RU2772733C2 - Hmgb1 mutants - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: polypeptide of high-mobility group protein B1 (hereinafter – HMGB1) is proposed, while in the specified HMGB1 polypeptide, at least one, preferably two, more preferably three, most preferably all four tyrosine residues are substituted with an amino acid residue. In the present invention, polynucleotide encoding polypeptide according to the present invention, a vector containing the specified polynucleotide, and a host cell containing the specified polypeptide, the specified polynucleotide and/or the specified vector are also proposed. In addition, within the present invention, methods, kits, and applications related to the specified inventions are also proposed.

EFFECT: group of inventions allows for obtaining HMGB1 polypeptide, the use of which causes increased death of cancer cells.

37 cl, 1 dwg, 1 tbl, 2 ex

Description

In accordance with the present invention, there is provided a high-mobility group box 1 B1 protein polypeptide (HMGB1; high-mobility group box 1), while in the specified HMGB1 polypeptide in positions corresponding to the positions of amino acids Y109, Y144, Y155 and / or Y162 of human HMGB1, at least one, preferably two, more preferably three, most preferably all four tyrosine residues are replaced with an amino acid residue independently selected from glutamic acid, glutamine, aspartic acid, asparagine, homoglutamic acid (2-aminohexanedioic acid), and homoglutamine (2,6-diamino -6-oxohexanoic acid). In addition, the present invention provides a polynucleotide encoding a polypeptide according to the present invention, a vector containing the specified polynucleotide, and a host cell containing the specified polypeptide, the specified polynucleotide and/or the specified vector. In addition, the present invention provides methods, kits and applications associated with these inventions.

The high mobility group B1 protein (HMGB1) belongs to the family of nuclear proteins, the High Mobility Group (HMG), so named because of the unusually high mobility of its members on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These proteins are the second most abundant chromatin-associated proteins after histones, these proteins play an architectural role in the nucleus of the eukaryotic cell, which is that they bend, deform, or otherwise modify the DNA conformation, thereby also modifying the binding of transcription factors to DNA. . HMG proteins have been associated with a number of different disorders, such as certain types of benign tumors and autoimmune diseases. In addition, the release of large amounts of HMGB1, in particular from natural killer (NK) cells, is a key factor in dendritic cell activation (Saidi et al. (2008), PloS one 3, e3601) and chemotaxis (Yang et al. (2007) , Journal of leukocyte biology 81, 59-66). HMGB1 also exhibits exceptional antimicrobial activity leading to rapid bacterial killing (Zetterstrom et al., (2002), Pediatric research 52, 148-154).

Endogenous HMGB1 is also actively involved in the energy metabolism of cells and organs. Mice with HMGB1 knockout are unable to use glycogen stores in hepatocytes and die due to perinatal hypoglycemia. Glucose may save them for a short time, but the animals still die after a few days due to severe atrophy of the internal organs, muscles and adipose tissue (Calogero et al. (1999), Nature genetics 22, 276-280). Incubation of mouse muscle tissue with HMGB1 ex vivo results in rapid wasting of muscle fibers, and HMGB1 is found to be elevated in the myoplasm of patients suffering from polymyositis (Grundtman et al. (2010), The FASEB journal 24, 570-578). Thus, both deficiency and excess of HMGB1 strongly affect the energy metabolism of cells.

Extracellular HMGB1 is a potent cytokine and a strong activating factor for macrophages and other cells of the immune system, leading to a massive inflammatory response. Because of this mechanism, HMGB1 is involved in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. On the other hand, high levels of HMGB1 in the blood indicate serious or life-threatening inflammatory conditions such as sepsis. To combat such pathological conditions associated with HMGB1, inhibitors of HMGB1 function have been described, for example, inhibitory antibodies or fragments thereof, mutated in the box A domain of HMGB1 variants or polymer conjugates of the box A domain (US 6,468,533, WO 02/074337, US 2003/0144201 , WO 2006024547 and WO 2008031612). At the same time, HMGB1 has been proposed as an anti-cancer agent (US 2011/0123483 A1, Gdynia et al. (2016), Nature Communications 7:10764. doi: 10.1038/ncomms 10764).

Several structural motifs have been described in HMGB1 proteins: two DNA-binding domains (box A and box B), two nuclear localization sequences, and a C-terminal acidic domain. HMGB1 proteins can undergo strong post-translational modification by acetylation, methylation, ADP-ribosylation, phosphorylation, or glycosylation. Acetylation of nuclear localization sites is a signal that leads to active secretion of the HMGB1 protein by activated cells of the immune system. In addition to active secretion, HMGB1 is also passively released from necrotic cells.

In addition to the surgical removal of tumor tissue, cancer treatment is essentially based on the use of drugs and/or therapies that have a negative effect on actively dividing cells. By itself, such treatment harms non-tumor dividing cells and tissues of the body, which leads to most of the well-known and serious side effects of chemotherapy and radiotherapy, including nausea, digestive disorders, fatigue, hair loss, etc. Thus, obtaining new therapeutic agents with novel, as yet unknown mechanisms of action that would potentially allow a reduction in the dose of chemotherapy and/or radiotherapy, while attenuating their side effects, is desirable. Providing such agents that engage novel pathways to kill cancer cells could potentially also help eliminate cancer stem cells that are able to survive chemotherapy into a dormant state and have recently been found to be responsible for at least a fraction of relapses and metastases.

In recent years, it has been shown that NK cells can potentially be used in the treatment of cancer. The main advantage of these cells is that they belong to the innate immune system and do not require antigen-specific activation. NK cells can be divided into three main subgroups, depending on their ability to bind and destroy sensitive target cells: free, binding and killer NK cells (Jewett et al. (1996), J Clin Immunol. 16(1):46 -54). Non-binding and non-killer NK cells are the most immature and can be activated to become binding and killer, and the ability of NK cells to secrete TNF-alpha has been found to correlate with the functional stage of NK cell maturation (Jewett et al., loc. cit).

As cancer immunotherapy becomes more promising, more effective, and more accessible, optimal control of its potential toxicity becomes more important (Lee et al. (2014), Blood 124(2): 188). In particular, Cytokine release syndrome (CRS), which is a syndrome associated with increased levels of cytokines in the circulation, is a potentially life-threatening condition that has been observed after the administration of natural and bispecific antibodies and, more recently, after therapy. cancer with T or NK cells (Lee et al., loc. cit.).

Therefore, there is a need for improved methods of treating cancer. This problem is solved by means and methods disclosed below in the framework of this application.

Thus, within the scope of the present invention, there is provided a high mobility group B1 protein polypeptide (polypeptide) HMGB1, wherein said HMGB1 polypeptide has at least one, preferably two, more preferably three, most preferably all four tyrosine residues in positions corresponding to amino acid positions. 22, 57, 68 and 75 of SEQ ID NO: 1 are replaced with an amino acid residue independently selected from glutamic acid, glutamine, aspartic acid, asparagine, homoglutamic acid (2-aminohexanedioic acid), and homoglutamine (2,6-diamino-6 -oxohexanoic acid).

As used below, terms such as "have", "comprise" or "include" or any arbitrary grammatical forms thereof are non-exclusive. Thus, these terms can refer both to a situation in which, apart from the feature introduced by these terms, there are no other features of the object in the given context, or to a situation in which one or more features may be present. For example, the expressions “A has B”, “A contains B”, and “A includes B” can refer to a situation in which A has no other elements than B (that is, a situation in which only and consists exclusively of B ), but also to the situation in which A has one or more additional elements besides B, such as element C, elements C and D, or even additional elements.

In addition, the terms "preferably", "more preferably", "most preferably", "specifically", "more specifically", "especially", "in particular" and the like, as used below, are used in conjunction with optional features, and do not limit additional features. Thus, all features described by these terms are optional and are not intended to limit the scope of the claims in any way. The person skilled in the art will appreciate that the invention may also be practiced using alternative features. Likewise, features that are described by the expression "in an embodiment of the invention" or similar expressions are intended to be used as optional features without any limitation as to other possible embodiments of the invention, without any limitation as to the scope of the invention, and without any or restrictions on the possibility of combining the features thus described with other optional or mandatory features of the present invention. In addition, unless otherwise indicated, the term "approximately" indicates that the indicated value has an accuracy interval of ±20%.

As used herein, the term "high mobility group B1 protein polypeptide" (HMGB1 polypeptide) refers to a member of the group of high mobility polypeptides that are known to those skilled in the art, and has the modifications indicated in the claims, and includes, but is not limited to, partial amino acid sequences or derivatives thereof as follows. Thus, according to the present invention, the HMGB1 polypeptide will preferably not be a naturally occurring HMGB1 polypeptide. Also preferably, the HMGB1 polypeptide of the present invention is not human HMGB1, and is not HMGB1 phosphorylated at amino acids Y109, Y144, Y155 and/or Y162. Preferably, the HMGB1 polypeptide according to the present invention has the activity described in the present invention, preferably cytotoxic activity and/or the ability to activate NK cells, preferably according to the Examples of this application. Preferably, the HMGB1 polypeptide is a derivative of the human HMGB1 polypeptide (Genbank ACC No: NP 002119.1 GL4504425, SEQ ID NO: 3). Methods of analysis suitable for measuring the above activities are also described in the attached Examples. The HMGB1 polypeptide may be purified from cells or tissues, or it may be chemically synthesized or, preferably, recombinantly produced. The HMGB1 polypeptide may contain additional amino acids that may serve as a label for purification or detection, and/or the HMGB1 polypeptide may be contained in a fusion polypeptide of the present invention.

Thus, in a preferred embodiment, the polypeptide or peptide of the present invention further comprises a detectable label. The term "detectable label" refers to a number of amino acids added or built into the structure of a polypeptide or peptide. Preferably, the label should be added to the C- or N-terminus of the polypeptide or peptide, the above-described series of amino acids may, for example, allow recognition of the peptide or polypeptide by an antibody that specifically recognizes the label, or it should participate in the formation of a functional conformation, such as a chelator , or it must provide visualization with fluorescent labels. Preferred tags are Myc-tag, FLAG-tag, 6-His-tag, HA-tag, GST-tag or GFP-tag. All of these labels are well known in the art.

A preferred embodiment is an HMGB1 polypeptide comprising the B-Box motif of an HMGB1 polypeptide, preferably Box B human HMGB1, more preferably comprising a derivative of SEQ ID NO: 1 with the mutations set forth herein, more preferably including a mutated Box B human HMGB1 as described in this application below.

In the HMGB1 polypeptide of the present invention, at least one, preferably two, more preferably three, most preferably all four tyrosine residues at positions corresponding to amino acid positions 22, 57, 68 and 75 of SEQ ID NO: 1 are replaced by an amino acid residue , independently selected from glutamic acid, glutamine, aspartic acid, asparagine, homoglutamic acid (2-aminohexanedioic acid), and homoglutamine (2,6-diamino-6-oxohexanoic acid). The person skilled in the art will understand that the above amino acid positions correspond to amino acids Y109, Y144, Y155 and Y162 of human HMGB1 (SEQ ID NO: 3). Preferably, in said HMGB1 polypeptide, at least one, preferably two, more preferably three, most preferably all four tyrosine residues at said positions are replaced with an amino acid residue independently selected from glutamic acid, glutamine, aspartic acid, and asparagine. More preferably, in said HMGB1 polypeptide, at least one, preferably two, more preferably three, most preferably all four tyrosine residues at said positions are replaced by glutamic acid residues or glutamine residues. Even more preferably, in said HMGB1 polypeptide, at least one, preferably two, more preferably three, most preferably all four tyrosine residues at said positions are replaced by glutamine residues; thus, preferably, the HMGB1 polypeptide preferably comprises the amino acid sequence of SEQ ID NO: 5, more preferably comprises the amino acid sequence of SEQ ID NO: 6. Most preferably, said HMGB1 polypeptide has at least one, preferably two, more preferably three, most preferably all four tyrosine residues in these positions were replaced by glutamic acid residues; thus, it is preferred that the HMGB1 polypeptide comprises the amino acid sequence of SEQ ID NO: 7, more preferably the amino acid sequence of SEQ ID NO: 8.

In an exemplary embodiment, the HMGB1 polypeptide or derivative thereof is an oligophosphorylated HMGB1 polypeptide, in particular an HMGB1 polypeptide in which at least one, preferably two, more preferably three, and most preferably all four, tyrosine residues are replaced by glutamine residue(s), asparagine or homoglutamine. In a more preferred embodiment, the HMGB1 polypeptide or derivative thereof is a phosphomimetic of an HMGB1 polypeptide, in particular an HMGB1 polypeptide in which at least one, preferably two, more preferably three, most preferably all four tyrosine residues are replaced by glutamic acid residue(s), aspartic acid or homoglutamic acid.

Preferably, the HMGB1 polypeptide or derivative thereof is an HMGB1 polypeptide as described herein above; preferably, the term also includes a polypeptide whose amino acid sequence is at least 70% identical to the HMGB1 or Box B polypeptide of the HMGB1 polypeptide as defined in the present invention, and, in addition, has the ability to enhance the death of cancer cells, for example, SW480 cells. Thus, preferably, the HMGB1 polypeptide at a concentration of 0.8 μM has the ability to cause increased cell death in SW480 cell culture compared to a polypeptide comprising or preferably consisting of the amino acid sequence of SEQ ID NO: 3. More preferably, the HMGB1 polypeptide at a concentration of 0.8 μM has the ability to cause increased cell death in SW480 cell culture compared to a polypeptide comprising or preferably consisting of the amino acid sequence of SEQ ID NO: 6. Also preferably, the term preferably also includes a polypeptide comprising the amino acid sequence, at least 70% identical to an HMGB1 or Box B polypeptide of an HMGB1 polypeptide according to the invention, and having the ability to stimulate NK cells to kill cancer cells; more preferably, the increase in the ability of NK-92 CI cells to kill SW480 cells in cell culture at a concentration of 0.8 μm is higher compared to a polypeptide comprising, preferably consisting of the amino acid sequence of SEQ ID NO: 3; more preferably, the increase in the ability of NK-92 CI cells to kill SW480 cells in cell culture at a concentration of 0.8 μM is higher compared to a polypeptide comprising, preferably consisting of the amino acid sequence of SEQ ID NO: 6. Also preferably, the term, preferably, also includes a polypeptide having an amino acid sequence at least 70% identical to the HMGB1 or Box B polypeptide of the HMGB1 polypeptide as described in this invention, and having the ability to stimulate the maturation of NK cells, and also, preferably, at a concentration of 0.8 μm to increase secretion tumor necrosis factor alpha (TNF-alpha) by a NK-92 CI cell culture is stronger than a polypeptide comprising or preferably consisting of the amino acid sequence of SEQ ID NO: 3. More preferably, said HMGB1 polypeptide at a concentration of 0.8 μM increases the secretion of the factor tumor necrosis alpha (TNF-alpha) by cultured NK-92 CI cells is stronger than polypeptide, BK consisting of, or preferably consisting of, the amino acid sequence of SEQ ID NO: 6.

The term "TNF-alpha" or "tumor necrosis factor alpha" is known to the person skilled in the art as a cytokine involved in the process of systemic inflammation, in particular in its acute phase reaction. Preferably, TNF-alpha is human TNF-alpha.

Preferably, the HMGB1 polypeptide contains a nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95% and most preferably at least 98 % sequence identity with SEQ ID NO: 1 and/or contains a nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95% and , most preferably at least 98% sequence identity with SEQ ID NO: 3, and more preferably has at least one, more preferably at least two, most preferably all three of the activities described above. More preferably, the HMGB1 polypeptide contains a nucleic acid sequence of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95%, and most preferably at least 98% sequence identity with SEQ ID NO: 5-8, preferably SEQ ID NO: 6 or 8 and more preferably has at least one, more preferably at least two, most preferably all three of the above activities.

The term "agent providing an HMGB1 polypeptide" as used herein refers to any agent or composition capable of providing or releasing an HMGB1 polypeptide, in accordance with the present invention, into a biological system. Preferably, the agent providing the HMGB1 polypeptide is used at a dose that induces a plasma concentration of 1 nM to 5 µM, more preferably 10 nM to 1 µM, most preferably 50 nM to 900 nM. Preferably, the term also includes an agent that specifically binds to a tumor cell containing the HMGB1 polypeptide. Preferably, said tumor cell specific binding agent is an antibody, an aptamer, a lectin, or the like. Preferably also, the term HMGB1 polypeptide providing agent refers to an HMGB1 secreting cell induced to secrete an HMGB1 polypeptide. Cells that can be induced to secrete HMGB1, as well as methods for providing said secretion, are known in the art and include, preferably, the methods given in the examples; preferred cell types that can be induced to secrete HMGB1 are macrophages and NK cells. Also preferably, the term HMGB1 polypeptide providing agent refers to an expressible polynucleotide encoding an HMGB1 polypeptide. As known to one of skill in the art, such a polynucleotide is preferably incorporated into a vector or host cell, preferably as described herein below.

The term "derivative" as used in the context of a chemical component of the present invention refers to a molecule having a structure associated with a chemical compound according to the present invention. Preferably, the derivative can be obtained from the chemical compound according to the present invention by no more than three, more preferably no more than two, most preferably one chemical derivatization reaction. Preferably, this derivative is a component that is metabolized in the body of a mammal, preferably a human, into a chemical compound according to the present invention. Also preferably, the derivative is a component from which the chemical compound of the present invention can be obtained by hydrolysis. In the event that said chemical compound is a peptide or polypeptide, then the corresponding derivative is preferably at least somewhat similar, as described in the present invention, to the component from which it is derived. In accordance with the present application, a derivative of the HMGB1 polypeptide has at least the activity in inhibiting cancer cells, as described here above.

The term "derivative" representing a polypeptide or peptide includes variants of the amino acid sequence of said polypeptide or peptide, these variants are at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity of the polypeptide or peptide, and these variants retain the function of the polypeptide or peptide according to the present invention. Percentages are preferably calculated from the entire amino acid sequence. A number of programs based on various algorithms are available to those skilled in the art for comparing different sequences. In this case, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. For sequence comparison, the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153), Gap and BestFit (Needleman and Wunsch (J. Mol Biol. 48; 443-453 (1970)) or Smith and Waterman Adv. Appl. Math. 2; 482-489 (1981) included in the software (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)). Sequence identity values given above in percent (%) should preferably be determined using the GAP program considering the entire sequence with the following settings: "Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000" .

In addition, derivatives of polypeptides or peptides also encompass possible variants of the specific amino acid sequences described above, which may represent orthologs, paralogs, or other homologues of certain polypeptides or peptides. These variants preferably contain an amino acid sequence, characterized in that the sequence can be derived from the aforementioned polypeptide or peptide sequences by at least one amino acid substitution and/or addition and/or deletion.

The term "derivative" also includes chemically modified polypeptides, for example, polypeptides containing modified amino acids or polypeptides that are, for example, biotinylated or linked to fluorophores such as fluorescein or Cy 3 , conformationally restricted, for example, by the introduction of a disulfide bridge or crosslinking (Walensky 2004, Science 305 (5689): 1466-1470), or associated with cell entry polypeptides or protein transduction domains (Snyder 2004, Pharm Res 21 (3): 389-393). Such modifications are capable of improving the biological properties of the polypeptides, eg cell entry, binding, stability, or may be used as detection labels.

Preferably, the HMGB1 polypeptide is provided as a pharmaceutically compatible formulation. The terms "pharmaceutically compatible composition" and "pharmaceutical composition" as used in this application refer to compositions comprising at least one compound according to the present invention and one or more pharmaceutically acceptable carriers. Compounds according to the present invention can be prepared in the form of pharmaceutically acceptable salts. Preferred salts are acetate, methyl ether, HCl, sulfate, chloride and the like. Pharmaceutical compositions are preferably administered topically or more preferably systemically. Suitable routes of administration commonly used for administering drugs are oral, intravenous or parenteral administration, as well as inhalation. However, depending on the nature and mode of action of the compound, pharmaceutical compositions may also be administered by other routes. In addition, the compounds can be administered in combination with other drugs, or as part of a commonly used pharmaceutical composition or as separate pharmaceutical compositions, as described in the present invention, while these individual pharmaceutical compositions can be provided in the form of a composite kit.

The compounds are preferably administered in conventional dosage forms obtained by combining drugs with standard pharmaceutical carriers, according to conventional procedures. These procedures may include mixing, granulating and compressing or dissolving the ingredients depending on the desired finished form. The form and type of the pharmaceutically acceptable carrier or diluent is determined by the amount of active ingredient to be combined with, by means of administration, and other well known variables.

The carrier(s) must be acceptable, which means that it is compatible with the other ingredients of the composition and does not endanger the recipient. The carrier used may be, for example, a solid, gel or liquid. Examples of solid carriers are lactose, gypsum, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, and the like. Examples of liquid carriers are sodium phosphate buffer, syrup, oil, including, for example, peanut and olive oil, water, emulsions, various types of wetting agents, sterile solutions, and the like. Similarly, the carrier or diluent may include any sustained release component well known in the art, such as glyceryl mono- or glyceryl distearate, alone or mixed with a wax. Suitable carriers named include those mentioned above and others well known in the art, according to, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

The diluent(s) are chosen so as to avoid affecting the biological activity of the active compound or compounds. Examples of such diluents are distilled water, saline, Ringer's solutions, dextrose solution and Hank's solution. In addition, the pharmaceutical composition may include other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

A therapeutically effective dose refers to the amount of compounds used in a pharmaceutical composition of the present invention that prevents, ameliorates, or cures the symptoms associated with the disease or condition of the present invention. The therapeutic efficacy and toxicity of these compounds can be determined by standard pharmaceutical approaches in cell cultures or in animal experiments, for example, semi-effective dose (ED50, ED50 - dose that is therapeutically effective for 50% of the population) and semi-lethal dose (LD50, LD50 - dose that is lethal for 50% of the population). The ratio between doses producing therapeutic and toxic effects is the therapeutic index, and it can also be expressed as the LD50/ED50 ratio.

The dosage regimen will be determined by the attending physician and other clinical factors, preferably in accordance with one of the methods described above. As is well known in the medical field, dosages for each individual patient depend on many factors, including size, body surface area, age, particular compound being administered, sex, time and route of administration, general health, and other co-administered drugs. Progress can be monitored through periodic evaluation. The usual dose may be, for example, in the range from 1 to 1000 μg; however, doses above or below this approximate range are also contemplated, especially considering the aforementioned factors. Typically, the regimen for regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, then it should also be in the range of 1 µg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be tracked through periodic evaluation. Preferred doses and concentrations of the compounds of this invention are indicated elsewhere in the document. The pharmaceutical compositions and preparations mentioned herein are preferably administered at least once to treat, ameliorate, or prevent the diseases or conditions listed herein. However, said pharmaceutical compositions may be administered more than once, for example, from one to four times a day for an unlimited number of days.

Specific pharmaceutical compositions are prepared by methods known in the pharmaceutical art and include at least one active compound of the present invention mixed or otherwise associated with a pharmaceutically acceptable carrier or diluent. For the manufacture of these specific pharmaceutical compositions, the active compound(s) will typically be mixed with a carrier or diluent, or enclosed or encapsulated in capsules, sachets, cachets, paper, and other suitable containers or carriers. The formulations obtained must be adapted to the mode of administration, ie presented in the form of tablets, capsules, suppositories, solutions, suspensions or the like. Dosage recommendations should be indicated in the prescribing or use instructions so that necessary dose adjustments can be made depending on the characteristics of the intended patient.

In the course of the work underlying the present invention, it was found that the mutated HMGB1 polypeptides of the present invention have increased activities compared to wild-type HMGB1, with Glu-HMGB1 being more active than Gln-HMGB1. In addition, it has been found that, by using different HMGB1 mutants, it is possible to regulate the level of cytotoxicity for cancer cells and the activation of the immune system as needed.

The definitions introduced above apply mutatis mutandis to the following: The additional definitions and explanations given below also apply mutatis mutandis to all described embodiments described in this application.

The present invention further relates to a polynucleotide y encoding a polypeptide according to the present invention.

The term "polynucleotide" as used herein refers to a polynucleotide containing a nucleotide sequence that encodes a polypeptide having the biological activity described above, preferably including a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 and/or 8 or their derivative, as described here above. It should be understood that a polypeptide with the amino acid sequence described in detail above, due to the degeneracy of the genetic code, may be encoded by more than one kind of polynucleotide. Moreover, the term "polynucleotide" as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides. Said polynucleotide variants may be orthologues, paralogs, or other homologues of a polynucleotide of the present invention. Polynucleotide variants preferably comprise a nucleotide sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequences by at least one nucleotide substitution, addition and/or deletion, while the variant nucleotide sequence must still encode a polypeptide having mutation(s) and activity as described above. Variants also include polynucleotides, with a nucleotide sequence that is capable of hybridizing to the aforementioned specific nucleotide sequences, preferably under stringent hybridization conditions. These stringent conditions are known to the person skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. Preferred Example of Stringent Conditions Hybridization Hybridization conditions in 6x concentrated sodium chloride/sodium citrate (=SSC) at approximately 45°C followed by single or multiple washes in 0.2x SSC, 0.1% SDS at 50-65°C . Those skilled in the art will recognize that these hybridization conditions differ depending on the type of nucleic acid and, for example, in the presence of organic solvents, temperature and buffer concentration. For example, under "standard hybridization conditions", the temperature varies, depending on the type of nucleic acid, from 42 to 58°C in an aqueous buffer with a concentration of 0.1 to 5x SSC (pH 7.2). If an organic solvent is present in the above buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C. Hybridization conditions for DNA:DNA hybrids are preferably, for example, 0.1 x SSC buffer and 20°C to 45°C, preferably 30 to 45°C. Conditions for the formation of DNA hybrids: RNA is preferably, for example, 0.1× SSC solution and from 30 to 55°C, preferably from 45 to 55°C. The above hybridization temperatures are defined, for example, for a nucleic acid of approximately 100 bp in length. (= pairs of nucleotides) in length and with a content of G+C (G+C) 50% in the absence of formamide. The person skilled in the art knows how to determine the necessary hybridization conditions by referring to reference books, incl. guide mentioned above.

Alternatively, polynucleotide variants can be generated by PCR based pathways such as DNA amplification based on mixed oligonucleotide primers, in particular the use of degenerate primers against the conserved domains of the polypeptides of the present invention. Conservative domains of the polypeptides of the present invention can be identified by comparing the nucleotide sequences of a polynucleotide or the amino acid sequence of a polypeptide as described above. Suitable PCR conditions are well known in the art. DNA or cDNA from AAV (adeno-associated virus) can be used as a template. In addition, polynucleotide variants comprising nucleotide sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acid sequences detailed above. Percent identity values are preferably calculated as stated above.

A polynucleotide comprising a fragment of any of the aforementioned nucleotide sequences is also included within the scope of a polynucleotide according to the present invention. The fragment must encode a polypeptide that will still have the biological activity described above. Accordingly, the polypeptide may contain or consist of domains of the polypeptide according to the present invention, responsible for the specified biological activity. A fragment as understood herein preferably comprises at least 50, at least 100, at least 250, or at least 500 consecutive nucleotides of any of the aforementioned nucleotide sequences, or encodes an amino acid sequence of at least 20, at least at least 30, at least 50, at least 80, at least 100 or at least 150 consecutive amino acids of any of the above amino acid sequences.

The polynucleotide of the present invention should preferably be provided either as an isolated polynucleotide (ie, isolated from its natural environment) or in a genetically modified form. The polynucleotide is preferably DNA, including cDNA, or RNA. The term encompasses both single-stranded and double-stranded polynucleotides. In addition, it also includes chemically modified polynucleotides, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified, such as biotinylated polynucleotides.

The polynucleotides of the present invention either essentially consist of the aforementioned nucleic acid sequences or contain the aforementioned nucleic acid sequences. Therefore, they may also contain additional nucleic acid sequences. In particular, the present invention also relates to a vector comprising the above-described polynucleotide according to the present invention.

The present invention also relates to a vector comprising a polynucleotide according to the present invention.

The term "vector" preferably encompasses phage, plasmid, viral or retroviral vectors, as well as artificial chromosomes such as bacterial and yeast artificial chromosomes. More preferably, the term refers to a vector derived from a virus, and said virus preferably targets tumor cells (oncotropic virus) or targets cancer cells (oncolytic virus). In addition, this term refers to targeted constructs that provide random or site-specific integration into genomic DNA. Such targeted constructs preferably contain DNA of sufficient length for either homologous or heterologous recombination. The vector comprising the polynucleotide of the present invention preferably also includes selectable markers for propagation and/or selection in the host. The vector can be incorporated into the host cell in a variety of ways well known in the art. For example, the plasmid vector can be delivered in a precipitate, such as calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon clusters, such as fullerenes. Alternatively, the plasmid vector can be introduced using heat shock or electroporation. In case the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line prior to use on host cells. Viral vectors can be replication competent or replication defective.

Preferably, in the vector of the present invention, the polynucleotide of the present invention is operably linked to sequences for expression in prokaryotic or eukaryotic cells or isolated fractions of said cells. Expression of said polynucleotide implies transcription of the polynucleotide, preferably into translational mRNA. Regulatory elements for expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They preferably include regulatory sequences to initiate transcription and optionally poly-A signals to terminate transcription and stabilize the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the lac, trp, or tac promoters in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoters for yeast or CMV-, SV40 - RSV promoters (Rous sarcoma virus), CMV enhancer, 8V40 enhancer and globin intron for mammalian and other animal cells. In addition, inducible expression control sequences can be used in the expression vector of the present invention. Such inducible vectors may preferably contain tet or lac operator sequences or sequences activated by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. In addition to elements responsible for starting transcription, such regulatory elements may also include transcriptional termination signals, such as the SV40-poly-A site or the tk-poly-A site, 5'-3' from the polynucleotide. In this context, suitable expression vectors known in the art are, for example, pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (InVitrogene) or pSPORTl (GIBCO BRL). Expression vectors derived from viruses, such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papillomavirus, can be used to deliver the polynucleotides or the vector of the present invention to target cell populations. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the methods described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).

The present invention also relates to a host cell containing a polypeptide, polynucleotide and/or vector according to the present invention.

The term "host cell" preferably refers to a cell compatible with administration to a subject. More preferably, said cell is immunologically compatible with the subject. Most preferably, the cell is a cell obtained from the specified subject. The host cell of the present invention is preferably a cell with a propensity to migrate to the location of cancer cells. More preferably, the host cell is an immune cell, most preferably an immune system cell capable of selectively recognizing specific tumor antigens, eg, a tumor antigen specific T cell.

Moreover, the host cell containing the polynucleotide or vector of the present invention is preferably a cell capable of producing the HMGB1 polypeptide of the present invention, such as a bacterial, yeast, insect or mammalian cell.

The present invention also relates to a polypeptide, polynucleotide, vector and/or host cell according to the present invention for use as a drug.

In addition, the present invention relates to a polypeptide, polynucleotide, vector and/or host cell according to the present invention for use in cancer treatment and/or immunomodulation.

The term "cancer" as used herein refers to a disease in an animal, preferably a human, characterized by inappropriate and/or uncontrolled growth of a group of body cells. This uncontrolled growth may be accompanied by penetration into the surrounding tissue and destruction of the surrounding tissue and possibly the spread of abnormally proliferating cells to other areas of the body. Thus, preferably, the polynucleotide, vector and/or host cell of the present invention is for use in the treatment of cancer.

Preferably, the type of cancer was selected from the following list: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid-rhabdoid tumor, basalioma, cholangiocarcinoma, bladder cancer, glioma brainstem, breast cancer, Burkitt's lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, optic tract glioma , sa Kaposi's carcinoma, larynx cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel's carcinoma, mesothelioma, oral cancer, multiple endocrine neoplasia, multiple myeloma, mycosis fungoides, nasal cavity and paranasal sinuses cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor (NE)/carcinoma , non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial tumor, ovarian germ cell tumor, ovarian tumor with low malignant potential, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, cancer parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, kidney cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Cesari's syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma cell carcinoma, squamous cell neck cancer, testicular cancer, throat cancer, thymus cancer, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

Most preferably, the cancer is leukemia, lymphoma, HPV-associated cancer, colorectal carcinoma, gastric cancer, pancreatic cancer, lung cancer, brain cancer, or breast cancer. Preferably, the colorectal carcinoma is a colon carcinoma. Even more preferably, the cancer is a leukemia, most preferably chronic lymphocytic leukemia (CLL).

The term "cancer therapy" includes all means and methods known to a person skilled in the art, suitable for the treatment of cancer. Preferably, the term represents a therapy approved for use in humans. The term "cancer cellular immunotherapy" preferably represents a method of treating cancer comprising administering cells, preferably autologous cells, to a subject, e.g., preferably B cells, T cells and/or NK cells, or progenitor cells thereof, e.g. . Preferably, said cells are NK cells or their progenitors. More preferably, said cells are tumor-recognizing NK cells or their precursors. Even more preferably, said cells are autologous tumor-recognizing NK cells.

Preferably, cancer treatment and immunomodulation includes cancer cell immunotherapy. Also preferably, cancer treatment and immunomodulation include stimulation of TNF-alpha secretion by NK cells, preferably stimulation of maturation of NK cells. In addition, preferably, cancer treatment and/or immunomodulation include the prevention of side effects, more preferably include the prevention of cytokine release syndrome (CRS).

The terms "NK cells" and "natural killer cells" (NK-cells) are known to those skilled in the art and represent cytotoxic lymphocytes that are part of the innate immune system and which, through receptors including NKG2D, NKp44, NKp46, NKp30, etc. ., recognize a number of ligands, including ULBP and MICA, commonly expressed on tumor cells. The term "NK cell maturation" is also understood by those skilled in the art. Preferably, NK cell maturation consists in increasing the fraction of NK cells capable of binding and/or killing tumor cells in the total NK cell fraction.

"Side effects" that relate to HMGB1 treatment and cellular immunotherapy, as well as related indicators, symptoms, and suitable treatments, are known to those skilled in the art (e.g., as reviewed by Teu, S-K (2014), Clinical and Translational Immunology 3 , el7; doi: 10.1038/cti.2014.11). Side effects preferably include neurotoxicity (leading, for example, to hearing loss, seizures or coma), vitiligo, colitis, elevated liver enzymes, acute pulmonary infiltrates, B cell depletion, hypogammaglobulinemia, and cytokine release syndrome (CRS). The term "cytokine release syndrome (CRS)" and its symptoms and markers are known to those skilled in the art, for example, from Lee et al. (loc. cit.). Preferably, the marker for advanced or delayed CRS is C-reactive protein, IL-6 and/or TNF-alpha, preferably TNF-alpha.

In addition, the present invention relates to a method of treating cancer and/or inducing immunomodulation in a subject, namely:

a) bringing the subject into contact with a polypeptide corresponding to any of embodiments 1-18, a polynucleotide corresponding to embodiment 19, a vector corresponding to embodiment 20, and/or a host cell corresponding to embodiment 21, and thus,

b) treating cancer and/or inducing immunomodulation in said subject.

The methods of the present invention are preferably in vivo methods. In addition, they may include steps in addition to those directly mentioned above. For example, the next step for a method of treating cancer and/or inducing immune modulation of the response may be, for example, surgical removal of tumor tissue before or after administration of said pharmaceutically active compounds. Preferably, the methods are carried out in such a way that the steps follow in the suggested order. In addition, one or more of these steps can be carried out on automated equipment.

The term "subject" as used herein refers to an animal, preferably a farm or domestic animal, more preferably a mammal, most preferably a human. Preferably, said subject is a subject suffering from cancer, more preferably as described above.

The term "treatment" refers to the alleviation of the diseases and conditions described in this application, or the symptoms that accompany them to a high degree. The specified treatment, according to the present application, also includes, preferably, the complete restoration of health from the relevant diseases or disorders according to the present application. It should be understood that the treatment, as used in accordance with the present invention, may not be effective for all subjects to be treated. However, the term should preferably require that a statistically significant proportion of subjects suffering from the disease and disorder presented here could be successfully treated. Whether this proportion is statistically significant can easily be determined by one of ordinary skill in the art using widely known statistical evaluation tools, e.g. 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 treatment should be effective in at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in a given cohort or population.

The term "immunomodulation" as used herein refers to inducing a change in the degree of response of a subject's immune system to an antigen. Preferably, the immune modulation is activation. Preferably, the antigen is a tumor antigen, more preferably a tumor-specific antigen, ie preferably expressed and/or presented only by the subject's tumor cells. More preferably, modulating the subject's immune response is to induce secretion of TNF-alpha by NK cells, most preferably to induce maturation of NK cells, preferably as described above.

The present invention also relates to a method of providing a subject with HMGB1 treatment, preventing a side effect, preferably preventing the induction of cytokine release syndrome (CRS), which includes

a) administering to said subject an agent providing HMGB1,

b) determining at least one side effect parameter, preferably cytokine release syndrome (CRS), in a sample from said patient,

c) continuing to administer the HMGB1 providing agent using an alternative HMGB1 providing agent,

wherein said alternative agent for providing HMGB1 provides an HMGB1 polypeptide having a reduced ability to stimulate TNF-alpha secretion and/or a reduced ability to stimulate cell killing by NK cells, if said parameter determined in step b) indicates increased likelihood of a side effect, preferably cytokine release syndrome (CRS); and/or

in the event that said alternative agent for providing HMGB1 provides an HMGB1 polypeptide with increased ability to stimulate TNF-alpha secretion and/or reduced ability to stimulate cell killing by NK cells in the event that said parameter determined in step b) indicates a reduced likelihood of a side effect effect, preferably cytokine release syndrome (CRS),

d) thereby providing treatment for HMGB1 in the subject, preventing the side effect, preferably preventing the induction of cytokine release syndrome (CRS).

Preferably, the HMGB1 polypeptide with reduced activity in promoting TNF-alpha secretion and/or reduced activity in promoting cell killing by NK cells is the HMGB1 polypeptide of Embodiment 5, preferably a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 6. Also preferably, the HMGB1 polypeptide with reduced activity in promoting TNF-alpha secretion and/or reduced activity in promoting cell killing by NK cells is the HMGB1 polypeptide of Embodiment 4, preferably a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8. Preferably, the CRS parameter is concentration of TNF-alpha.

Also, the present invention relates to a method for modulating the immune response of a subject, including:

a) administering an agent providing an HMGB1 polypeptide to said subject, said HMGB1 polypeptide being a polypeptide according to the present invention and thus

b) modulating the immune response of said subject.

Preferably, said modulation of the subject's immune response is to induce secretion of TNF-alpha by NK cells, preferably to induce maturation of NK cells, preferably as defined above.

In addition, the present invention relates to a kit containing a polypeptide, polynucleotide, vector and/or host cell, in accordance with the present invention, in a housing (case).

The term "kit", as used in this application, refers to a collection that includes at least one of the above tools, provided separately or in combination, preferably in one container. The container also preferably further includes instructions for carrying out the method of the present invention. The components of the kit are preferably provided in a "ready to use" form, eg concentrations adjusted accordingly, etc. Preferably, NK cells and/or means for isolating these cells are also included. NK cell isolation agents are agents for specific use in the isolation of NK cells and include, in particular, antibodies that specifically bind to NK cells.

In view of the above, the following embodiments of the invention are preferred.

1. A high mobility group B1 protein (HMGB1) polypeptide, wherein said HMGB1 polypeptide has at least one, preferably two, more preferably three, most preferably all four tyrosine residues at positions corresponding to amino acid positions 22, 57, 68 and 75 the sequences of SEQ ID NO: 1 are replaced with an amino acid residue independently selected from glutamic acid, glutamine, aspartic acid, asparagine, homoglutamic acid (2-aminohexanedioic acid) and homoglutamine (2,6-diamino-6-oxohexanoic acid).

2. An HMGB1 polypeptide according to embodiment 1, characterized in that in said HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues at said positions are replaced by an amino acid residue independently selected from glutamic acid, glutamine, aspartic acid and asparagine.

3. An HMGB1 polypeptide according to embodiment 1 or 2, wherein in said HMGB1 polypeptide at least one, preferably two, more preferably three, or most preferably all four tyrosine residues at said positions are replaced by a glutamic acid residue glutamine.

4. An HMGB1 polypeptide according to any one of embodiments 1-3, wherein said HMGB1 polypeptide has at least one, preferably two, more preferably three, most preferably all four tyrosine residues at said positions replaced by glutamic acid residues.

5. An HMGB1 polypeptide according to any one of embodiments 1-4, wherein said HMGB1 polypeptide has at least one, preferably two, more preferably three, most preferably all four tyrosine residues at said positions replaced by glutamine residues.

6. An HMGB1 polypeptide according to any one of embodiments 1-5, wherein said HMGB1 polypeptide contains at least 70% nucleic acid sequence, preferably at least 80%, more preferably at least 90%, still more preferably 95%, most preferably at least 98% identical to SEQ ID NO: 1 and/or contains at least 70% nucleic acid sequence, preferably at least 80%, more preferably at least 90% %, even more preferably 95%, most preferably at least 98% identical to SEQ ID NO: 3.

7. An HMGB1 polypeptide according to any one of embodiments 1-6, wherein said HMGB1 polypeptide contains a nucleic acid sequence of at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably 95%, most preferably at least 98% identical to at least one of the sequences of SEQ ID NO: 5-8, preferably SEQ ID NO: 6 or 8.

8. An HMGB1 polypeptide according to any one of embodiments 1-7, wherein said HMGB1 polypeptide is derived from a human HMGB1 polypeptide.

9. An HMGB1 polypeptide according to any one of embodiments 1-8, wherein said HMGB1 polypeptide does not occur naturally.

10. An HMGB1 polypeptide according to any one of embodiments 1-9, wherein said HMGB1 polypeptide causes increased cell death in SW480 cell culture at a concentration of 0.8 μM compared to a polypeptide containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 3.

11. An HMGB1 polypeptide according to any one of embodiments 1-10, wherein said HMGB1 polypeptide causes increased cell death in SW480 cell culture at a concentration of 0.8 μM compared to a polypeptide containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 6.

12. An HMGB1 polypeptide according to any one of embodiments 1-11, wherein said HMGB1 polypeptide increases said cell death by at least 10%, preferably at least 20%, more preferably at least 30%, and most preferably at least 40%.

13. An HMGB1 polypeptide according to any one of embodiments 1-12, wherein said HMGB1 polypeptide causes increased killing of cultured SW480 NK-92 CI cells at a concentration of 0.8 μM compared to a polypeptide containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 3.

14. An HMGB1 polypeptide according to any one of embodiments 1-13, wherein said HMGB1 polypeptide causes increased killing of cultured SW480 NK-92 CI cells at a concentration of 0.8 μM compared to a polypeptide containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 6.

15. An HMGB1 polypeptide according to any one of embodiments 1-14, wherein said HMGB1 polypeptide increases said cell killing by at least 5%, preferably by at least 10%, more preferably by at least 15%, and most preferably by at least 20%.

16. An HMGB1 polypeptide according to any one of embodiments 1-15, wherein said HMGB1 polypeptide causes increased secretion of tumor necrosis factor alpha (TNF-alpha) by a NK-92 CI cell culture at a concentration of 0.8 μM compared to a polypeptide comprising or preferably consisting of the amino acid sequence of SEQ ID NO: 3.

17. An HMGB1 polypeptide according to any one of embodiments 1-16, wherein said HMGB1 polypeptide causes increased secretion of tumor necrosis factor alpha (TNF-alpha) by a NK-92 CI cell culture at a concentration of 0.8 μM compared to a polypeptide comprising or preferably consisting of the amino acid sequence of SEQ ID NO: 6.

18. An HMGB1 polypeptide according to any one of embodiments 1-17, wherein said HMGB1 polypeptide has said tumor necrosis factor alpha (TNF-alpha) secretion increased by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50%.

19. A polynucleotide encoding a polypeptide according to any one of embodiments 2-18.

20. A vector containing a polynucleotide according to embodiment 19.

21. A host cell comprising a polypeptide according to any one of embodiments 1-18, a polynucleotide according to embodiment 19, and/or a vector according to embodiment 20.

22. A polypeptide according to any one of Embodiments 1-18, a polynucleotide according to Embodiment 19, a vector according to Embodiment 20, and/or a host cell according to Embodiment 21, used as a medicine.

23. A polypeptide according to any of Embodiments 1-18, a polynucleotide according to Embodiment 19, a vector according to Embodiment 20, and/or a host cell according to Embodiment 21, for use in cancer treatment and/or immunomodulation.

24. A polypeptide, polynucleotide, vector and/or host cell according to embodiment 23, characterized in that said use for cancer treatment and for immunomodulation comprises cancer cellular immunotherapy.

25. A polypeptide, polynucleotide, vector and/or host cell according to embodiment 23, characterized in that said use for cancer treatment and/or immunomodulation comprises inducing secretion of TNF-alpha by NK cells, preferably inducing maturation of NK cells.

26. A polypeptide, polynucleotide, vector and/or host cell according to embodiment 23, characterized in that said use for cancer treatment and/or immunomodulation includes prevention of a side effect, preferably includes prevention of the induction of cytokine release syndrome (CRS).

27. A method for treating cancer and/or immunomodulation in a subject, comprising:

a) contacting said subject with a polypeptide according to any one of embodiments 1-18, a polynucleotide according to embodiment 19, a vector according to embodiment 20, and/or a host cell according to embodiment 21, and thus,

b) treating cancer and/or inducing immunomodulation in said subject.

28. A method of providing a subject with HMGB1 treatment to prevent a side effect, preferably preventing the induction of cytokine release syndrome (CRS), comprising:

a) administering to said subject an agent providing HMGB1,

b) determining at least one side effect parameter, preferably cytokine release syndrome (CRS), in a sample from said patient,

c) continuing to administer the HMGB1 providing agent via an alternative HMGB1 providing agent,

in the event that said alternative agent for providing HMGB1 provides an HMGB1 polypeptide characterized by a reduced ability to stimulate TNF-alpha secretion and/or a reduced ability to stimulate cell killing by NK cells in the event that said parameter determined in step b) indicates an increased likelihood side effect, preferably cytokine release syndrome (CRS); and/or

in the event that said alternative agent for providing HMGB1 provides an HMGB1 polypeptide with increased ability to stimulate TNF-alpha secretion and/or reduced ability to stimulate cell killing by NK cells in the event that said parameter determined in step b) indicates a reduced likelihood of a side effect effect, preferably cytokine release syndrome (CRS);

d) thereby providing the HMGB1 subject with treatment to prevent the side effect, preferably Cytokine Release Syndrome (CRS).

29. The method according to embodiment 28, wherein said HMGB1 polypeptide with reduced activity in promoting TNF-alpha secretion and/or reduced activity in promoting cell killing by NK cells is an HMGB1 polypeptide according to embodiment 5.

30. The method according to embodiment 28 or 29, wherein said HMGB1 polypeptide with increased activity in promoting TNF-alpha secretion and/or increased activity in promoting cell killing by NK cells is the HMGB1 polypeptide of embodiment 4.

31. The method of any one of embodiments 28-30, wherein said CRS parameter is the concentration of TNF-alpha.

32. A method for modulating the immune response of a subject, comprising:

a) administering an agent providing an HMGB1 polypeptide to said subject, wherein said HMGB1 polypeptide is a polypeptide according to any one of Embodiments 1-18 and thus,

b) modulating the immune response of said subject.

33. The method according to embodiment 32, wherein said modulation of the subject's immune response is to induce secretion of TNF-alpha by NK cells, preferably to induce maturation of NK cells.

34. A kit containing in the body a polypeptide according to any one of embodiments 1-18, a polynucleotide according to embodiment 19, a vector according to embodiment 20, and/or a host cell according to embodiment 21.

35. A kit according to embodiment 34, characterized in that said kit further comprises natural killer (NK) cells and/or a means for isolating said cells.

36. A kit according to embodiment 34 or according to embodiment 35, wherein said killer cells are tumor-recognizing NK cells.

37. The use of a polypeptide according to any one of embodiments 1-18, a polynucleotide according to embodiment 19, a vector according to embodiment 20, and/or a host cell according to embodiment 21, to obtain a drug, preferably a drug for the treatment of cancer and/or for immunomodulation.

All sources used in this document are incorporated herein by reference in relation to all of their available content, as well as available content specifically mentioned in this text.

Figure descriptions

Figure 1 Strong activation of NK cells (NK-92 CI) with Glu-HMGB1 (n=3, p<0.05). WT=HMGB1 WT, E-Variant=Glu-HMGBl (SEQ ID NO: 8), Q-Variant=Gln-HMGBl (SEQ ID NO: 6).

The following examples are intended solely to illustrate the present invention. However, the following examples should in no way be used to limit the scope of the present invention.

Example 1 Methods

Cr-51 release test

SW480 colorectal cancer cells were cultured in 96-well plates (20,000 cells/well) and labeled with ⁵¹ Cr (25 μCi/well) for 2 hours. Thereafter, the cells were treated for 24 hours with HMGB1 and NK cells. The culture medium of these cells was removed and used to measure radioactivity (cpm1). The cells were washed 4 times with the medium and dissolved in 100 µl of 0.5 N sodium hydroxide solution NaOH. The resulting lysates were also used to measure radioactivity (cpm2).

Measurement of radioactivity

Samples were mixed with 10 ml UltimaGold and measured with a liquid scintillation counter.

Obtaining Wild Type GluHMGB1, GlnHMGB1 and HMGB1

Plasmids encoding the HMGB1 polypeptide, in which all four tyrosine residues of the B-Box domain were replaced by glutamate or glutamine residues, respectively, were transfected into HEK cells (cell culture in suspension, without serum, 1000 ml (approximately 2.5×10 ⁶ cells/ml, to which valproic acid was added afterwards).To obtain wild-type HMGB1, tyrosine residues in the B-Box domain were left unchanged.The cell pellet was homogenized and purified using IMAC and TALON Resins (Clontech), elution was using imidazole.The eluates thus obtained (15 samples) were analyzed by SDS-PAGE (Coomassie staining).After all the positive eluates were combined, the protein was subjected to gel filtration (Superdex) and finally analyzed by SDS-PAGE. experiments used 800 nm of purified protein.

NK cell activation

Activation of the NK cell line NK-92 CI was measured by detecting the release of TNF-alpha into the supernatant after 24 hours of stimulation of 400,000 of these cells with 800 nM of the indicated HMGB1 protein. TNF-alpha detection was performed using an ELISA kit (Avia Systems Biology, Catalog No. OKAA00027_96W) according to the manufacturer's instructions.

Example 2 Results and Discussion

Different forms of HMGB1 exhibit pronounced cytotoxicity against cancer cells and different activities in enhancing NK cell cytotoxicity against cancer cells and activating NK cells.

The inventors used both a very short time period of 24 hours to assess HMGB1 cytotoxicity and a highly sensitive assay based on the release of ⁵¹ Cr (chromium 51), which is the gold standard among tests for determining cell death, and found unexpected new forms of cytotoxicity of various modifications HMGB1 modified in the B-Box domain. The effect of Gln-HMGB1 on cancer cell death was 43% greater than that of wild-type HMGB1, while Glu-HMGB1 showed 83% greater cell death than wild-type HMGB1 (Table 1).

Next, effector cells (E, NK cell line NK-92 CI) were incubated with target cells (T, SW480 colon cancer cells) labeled with ⁵¹ Cr. Incubation was carried out at various ratios of E:T, namely 5:1, 10:1 and 20:1 (Table 1). In the presence of Gln-HMGB1, NK cells caused cancer cell death 55% more efficiently than the same NK cells without stimulation. In the presence of Glu-HMGB1, however, a 94% increased cancer cell death was demonstrated compared to the lytic activity of NK cells without stimulation (with an appropriate E:T ratio of 5:1) (Table 1). In addition, NK cells in the presence of Gln-HMGB1 caused 13% more cancer cell death compared to the lytic activity of NK cells after their stimulation with wild-type HMGB1. At the same time, Glu-HMGB1 was 25% more effective in terms of cell death compared to the lytic activity of NK cells after their stimulation with wild-type HMGB1 (at an appropriate E:T ratio of 5:1) (Table 1).

In summary, the three different isoforms of HMGB1 have different cytotoxicity profiles. Glu-HMGB1 is superior to both wild-type HMGB1 and Gln-HMGB1 in terms of cytotoxicity against cancer cells and the ability to enhance the lytic activity of NK cells against cancer cells. Gln-HMGB1 is superior to wild-type HMGB1 in terms of cytotoxicity against cancer cells and the ability to enhance the lytic activity of NK cells against cancer cells. Thus, wild-type HMGB1 shows the weakest cytotoxicity compared to Glu-HMGB1 and Gln-HMGB1. Gln-HMGB1 still exhibits strong cytotoxicity against cancer cells (albeit less than Glu-HMGB1) with a modest increase in NK cell lytic activity ranging between wild-type HMGB1 (low NK cell lytic activity) and Glu-HMGB1 ( high lytic activity of NK cells).

Moreover, three different isoforms of HMGB1 show different activity in relation to the activation of NK cells. The inventors measured the secretion of tumor necrosis factor-alpha (TNF-alpha) by NK-92 CI cells after HMGB1 stimulation. It turned out that Glu-HMGB1 most strongly stimulated the secretion of TNF-alpha and, thus, activated NK cells more strongly than others with their differentiation into binders and killers.

Thus, by using various forms of HMGB1, Glu-HMGB1 and Gln-HMGB1 in cancer treatment, one can regulate (i) direct cytotoxicity to cancer cells, (ii) cytotoxicity of tumor-proximate NK cells to cancer cells, and (iii) general activation of circulating NK cells (switching them to binders and killers) and, thus, increase the recruitment of NK cells to the tumor. The results presented indicate that Glu-HMGB1 and Gln-HMGB1 alone or in combination with cellular immunotherapy (eg, NK cells) exhibit increased cytotoxicity against cancer cells compared to wild-type HMGB1.

With GluHMGB1 and GlnHMGB1, physicians have a tool with which they can regulate the general activation of the immune system (for example, the activation of NK cells that secrete various cytokines, including TNF-alpha) in HMGB1-based cancer therapy. Depending on the desired degree of activation or the desired level of plasma cytokines (e.g. TNF-alpha), Glu-HMGB1 can be used for strong immune system activation and increased cytokine release, while Gln-HMGB1 can be used for moderate activation and moderate cytokine release. (particularly from NK cells). By combining Glu-HMGB1 with Gln-HMGB1, it is possible to increase or decrease the level of cytokine release, while having consistently increased direct cytotoxicity towards cancer cells and increased cytotoxicity of tumor-proximal NK cells directed to cancer cells, compared to wild-type HMGB1 .

Claims (40)

1. A high mobility group B1 (HMGB1) protein polypeptide having cytotoxicity against cancer cells, wherein said HMGB1 polypeptide has at least one, preferably two, more preferably three, or most preferably all four tyrosine residues in positions corresponding to the positions amino acids 22, 57, 68 and 75 of SEQ ID NO: 1 are replaced with an amino acid residue independently selected from glutamic acid, glutamine, aspartic acid, asparagine, homoglutamic acid (2-aminohexanedioic acid), and homoglutamine (2,6-diamino- 6-oxohexanoic acid). 2. The HMGB1 polypeptide according to claim 1, characterized in that in said HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues at said positions are replaced by an amino acid residue independently selected from glutamic acid, glutamine , aspartic acid and asparagine. 3. The HMGB1 polypeptide according to claim 1 or 2, characterized in that in said HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues in the indicated positions are replaced by glutamic acid residues or replaced by glutamine residues . 4. The HMGB1 polypeptide according to any one of paragraphs. 1-3, characterized in that in the specified HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues in the indicated positions are replaced by glutamic acid residues. 5. The HMGB1 polypeptide according to any one of paragraphs. 1-4, characterized in that in the specified HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues in the indicated positions are replaced by glutamine residues. 6. The HMGB1 polypeptide according to any one of paragraphs. 1-5, characterized in that said HMGB1 polypeptide contains the sequence of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95%, most preferably at least 98% identical to SEQ ID NO: 1 and/or contains a sequence of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95%, most preferably at least 98% identical to SEQ ID NO: 3. 7. The HMGB1 polypeptide according to any one of paragraphs. 1-6, characterized in that said HMGB1 polypeptide contains the sequence of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95%, most preferably at least 98% identical to at least one of the sequences of SEQ ID NO: 5-8, preferably SEQ ID NO: 6 or 8. 8. The HMGB1 polypeptide according to any one of paragraphs. 1-7, characterized in that said HMGB1 polypeptide is a derivative of a human HMGB1 polypeptide. 9. The HMGB1 polypeptide according to any one of paragraphs. 1-8, characterized in that said HMGB1 polypeptide does not occur in nature. 10. The HMGB1 polypeptide according to any one of paragraphs. 1-9, characterized in that the specified HMGB1 polypeptide causes increased cell death in SW480 cell culture at a concentration of 0.8 μm compared to a polypeptide containing the amino acid sequence of SEQ ID NO: 3, preferably consisting of the specified sequence. 11. A high mobility group B1 (HMGB1) protein polypeptide for activating the immune system, wherein said HMGB1 polypeptide has at least one, preferably two, more preferably three, or most preferably all four tyrosine residues at positions corresponding to amino acid 22 positions, 57, 68 and 75 of SEQ ID NO: 1 are replaced with an amino acid residue independently selected from glutamic acid, glutamine, aspartic acid, asparagine, homoglutamic acid (2-aminohexanedioic acid), and homoglutamine (2,6-diamino-6-oxohexanoic acid). acids). 12. An HMGB1 polypeptide according to claim 11, characterized in that in said HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues at said positions are replaced by an amino acid residue independently selected from glutamic acid, glutamine , aspartic acid and asparagine. 13. The HMGB1 polypeptide according to claim 11 or 12, characterized in that in said HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues in the indicated positions are replaced by glutamic acid residues or replaced by glutamine residues . 14. The HMGB1 polypeptide according to any one of paragraphs. 11-13, characterized in that in the specified HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues in the indicated positions are replaced by glutamic acid residues. 15. The HMGB1 polypeptide according to any one of paragraphs. 11-14, characterized in that in the specified HMGB1 polypeptide at least one, preferably two, more preferably three, most preferably all four tyrosine residues in the indicated positions are replaced by glutamine residues. 16. The HMGB1 polypeptide according to any one of paragraphs. 11-15, characterized in that said HMGB1 polypeptide contains the sequence of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95%, most preferably at least 98% identical to SEQ ID NO: 1 and/or contains a sequence of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95%, most preferably at least 98% identical to SEQ ID NO: 3. 17. The HMGB1 polypeptide according to any one of paragraphs. 11-16, characterized in that said HMGB1 polypeptide contains the sequence of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95%, most preferably at least 98% identical to at least one of the sequences of SEQ ID NO: 5-8, preferably SEQ ID NO: 6 or 8. 18. The HMGB1 polypeptide according to any one of paragraphs. 11-17, characterized in that said HMGB1 polypeptide is a derivative of a human HMGB1 polypeptide. 19. The HMGB1 polypeptide according to any one of paragraphs. 11-18, characterized in that said HMGB1 polypeptide does not occur in nature. 20. The HMGB1 polypeptide according to any one of paragraphs. 11-19, characterized in that the specified HMGB1 polypeptide causes increased cell death in SW480 cell culture at a concentration of 0.8 μm compared to a polypeptide containing the amino acid sequence of SEQ ID NO: 3, preferably consisting of the specified sequence. 21. Polynucleotide encoding a polypeptide according to any one of paragraphs. 2-10, 12-20. 22. An expression vector containing a polynucleotide according to claim 21. 23. A host cell capable of producing an HMGB1 polypeptide according to the present invention, containing a polypeptide according to any one of paragraphs. 1-20, a polynucleotide according to claim 21 and/or a vector according to claim 22. 24. A method of treating cancer in a subject, comprising: a) bringing into contact the specified subject with the polypeptide according to any one of paragraphs. 1-20, a polynucleotide according to claim 21, a vector according to claim 22 and/or a host cell according to claim 23, and such b) providing treatment for cancer in said subject. 25. The method of claim 24 wherein said cancer treatment comprises cancer cell immunotherapy. 26. The method according to claim 24 or 25, wherein said cancer treatment comprises inducing secretion of TNF-alpha by NK cells, preferably inducing maturation of NK cells. 27. The method according to any one of paragraphs. 24-26, characterized in that said cancer treatment includes preventing the occurrence of a side effect, preferably includes preventing the occurrence of cytokine release syndrome (CRS). 28. A method for inducing immunomodulation in a subject, comprising: a) bringing into contact the specified subject with the polypeptide according to any one of paragraphs. 1-20, a polynucleotide according to claim 21, a vector according to claim 22 and/or a host cell according to claim 23, and thus b) providing immunomodulation in said subject. 29. The method of claim 28, wherein said immunomodulation comprises cancer cell immunotherapy. 30. The method according to claim 28 or 29, wherein said immunomodulation comprises inducing secretion of TNF-alpha by NK cells, preferably inducing maturation of NK cells. 31. The method according to any one of paragraphs. 28-30, characterized in that said immunomodulation includes preventing the occurrence of a side effect, preferably includes preventing the occurrence of cytokine release syndrome (CRS). 32. A kit for use in a method for treating cancer in a subject in need thereof, comprising in the body a polypeptide according to any one of paragraphs. 1-20, a polynucleotide according to claim 21, a vector according to claim 22 and/or a host cell according to claim 23, and instructions for carrying out said method. 33. The kit according to claim 32, characterized in that said kit additionally contains natural killer cells (NK cells) and/or a means for isolating said cells. 34. A kit according to claim 32 or 33, characterized in that said killer cells are tumor-recognizing NK cells. 35. A kit for use in a method of immunomodulation in a subject in need thereof, containing in the body a polypeptide according to any one of paragraphs. 1-20, a polynucleotide according to claim 21, a vector according to claim 22 and/or a host cell according to claim 23, and instructions for carrying out said method. 36. The kit according to claim 35, characterized in that said kit additionally contains natural killer cells (NK cells) and/or a means for isolating said cells. 37. A kit according to claim 35 or 36, characterized in that said killer cells are tumor-recognizing NK cells.

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