure after administration of the fusion polypeptide, and, if necessary, administering the fusion polypeptide or the polynucleotide encoding it again or in different doses.

[0056] In view of the above, the following embodiments are particularly conceivable: 1. A PD-L1 polypeptide comprising at least a first amino acid sequence that is at least 70% identical to SEQ ID NO: 8, and a second sequence that is at least 70% identical to SEQ ID NO: 10, wherein the polypeptide carries an amino acid substitution at at least one of the following positions: V54, Y56, Q63, Q66, V68, A69, P76, I115, A121, D122, Y123, K124, and R125, and if the polypeptide includes an amino acid substitution at position Y56, C113, or I115, the polypeptide further carries at least one of the above substitutions; the amino acid positions are based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6), for a PD-L1 polypeptide. 2. A PD-L1 polypeptide comprising at least a first amino acid sequence that is at least 70% identical to SEQ ID NO: 8, and a second sequence that is at least 70% identical to SEQ ID NO: 10, wherein the polypeptide carries amino acid substitutions at least at amino acid positions Y56 and P76, and the amino acid positions are based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6). 3. A PD-L1 polypeptide comprising at least a first amino acid sequence that is at least 70% identical to SEQ ID NO: 8, and a second sequence that is at least 70% identical to SEQ ID NO: 10, wherein the polypeptide carries at least one amino acid substitution at at least one of the following amino acid positions in the first amino acid sequence: V54, Y56, Q63, Q66, V68, A69, P76; and at least one amino acid substitution at at least one of the following amino acid positions in the second amino acid sequence: I115, A121, D122, Y123, K124, and R125, the amino acid positions being based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6). 4. A PD-L1 polypeptide according to any one of Embodiments 1 to 3, wherein the first amino acid sequence is at least 80%, preferably at least 90%, and more preferably at least 95%, identical to SEQ ID NO: 8, and / or the second amino acid sequence is at least 80%, preferably at least 90%, and more preferably at least 95%, identical to SEQ ID NO: 10. 5. A PD-L1 polypeptide according to any one of Embodiments 1 to 4, wherein the polypeptide comprises at least one amino acid sequence selected from SEQ ID NOs: 7 and SEQ ID NOs: 8, which includes the amino acid substitutions (one or more), and a second sequence selected from SEQ ID NOs: 9 and SEQ ID NOs: 10. 6. A PD-L1 polypeptide according to any one of Embodiments 1 to 5, wherein the polypeptide contains, and preferably comprises, an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 5. 7. A PD-L1 polypeptide according to any one of Embodiments 1 to 6, wherein the polypeptide comprises, preferably, at least 80%, preferably at least 90%, and more preferably at least 95%, an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 5. 8. A PD-L1 polypeptide according to any one of Embodiments 1 to 7, wherein the polypeptide comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. 9. A PD-L1 polypeptide according to any one of Embodiments 1 to 8, wherein the polypeptide comprises at least one amino acid substitution at position P76. 10. A PD-L1 polypeptide according to any one of Embodiments 1 to 9, wherein the polypeptide carries at least one amino acid substitution at at least one of the following amino acid positions in the first amino acid sequence: V54, Y56, Q63, Q66, V68, A69, P76; and at least one amino acid substitution at at least one of the following amino acid positions in the second amino acid sequence: I115, A121, D122, Y123, K124, and R125. 11. A PD-L1 polypeptide according to any one of Embodiments 1 to 10, wherein the polypeptide supports amino acid substitutions at least at amino acid positions Y56 and P76. 12. A PD-L1 polypeptide according to any one of Embodiments 1 to 11, wherein the PD-L1 polypeptide is a variant of PD-L1. 13. A PD-L1 polypeptide according to any one of Embodiments 1 to 12, wherein the PD-L1 polypeptide supports at least a further substitution at amino acid position C113. 14. A PD-L1 polypeptide according to any one of Embodiments 1 to 13, wherein the PD-L1 polypeptide carries at least one further substitution at at least one of the amino acid positions V54, Q66, V68, A69 and / or I115. 15. Any one of Embodiments 1 to 14, wherein (i) the Q63 substitution is not a Q63N substitution, (ii) the A69 substitution is not an A69H substitution, (iii) the P76 substitution is not a P76V substitution, and / or (iv) the I115 substitution is not an I115M substitution. 16. A PD-L1 polypeptide according to any one of Embodiments 1 to 16, wherein (i) the V54 substitution is a V54L substitution, (ii) the Y56 substitution is a Y56G, Y56A, Y56D, or Y56S substitution, (iii) the Q63 substitution is a Q63H substitution, (iv) the Q66 substitution is a Q66R substitution, (v) the V68 substitution is a V68E substitution, (vi) the A69 substitution is an A69T or A69S substitution, (vii) the P76 substitution is a P76F or P76H substitution, and / or (viii) the I115 substitution is an I115L substitution. 17. Any one of the PD-L1 polypeptides of Embodiments 1 to 16, wherein the polypeptide comprises substitutions (i) Y56S, P76F, and I115L; (ii) V54L, Y56D, Q66R, V68E, A69S, and P76H; (iii) Y56G, Q63H, P76F, and I15L; (iv) Y56A, Q63H, A69T, and P76F; or (v) Y56A, Q63H, and P76H. 18. A PD-L1 polypeptide according to any one of Embodiments 1 to 17, wherein the PD-L1 polypeptide comprises, preferably, one of the amino acid sequences shown in SEQ ID NOs: 16 to 20; preferably, the PD-L1 polypeptide comprises, preferably, one of the amino acid sequences shown in SEQ ID NOs: 16 or 17. 19. A PD-L1 polypeptide according to any one of Embodiments 1 to 18, wherein the PD-L1 polypeptide is included in a fusion polypeptide and / or polypeptide complex. 20. A PD-L1 polypeptide according to any one of Embodiments 1 to 19, wherein the fusion polypeptide further comprises at least one polypeptide that extends the in vivo half-life of the fusion polypeptide. 21. Any one of the PD-L1 polypeptides of Embodiments 1 to 20, wherein the fusion polypeptide further comprises at least one antibody fragment and / or ovalbumin or a fragment thereof; preferably, the fusion polypeptide further comprises an Fc fragment of an immunoglobulin. 22. A PD-L1 polypeptide according to any one of Embodiments 1 to 21, wherein the polypeptide comprises, and preferably consists of, the amino acid sequence shown in SEQ ID NO: 21 or 22, which is preferably encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23 or 24. 23. Any one of the PD-L1 polypeptides of Embodiments 1 to 22, wherein the PD-L1 polypeptide additionally comprises a portion, preferably an immunoglobulin or a fragment thereof, that is capable of specifically binding to cytotoxic T cells. 24. The PD-L1 polypeptide of Embodiment 23, wherein the immunoglobulin is human IgG. 25. A PD-L1 polypeptide according to any one of Embodiments 1 to 24, wherein a portion capable of specifically binding to the cytotoxic T cells comprises a polypeptide containing a portion of an MHC-I complex capable of binding to CD8, a portion of CD80 capable of binding to CD28, a polypeptide which is an antibody or a fragment thereof capable of specifically binding to CD8, and a polypeptide which is an antibody or a fragment thereof capable of specifically binding to CD28. 26. A polynucleotide encoding the PD-L1 polypeptide described in any one of Embodiments 1 to 25. 27. The polynucleotide of Embodiment 26, wherein the polynucleotide comprises, preferably, a sequence that is at least 60% identical to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; preferably, the polynucleotide comprises, preferably, the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12. 28. A PD-L1 polypeptide according to any one of Embodiments 1 to 25 or a polynucleotide according to Embodiment 26 or 27, for use as a pharmaceutical. 29. A PD-L1 polypeptide according to any one of Embodiments 1 to 25 or a polynucleotide according to Embodiment 26 or 27, for use in the treatment and / or prevention of organ failure in subjects suffering from sepsis, for use in the treatment of immunodeficiency, preferably lupus erythematosus, and / or for use in immuno-oncological treatment. 30. The PD-L1 polypeptide or polynucleotide for use according to Embodiment 29, wherein the organ failure is CD8 cytotoxic T cell-dependent multiple organ failure and / or is caused by an inflammatory response, preferably by systemic inflammatory response syndrome (SIRS) or sepsis. 31. The PD-L1 polypeptide or polynucleotide for use according to Embodiment 29 or 30, wherein the subject is a mammal, preferably a human. 32. The PD-L1 polypeptide or polynucleotide for use according to any one of Embodiments 29 to 31, wherein the polypeptide or polynucleotide inhibits cytotoxic T cells induced by sepsis in the subject upon administration. 33. The PD-L1 polypeptide or polynucleotide for use according to any one of Embodiments 29 to 32, wherein, upon administration, the PD-L1 polypeptide or polynucleotide induces long-term tolerance in cytotoxic T cells in the subject to activation induced by sepsis. 34. A vector comprising the polynucleotide described in Embodiment 26 or 27. 35. A host cell comprising a PD-L1 polypeptide according to any one of Embodiments 1 to 26, a polynucleotide according to Embodiment 26 or 27, and / or a vector according to Embodiment 34. 36. A non-human transgenic organism comprising the host cell described in Embodiment 35, the PD-L1 polypeptide described in any one of Embodiments 1 to 26, the polynucleotide described in Embodiment 26 or 27, and / or the vector described in Embodiment 34. 37. The following steps: (i) the step of expressing the polynucleotide described in Embodiment 26 or 27 in a host cell; and (ii) Steps to obtain PD-L1 polypeptide A method for producing a PD-L1 polypeptide according to any one of the embodiments, including the following: 38. PD-L1 polypeptide obtainable by the method of Embodiment 37.

[0057] All references cited herein are incorporated herein by reference with respect to their entirety and to the disclosures specifically referred to herein. [Brief explanation of the drawing]

[0058] [Figure 1] This figure shows a competitive phage ELISA. Five PD-L1 variants (214, 243, 291, 246, and 258) and unmodified PD-L1 wt were presented on phages and tested for binding affinity to surface-immobilized PD-1 (black). In similar experiments, binding affinity was induced using either 50 nM (dark gray) or 500 nM (light gray) PD-1 in solution to estimate binding affinity. The recorded signals correspond to optical density (OD) at 450 nm. [Figure 2-1]This figure shows a structural comparison of PD-L1 mutant 0258, which possesses the PD-L1 Ig variable domain (PDB:3BIK). (A) Parallel comparison of amino acid positions in the mutated wt PD-L1 (left panel) in clone 0258 (right panel). Amino acids are indicated and shown as rods. (B) Structural alignment shows identical structural conformation. (C)~(E) Representative biolayer interferometry sensorograms for PD-L1 wt (C) and two shown genetically modified PD-L1 mutants (D, E). Raw data (thick line) was fitted using a 1:1 Langmuir interaction model (thin line). [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-2. [Figure 3] This figure shows pull-down experiments using GST fusions of the distal Ig-like domain of PD-L1 wild-type and two genetically modified mutants (214 and 258). GST fusions expressed in bacteria were immobilized on glutathione beads and incubated with cell lysates of Jurkat cells stably expressing the mouse PD-1-EGFP construct. WB: Western blot. [Figure 4] This figure shows the Fc fusion protein of the Ig-like V domain of PD-L1. (A) SDS-PAGE analysis of purified PD-L1-Fc fusion protein. From left to right: unreduced, i.e., disulfide-linked dimer (78 kDa) and reduced monomers (39 kDa) of wt(200) and two genetically modified PD-L1 mutants (214 and 258). (B) Domain configuration of the disulfide-linked dimer of the PD-L1 Fc fusion protein. [Figure 5]This figure shows that recombinant PD-L1-Fc chimeras inhibit CTL-dependent cytotoxicity. Cytotoxic T cell-dependent hepatocyte killing was determined using Hepa1-6 cells as target cells and CD8+ T cells derived from OT-I mice as effector cells. CellTrackerOrange-stained Hepa1-6 cells were pulsed with OVA257-264 peptide for 2 hours. Subsequently, Hepa1-6 cells were co-cultured with enriched CD8+ T cells derived from the spleen of OT-I mice in a 5:1 ratio (effector:target cells). In parallel, recombinant PD-L1-Fc chimeras (WT, 214, and 258) were added at the indicated concentrations. The number of viable target cells was examined by FACS analysis. Data from four independent experiments are provided. Data are expressed as mean ± SD (*p<0.05, **p<0.01). [Figure 6] This figure shows the effect of PHI258 on cellular parameters in MLR. Four types of buffy coat-derived PBMCs were isolated, and PBMCs from two donors (2 × 10⁵ cells each) were co-cultured in T cell medium for 6 days with or without the addition of various concentrations of PHI258 (50 ng / mL to 50 μg / mL) on day 0. The cells were then analyzed by flow cytometry. (A, B) PD-1 expression on CD4+ (A) and CD8+ (B) T cells. (C) Surviving cells in the total cell population. (D) CD127 low CD25 high Treg relative to total CD4+ T cells. Individual data points and mean ± SEM are shown. Dunn's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001. [Figure 7] This figure shows the effect of PHI258 on cytokine production in MLRs. Four types of buffy coat-derived PBMCs were isolated, and PBMCs from two donors (2 × 10⁵ cells each) were co-cultured in T cell medium for 6 days with or without the addition of various concentrations of PHI258 (50 ng / mL to 50 μg / mL) on day 0. Cytokine levels in the supernatant were analyzed on day 6 using a Cytometric Bead Array. Individual data points and mean ± SEM are shown. Dunn's multiple comparison test. *p<0.05; **p<0.01. [Figure 8] This is a diagram showing PHI258 versus PHI200 (wild type) in MLR. PBMCs derived from four types of buffy coats were isolated, and PBMCs from two donors (2×105 each) were co-cultured in T cell medium for 6 days with or without the addition of PHI258 or PHI200 (10 μg / mL each) on day 0. (A) Cytokine levels in the supernatant were analyzed by Cytometric Bead Array, and viable cells within the total cell population were determined by FACS. Individual data points and mean ± SEM are shown. Dunn's multiple comparison test. **p<0.01; ***p<0.001.

Mode for Carrying Out the Invention

Examples

[0059] The present invention is merely illustrated by the following examples. The examples will not be construed in any way as limiting the scope of the present invention.

[0060] Example 1 A library of 2×10 9 species of mutants of PD-L1 presented on the surface of filamentous phage M13 was constructed. Using the published structure of the crystal complex of wild-type PD-1 and PD-L1 as a guide, the inventors genetically engineered only the distal Ig-like domain of PD-L1 (amino acids 18 - 132 on mouse PD-L1), and to improve the presentation level, the inventors additionally introduced a C113R mutation into the mouse PD-L1 portion prior to library construction. Based on the crystal structure, the inventors selected 14 amino acid residues of PD-L1 that are buried in the interface and whose side chains contact PD-1 for randomization.

[0061] Phage selection was performed using recombinant mouse PD-1 protein (amino acids 31-150) with a C83S substitution, fused to the AviTag peptide, biotinylated in vivo, and purified from E. coli, as an antigen. After one selection round, the binding affinity of enriched phage clones was analyzed by ELISA, and the selected PD-L1 variants were sequenced. Competitive phage IC 50 In ELISA, five PD-L1 mutants were tested for their binding properties by competing with PD-1-Fc at 500 nM or 50 nM concentrations in solution (Figure 1). All five clones were selected for further characterization and expressed in E. coli as His-tagged fusions. The sequences of the selected mutants and their affinity for PD-1-Fc (measured on an Octet device by biolayer interferometry BLI) showed that mutants 214 and 258 exhibited up to a 30-fold advantage in binding to surface-immobilized mouse PD-1-Fc compared to PD-L1 wt expressed in the same form.

[0062] Tables 1 and 2 show the amino acid sequences and affinities of the selected PD-L1 mutants. Amino acid positions 54, 56, 63, 66, 68, 69, 76, 113, 115, and 121-125 were randomized. For the selected mutants, a dash indicates no amino acid change compared to the wild-type sequence. Kd values ​​for the interaction between mutants and mouse PD-1 were obtained by biolayer interferometry; n / a: unavailable. Since the original C113 of the mouse sequence had already been substituted prior to library construction as shown above, Table 2 shows R113.

[0063] [Table 1]

[0064] [Table 2]

[0065] Clones 0258 (hereinafter also referred to as PHI258) and 0214 were further analyzed. Representative BLI sensorograms for these two variants are shown in Figure 2. The 0.762 Å structural overlay of clone 0258 with the PD-L1 Ig-like variable domain is also shown. 2 The low RMSD value indicates that the mutation does not alter the innate conformation. This result suggests that the increased affinity is due to the optimization of intermolecular contact and not to a change in the binding topology of clone 0258 to PD-1 compared to the unmodified PD-L1 Ig-like variable domain (Figure 2A, B, C). Furthermore, using lysates of Jurkat cells stably expressing the mouse PD-1-EGFP fusion construct, we performed pull-down assays using wt PD-L1 and mutants 0214 and 0258. Both genetically modified mutants interacted with PD-1-EGFP much more strongly than the wild-type construct or GST control (Figure 3).

[0066] On the other hand, the inventors cloned and expressed PD-L1 mutants as fusion proteins containing human Fc for expression in mammalian cells. The establishment of PD-L1wt and PD-L1 mutants as Fc fusion proteins improves bioavailability in subsequent in vivo experiments. In addition, the mammalian expression system enables optimal formation of intermolecular disulfide bonds with better yield than bacteria.

[0067] Following the expression and purification of the Fc fusion protein, the inventors obtained good purity and an average yield of 200 mg / L Expi293F cultures using one-step purification on a Protein A column (Figure 4). To confirm whether the purified protein still bound to PD-1, the inventors determined the PD-1 / PD-L1 affinity as described above. Due to the binding avidity resulting from the dimerization of the Fc fusion, the affinities of the mutants increased from 2.54 and 2.14 nM to 88 and 77 pM, respectively. In similar experiments, the inventors observed an affinity of approximately 600 pM for the wt protein, indicating that the PD-L1 mutant has up to approximately 8-fold improved affinity for PD-1 in vitro.

[0068] The inventors confirmed the immunosuppressive effect of the PD-L1 Fc fusion protein by testing the cytotoxicity of T cells in the presence of mutants and wt controls. Cytotoxic T cells were induced from OT-1 mice and were sensitive to the presentation of ovalbumin-derived peptides by MHC on surface mouse cells. To induce T cell activity, ovalbumin was pulsed into the mouse-derived hepatocyte line Hepa1, and cytotoxicity was measured by monitoring cell death using FACS (Figure 5).

[0069] Example 2 To analyze the effect of PHI258 (i.e., the aforementioned PD-L1 variant 258) on allogeneic lymphocyte activation under human conditions, we performed a mixed lymphocyte reaction (MLR) assay using PBMCs derived from the buffy coat of healthy, anonymous human blood donors. PBMCs from two donors were mixed and maintained in T cell proliferation medium for up to 6 days with or without the addition of PHI258 at various concentrations (50 ng / mL to 50 μg / mL).

[0070] First, the cells were analyzed by flow cytometry on day 6, and CD4 + and CD8 +PD-1 expression on T cells was determined. Even incubation with very low doses of PHI258 strongly interfered with the detection of PD-1 on the surface of both T cell subsets (Figure 6A, B), indicating either competition for PD-1 binding with the FACS antibody or PD-1 internalization. Next, the percentage of viable cells was determined as a function of MLR. PHI258 significantly increased the number of viable cells at concentrations greater than 5 μg / mL (Figure 6C). In other words, PHI258 appears to inhibit cell killing during MLR. The relative cell composition did not change, as illustrated exemplified in Figure 6D for regulatory T cells.

[0071] Next, cytokine production indicating leukocyte activation in the MLR was determined by cytometric bead array. PHI258 significantly suppressed the secretion of inflammatory cytokines IFN-γ and TNF-α at concentrations greater than 5 μg / mL, but its effects at relatively low concentrations were more heterogeneous (Figure 7). Importantly, PHI258 did not suppress the production of the anti-inflammatory cytokine IL-10 (Figure 7).

[0072] PD-1 is upregulated upon T cell activation, which occurs approximately 3 days after T cell activation. To analyze whether PD-1 expression is required for PHI258 to act, IFN-γ levels in MLR cells were compared on days 3 and 6. Furthermore, unmodified PD-L1 (PHI200) was used as a control. Neither PHI258 nor PHI200 affected IFN-γ levels on day 3. However, PHI258 strongly suppressed IFN-γ levels on day 6, while PHI200 did not (Figure 8A). Corresponding to this lack of efficacy in limiting inflammatory cytokine production, PHI200 also did not affect cell viability in MLR culture compared to PHI258 (Figure 8B).

[0073] In conclusion, PHI258 effectively bound to PD-1 on human T cells in MLR cultures and suppressed inflammatory cytokine levels and cell killing, but did not suppress anti-inflammatory features such as IL-10 expression and regulatory T cell levels. This specification includes the following embodiments. [1] A PD-L1 polypeptide comprising at least a first amino acid sequence that is at least 70% identical to SEQ ID NO: 8, and a second sequence that is at least 70% identical to SEQ ID NO: 10, wherein it carries amino acid substitutions at least at amino acid positions Y56 and P76, and the amino acid positions are based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6). [2] The PD-L1 polypeptide according to [1], comprising at least a first amino acid sequence selected from SEQ ID NOs. 7 and SEQ ID NOs. 8, and a second sequence selected from SEQ ID NOs. 9 and SEQ ID NOs. 10, including the aforementioned amino acid substitutions (one or more). [3] The PD-L1 polypeptide described in [1] or [2], comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. [4] The PD-L1 polypeptide according to any one of [1] to [3], wherein at least a further substitution at amino acid position C113 is supported. [5] The PD-L1 polypeptide according to any one of the items [1] to [4], wherein (i) the V54 substitution is a V54L substitution, (ii) the Y56 substitution is a Y56G, Y56A, Y56D, or Y56S substitution, (iii) the Q63 substitution is a Q63H substitution, (iv) the Q66 substitution is a Q66R substitution, (v) the V68 substitution is a V68E substitution, (vi) the A69 substitution is an A69T or A69S substitution, (vii) the P76 substitution is a P76F or P76H substitution, and / or (viii) the I115 substitution is an I115L substitution. [6] Substitution (i) Y56S, P76F, and I115L; (ii) V54L, Y56D, Q66R, V68E, A69S, and P76H; (iii) Y56G, Q63H, P76F, and I15L; (iv) Y56A, Q63H, A69T, and P76F; or (v) Y56A, Q63H, and P76H A PD-L1 polypeptide as described in any one of items [1] to [5], including the following: [7] A PD-L1 polypeptide according to any one of the amino acid sequences shown in SEQ ID NOs: 16 to 20, preferably comprising one of them; preferably comprising one of the amino acid sequences shown in SEQ ID NOs: 16 or 17, preferably comprising one of them, preferably comprising one of them, according to any one of [1] to [6]. [8] A PD-L1 polypeptide according to any one of [1] to [7], which is included in a fusion polypeptide and / or polypeptide complex, preferably the fusion polypeptide further comprising at least one antibody fragment and / or ovalbumin or a fragment thereof; preferably the fusion polypeptide further comprising an Fc fragment of an immunoglobulin. [9] Preferably comprising, and preferably consisting of, the amino acid sequence shown in SEQ ID NO: 21 or 22, encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23 or 24, according to any one of [1] to [8]. A polynucleotide encoding the PD-L1 polypeptide described in any one of items

[10] [1] to [9].

[11] PD-L1 polypeptide as described in any one of [1] to [9] or polynucleotide as described in

[10] for use as a pharmaceutical.

[12] A PD-L1 polypeptide as described in any one of [1] to [9] or a polynucleotide as described in

[10] for use in the treatment and / or prevention of organ failure in subjects suffering from sepsis, for use in the treatment of immunodeficiency, preferably lupus erythematosus, and / or for use in immuno-oncological treatment.

[13] The PD-L1 polypeptide or polynucleotide for use according to

[12] , wherein the organ failure is caused by an inflammatory response, preferably by systemic inflammatory response syndrome (SIRS) or sepsis. A host cell comprising a PD-L1 polypeptide as described in any one of claims

[14] [1] to [9], and / or a polynucleotide as described in claim

[10] .

[15] (i) expressing the polynucleotide described in claim 10 in a host cell; and (ii) Steps to obtain PD-L1 polypeptide A method for producing the PD-L1 polypeptide described in any one of [1] to [9], including the following:

[0074] citation document Hotchkiss and Opal (2010) N Engl J Med 363:87-89 Meisel et al. (2009) Am J Respir Crit Care Med 180:640-648 Otto et al. (2011) Crit Care 15:R183 Reinhart et al. (2001) Crit Care Med 29:765-769 Vincent et al. (2013) Lancet 381:774-775 von Knethen et al. (2019), Theranostics 9(7):2003 International Publication No. 2017 / 029389A1

Claims

1. A PD-L1 polypeptide comprising a first amino acid sequence that is at least 90% identical to SEQ ID NO: 5, or comprising a first amino acid sequence that is at least 90% identical to SEQ ID NO: 5, wherein the following amino acid substitutions are made: (i) The amino acids Y, P, and I at positions 56, 76, and 115 are replaced with S, F, and L, respectively; (ii) The amino acids V, Y, Q, V, A, and P at positions 54, 56, 66, 68, 69, and 76 are replaced with L, D, R, E, S, and H, respectively; (iii) The amino acids Y, Q, P, and I at positions 56, 63, 76, and 115 are replaced with G, H, F, and L, respectively; (iv) The amino acids Y, Q, A, and P at positions 56, 63, 69, and 76 are replaced with A, H, T, and F, respectively; or (v) The amino acids Y, Q, and P at positions 56, 63, and 76 are substituted with A, H, and H, respectively. Carrying, The amino acid position is based on the mouse PD-L1 amino acid sequence (SEQ ID NO: 6) of the PD-L1 polypeptide.

2. A PD-L1 polypeptide containing any of the amino acid sequences shown in SEQ ID NOs. 16 to 20, or consisting of any of the amino acid sequences shown in SEQ ID NOs. 16 to 20.

3. The PD-L1 polypeptide according to claim 1, comprising at least a variant of a first amino acid sequence selected from SEQ ID NOs. 7 and SEQ ID NOs. 8, and a variant of a second amino acid sequence selected from SEQ ID NOs. 9 and SEQ ID NOs. 10, and supporting at least the amino acid substitutions.

4. A variant of the amino acid sequence of SEQ ID NO: 4, comprising an amino acid sequence that supports at least the amino acid substitution, according to claim 1, for the PD-L1 polypeptide.

5. The PD-L1 polypeptide according to any one of claims 1 to 4, wherein it supports at least a further substitution at amino acid position 113.

6. A PD-L1 polypeptide according to any one of claims 1 to 5, which is included in a fusion polypeptide and / or polypeptide complex.

7. The PD-L1 polypeptide according to claim 6, wherein the fusion polypeptide further comprises at least one antibody fragment and / or ovalbumin or a fragment thereof.

8. The PD-L1 polypeptide according to claim 6 or 7, wherein the fusion polypeptide further comprises an Fc fragment of immunoglobulin.

9. A PD-L1 polypeptide according to any one of claims 1 to 8, comprising the amino acid sequence shown in SEQ ID NO: 21 or 22, encoded by a polynucleotide including the nucleotide sequence of SEQ ID NO: 23 or 24.

10. A polynucleotide encoding the PD-L1 polypeptide according to any one of claims 1 to 9.

11. A PD-L1 polypeptide according to any one of claims 1 to 9 or a polynucleotide according to claim 10, for use as a pharmaceutical.

12. A PD-L1 polypeptide according to any one of claims 1 to 9 or a polynucleotide according to claim 10, for use in the treatment and / or prevention of organ failure in subjects suffering from sepsis, for use in the treatment of immunodeficiency, and / or for use in immuno-oncological treatment.

13. The PD-L1 polypeptide or polynucleotide for use according to claim 12, wherein the immunodeficiency is lupus erythematous.

14. The PD-L1 polypeptide or polynucleotide for use according to claim 12, wherein the organ failure is caused by an inflammatory response.

15. The PD-L1 polypeptide or polynucleotide for use according to claim 14, wherein the inflammatory response is systemic inflammatory response syndrome (SIRS) or sepsis.

16. A host cell comprising a PD-L1 polypeptide according to any one of claims 1 to 9, and / or a polynucleotide according to claim 10.

17. (i) the step of expressing the polynucleotide described in claim 10 in a host cell; and (ii) Steps to obtain PD-L1 polypeptide A method for producing the PD-L1 polypeptide according to any one of claims 1 to 9, comprising:

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