WO2023110942A1

WO2023110942A1 - Prevention of impaired fracture healing

Info

Publication number: WO2023110942A1
Application number: PCT/EP2022/085720
Authority: WO
Inventors: Georg Duda; Hans-Dieter Volk; Katharina SCHMIDT-BLEEK; Christian Bucher
Original assignee: Charité-Universitätsmedizin Berlin
Priority date: 2021-12-14
Filing date: 2022-12-13
Publication date: 2023-06-22

Abstract

The present invention relates to a non-agonist ligand or an inhibitory nucleic acid molecule suppressing the biological activity of a target selected from IL-22; IL-22 receptor; IL-3; IL-3 receptor; IL-5; IL-5 receptor; GM-CSF; GM-CSF receptor; IL-1β; IL-1 receptor; CD121a; CD121b; IL-1RAcP; common beta chain receptor; α1 subunit of the IL-22 receptor; CDW210B; CD123; CD125; CD116; and IL-22 binding protein, in prevention or treatment of impaired bone fracture healing.

Description

Prevention of impaired fracture healing

The present invention relates to a non-agonist ligand or an inhibitory nucleic acid molecule in prevention or treatment of impaired bone fracture healing.

This application claims benefit of priority of European Application EP21214462.0, incorporated herein by reference.

Background of the Invention

Musculoskeletal conditions are accounted as the highest contributor to global disability and half of all American adults reported musculoskeletal conditions, the same number as those with cardiovascular or chronic respiratory diseases combined, as reported by the Global Burden of Disease Study. While the prevalence of musculoskeletal conditions varies by age and diagnosis, between 20%-33% of people across the globe live with a painful musculoskeletal condition. Musculoskeletal diseases and disorders are on the continuous rise due to the increased physical activity of the elderly and the elderly population is rapidly growing worldwide. In addition, musculoskeletal disorders and hematologic disorders are exponentially growing with aging, without an equivalent acceptable growth in the therapeutic management of these diseases in the aged. Not only the fracture incidence increases with advancing age, also the prevalence of age-related fractures and the financial burden of health care will further increase due to the demographic changes with a growing proportion of the elderly population. Furthermore, the survival probability after bone fracture is dependent on sex, type of fracture and age, with hip fractures displaying the lowest survival probability of only 20% in men above 80 years.

Currently, the treatment strategy for bone injury is classified by location or geometry and surgical strategies follow standardized concepts. Thus, a specific type of fracture is treated in the same way in young and old or immunologically impaired patients. These treatment strategies ignore the growing diversity of biologically challenging healing situations, with which patients nowadays come to the clinics, not least because of the substantial lack of understanding how endogenous healing processes of bone are altered in these individuals. Many age-related alterations in tissue regeneration and tissue maintenance seem to converge on inflammatory processes. Bone fracture healing is a multifaceted and age-depending process highly relying on an initial inflammatory process. Fracture healing is initiated with a pro-inflammatory response activating and attracting cells of the surrounding tissue. The hematoma is the initial immune-cell-rich tissue, forming a first replacement tissue, which is further organized and structured to form a more mechanically stable replacement tissue by attracting and stimulating progenitor cells to form extracellular matrix and mineral deposition.

The timely resolution of the pro-inflammatory signals is essential for successful healing. A prolonged pro-inflammatory process may significantly delay the healing process, as a tight intercalation of pro- and anti-inflammatory signalling cascades orchestrate the healing process and the timely switch of signalling molecules is essential for the successful fracture healing. In previous studies the inventors could show that the individual immune profile, especially in the adaptive immune cell compartment, significantly impacts the fracture healing potential. The local immune phenotype and the immune cells attracted to the site of injury dramatically change the signalling cascades and the diverging cytokine milieu guiding tissue repair, consequently having a significant impact on the healing cascade. Both innate and adaptive immune system undergo age-related alterations in terms of cell numbers and functions during aging. The immune system is a highly independent organ during aging, which does not primarily follow chronological processes: Alterations in the immune system rather rely on biological processes, which are highly influenced among others by dietary habits, lifestyle, genetic and socio-economic factors. Multiple studies revealed that the cells of innate immunity such as neutrophils, monocytes, macrophages, dendritic cells and NK cells display impaired receptor expression, chemotaxis, phagocytosis, antigen presentation, cytotoxicity, reactive oxygen species (ROS) and cytokine production upon aging. Antigen challenge of the adaptive immunity forms more reactive effector and memory cells eventually leading to long lasting memory cells. Adaptive immune cells (B cells and T cells) significantly shift in sub-populations resulting in a decrease of the naive cell pool and an accumulation of late-differentiated effector and memory cells with prolonged or repetitive antigen exposure. Memory and effector cells of the adaptive immune system show a heightened state of basal inflammation and diminished or hyperactive inflammatory responses compared to the young more naive immune composition. Thus, increased experience in the immune system creates a slight pro-inflammatory cytokine and chemokine milieu, termed inflammaging, a state of sterile, chronic and systemic low-grade inflammation. Inflammaging is a factor underlying most age-related pathologies. Age-associated functional changes in the immune system and inflammaging alters many endogenous processes like metabolism, vascularization or also bone homeostasis. Experience in the adaptive immune system changes the behaviour of osteoblastic, osteoclastic and progenitor cells in homeostasis of the bone. During biological aging, the expression of the costimulatory molecule CD28 is critically diminished in T cells and end-stage effector cells (CD8+CD57+CD28-) are accumulated. The accumulation of these effector cells has be linked to a delayed regenerative process during bone fracture healing. An inflammaged cytokine milieu in homeostasis and the increased pro-inflammatory response upon activation are hallmarks of age- related decline of the regenerative capacity. Rejuvenation of the immune system by cell transfer has been shown to ameliorate the regenerative capacity. The current standard treatment strategy for bone healing does not include pharmacological or immunomodulatory therapies. Surgical interventions, especially revision surgeries after unsatisfactory outcome of the initial treatment strategy, remain the unavoidable treatment for patients with compromised fracture healing. Analysing the local immune phenotype, immune cell activation and secreted cytokines in the aged organism may not only reveal useful marker for predicting patients at risk of biologically/immunologically compromised fracture healing, but may result in a promising target for an early intervention strategy. A focused understanding of the influence of the immunological aging/increased experience in the fracture healing process and new therapeutic approaches are, therefore, mandatory. In this study, the inventors analysed the contribution and the complex consequences of the aging immune system on the course of bone fracture healing. The inventors analysed the immune cell phenotype changes and resulting altered cytokine pattern in an advanced animal model that can mimic the healing in inflammaged patients. Our aim to identify the causes of age-associated diminished capacity of healing, an aspect that is linked to the age-associated challenges of fracture patient treatment. Ideally, the inventors would aim at re-juvenating bone healing in the aged by investigating new therapeutic approaches for age- and immune experience related disturbed regeneration.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to prevent or treat impaired bone fracture healing. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

Summary of the Invention

A first aspect of the invention relates to an agent for use in treatment or prevention of delayed bone fracture healing and/or non-union/fusion of a bone or spine segments, wherein the agent is selected from a. a non-agonist ligand capable of specifically binding to and abrogating (or shielding or blocking) the biological activity and/or effect of a target moiety; and b. a nucleic acid molecule capable of specifically suppressing expression and signal transduction of a target moiety; wherein the target moiety is selected from i. IL-22; ii. IL-22 receptor; (heterodimer of a1 and IL-10Rp2 subunit) iii. IL-3; iv. IL-3 receptor (heterodimer of CD123 and CD131); v. IL-5; vi. IL-5 receptor (heterodimer of CD125 and CD131); vii. GM-CSF; viii. GM-CSF receptor (heterodimer of CD116 and CD131 ); ix. IL-1 P; x. IL-1 receptor (IL-1 R); xi. CD121 a; xii. CD121 b; xiii. IL-1 RAcP (IL-1 receptor accessory protein); xiv. common beta chain receptor (CD131); xv. a1 subunit of the IL-22 receptor; xvi. CDW210B; xvii. CD123; xviii. CD125; xix. CD116; and xx. IL-22 binding protein (IL-22BP).

A second aspect of the invention relates to a nucleic acid molecule encoding the agent according to the first aspect and its embodiments for use in treatment or prevention of delayed bone fracture healing and/or non-union/fusion of a bone or spine segments.

A third aspect of the invention relates to a nucleic acid expression vector comprising the nucleic acid molecule of the second aspect for use in treatment or prevention of delayed bone fracture healing and/or non-union/fusion of a bone or spine segments.

A fourth aspect of the invention relates to a pharmaceutical composition comprising the agent according to the first aspect and its embodiments, the nucleic acid molecule according to the second aspect, or the nucleic acid expression vector according to the third aspect, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention relates to a pharmaceutical composition comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term non-agonist ligand refers to a ligand, particularly a human or humanized monoclonal antibody, capable of specifically binding to its target, at a ko of 10'⁸ mol/L or lower, particularly at a ko (equal to or lower than) 10'⁹ mol/L or even at a ko S10'¹⁰ mol/L. A “non-agonist” ligand interacts with its target without producing the biological effect of the target’s physiological ligand. As an example, a non-agonist ligand to an interleukin receptor binds to the interleukin receptor (ILR, the target) without producing the effect of the interleukin-ILR interaction, and inhibits binding of the interleukin.

General Biochemistry: Peptides, Amino Acid Sequences

The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

The term peptide in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly 8 to 15amino acids, that form a linear chain wherein the amino acids are connected by peptide bonds.

General Molecular Biology: Nucleic Acid Sequences, Expression

The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.

The term Nucleotides in the context of the present specification relates to nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with RNA or DNA oligomers on the basis of base pairing. The term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphotioates, 2’0-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2’0, 4’C methylene bridged RNA building blocks). Wherever reference is made herein to a hybridizing sequence, such hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.

The terms capable of forming a hybrid or hybridizing sequence in the context of the present specification relate to sequences that under the conditions existing within the cytosol of a mammalian cell, are able to bind selectively to their target sequence. Such hybridizing sequences may be contiguously reverse-complimentary to the target sequence, or may comprise gaps, mismatches or additional non-matching nucleotides. The minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T/U nucleotides, and on the backbone chemistry.

In the context of the present specification, the term hybridizing sequence encompasses a polynucleotide sequence comprising or essentially consisting of RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphothioate deoxyribonucleotides, 2’-0-methyl-modified phosphothioate ribonucleotides, LNA and/or PNA nucleotide analogues. In certain embodiments, the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof. The term antisense oligonucleotide in the context of the present specification relates to an oligonucleotide having a sequence substantially complimentary to, and capable of hybridizing to, an RNA. Antisense action on such RNA will lead to modulation, particular inhibition or suppression of the RNA’s biological effect. If the RNA is an mRNA, expression of the resulting gene product is inhibited or suppressed. Antisense oligonucleotides can consist of DNA, RNA, nucleotide analogues and/or mixtures thereof. The skilled person is aware of a variety of commercial and noncommercial sources for computation of a theoretically optimal antisense sequence to a given target. Optimization can be performed both in terms of nucleobase sequence and in terms of backbone (ribo, deoxyribo, analogue) composition. Many sources exist for delivery of the actual physical oligonucleotide, which generally is synthesized by solid state synthesis.

The term siRNA (small/short interfering RNA) in the context of the present specification relates to an RNA molecule capable of interfering with the expression (in other words: inhibiting or preventing the expression) of a gene comprising a nucleic acid sequence complementary or hybridizing to the sequence of the siRNA in a process termed RNA interference. The term siRNA is meant to encompass both single stranded siRNA and double stranded siRNA. siRNA is usually characterized by a length of 17-24 nucleotides. Double stranded siRNA can be derived from longer double stranded RNA molecules (dsRNA). According to prevailing theory, the longer dsRNA is cleaved by an endo-ribonuclease (called Dicer) to form double stranded siRNA. In a nucleoprotein complex (called RISC), the double stranded siRNA is unwound to form single stranded siRNA. RNA interference often works via binding of an siRNA molecule to the mRNA molecule having a complementary sequence, resulting in degradation of the mRNA. RNA interference is also possible by binding of an siRNA molecule to an intronic sequence of a pre-mRNA (an immature, non-spliced mRNA) within the nucleus of a cell, resulting in degradation of the pre-mRNA.

The term shRNA (small hairpin RNA) in the context of the present specification relates to an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi).

The term sgRNA (single guide RNA) in the context of the present specification relates to an RNA molecule capable of sequence-specific repression of gene expression via the CRISPR (clustered regularly interspaced short palindromic repeats) mechanism.

The term miRNA (microRNA) in the context of the present specification relates to a small noncoding RNA molecule (containing about 22 nucleotides) that functions in RNA silencing and post- transcriptional regulation of gene expression.

The term nucleic acid expression vector in the context of the present specification relates to a polynucleotide, for example a plasmid, a viral genome or a synthetic RNA molecule, which is used to transfect (in case of a plasmid or an RNA) or transduce (in case of a viral genome) a target cell with a certain gene of interest. In the case of a DNA expression construct, the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell, thus, the gene of interest is transcribed either constitutively or in response to a stimulus or dependent on the cell’s status. In the case of an RNA expression construct, it is understood that the term expression relates to translation of the RNA and the construct can be employed by the target cell as an m-RNA. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell.

Binding; Binders, Ligands, Antibodies:

If not specified more narrowly in the Detailed Description of the Invention, reference to binders and ligands encompasses antibodies, antibody-like molecules and aptamers as defined in the following paragraphs.

The term specific binding in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of < 10'⁸mol/L when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure.

The term aptamer relates an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers can be created by selecting them from a large random sequence pool. Nucleic acid aptamers can be generated through repeated rounds of in-vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to molecular targets such as small molecules, proteins or nucleic acids through non-covalent interactions. Aptamers offer molecular recognition properties that rival that of antibodies.

In the context of the present specification, the term antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region of IgG is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, the term encompasses a so-called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain.

In the context of the present specification, the term humanized antibody refers to an antibody originally produced by immune cells of a non-human species, the protein sequences of which have been modified to increase their similarity to antibody variants produced naturally in humans. The term humanized antibody as used herein includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of another mammalian species.

The term antibody-like molecule in the context of the present specification refers to a molecule capable of specific binding to another molecule or target with high affinity / a Kd < 10E-8 mol/l. An antibody-like molecule binds to its target similarly to the specific binding of an antibody. The term antibody-like molecule encompasses a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zurich), an engineered antibody mimetic protein exhibiting highly specific and high-affinity target protein binding (see US2012142611 , US2016250341 , US2016075767 and US2015368302, all of which are incorporated herein by reference). The term antibody-like molecule further encompasses, but is not limited to, a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins and a polypeptide derived from tetratricopeptide repeat proteins. The term antibody-like molecule further encompasses a specifically binding polypeptide derived from a protein A domain, a fibronectin domain FN3, a consensus fibronectin domain, a lipocalin (see Skerra, Biochim. Biophys. Acta 2000, 1482(1- 2):337-50), a polypeptide derived from a Zinc finger protein (see Kwan et al. Structure 2003, 11 (7):803-813), a Src homology domain 2 (SH2) or Src homology domain 3 (SH3), a PDZ domain, a gamma-crystallin, ubiquitin, a cysteine knot polypeptide or a knottin, cystatin, Sac7d, a triple helix coiled coil (also known as alphabodies), a Kunitz domain or a Kunitz-type protease inhibitor and a carbohydrate binding module 32-2.

In the context of the present specification, the term fragment crystallizable (Fc) region is used in its meaning known in the art of cell biology and immunology; it refers to a fraction of an antibody comprising, if applied to IgG, two identical heavy chain fragments comprised of a CH2 and a CH3 domain, covalently linked by disulfide bonds.

In the context of the present specification, the term dissociation constant (KD) is used in its meaning known in the art of chemistry and physics; it refers to an equilibrium constant that measures the propensity of a complex composed of [in most cases, two] different components to dissociate reversibly into its constituent components. The complex can be e.g. an antibody-antigen complex AbAg composed of antibody Ab and antigen Ag. KD is expressed in molar concentration [mol/l] and corresponds to the concentration of [Ab] at which half of the binding sites of [Ag] are occupied, in other words, the concentration of unbound [Ab] equals the concentration of the [AbAg] complex. The dissociation constant can be calculated according to the following formula:

[Ab]: concentration of antibody; [Ag]: concentration of antigen; [AbAg]: concentration of antibodyantigen complex

As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.

As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

As used herein, the term treating or treatment of any disease or disorder (e.g. impaired bone fracture healing) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.

Detailed Description of the Invention

A first aspect of the invention relates to an agent for use in treatment or prevention of non-fusion of a bone and/or spine segment and/or delayed bone fracture healing.

In certain embodiments, the agent is a non-agonist ligand capable of specifically binding to and abrogating (or shielding or blocking) the biological activity and/or effect of a target moiety. In certain embodiments, the agent is a nucleic acid molecule capable of specifically suppressing expression and signal transduction of a target moiety.

In certain embodiments, the target moiety is IL-22 (UniProt-ID Q9GZX6). In certain embodiments, the target moiety is the IL-22 receptor (heterodimer of a1 and IL-10RJ32 subunit, UniProt-ID Q8N6P7 and Q08334). In certain embodiments, the target moiety is IL-3 (UniProt-ID P08700). In certain embodiments, the target moiety is the IL-3 receptor (heterodimer of CD123 and CD131 , UniProt-ID P26951 and P32927). In certain embodiments, the target moiety is IL-5 (UniProt-ID P05113). In certain embodiments, the target moiety is the IL-5 receptor (heterodimer of CD125 and CD131 , UniProt-ID Q01344 and P32927). In certain embodiments, the target moiety is GM-CSF (UniProt-ID P04141). In certain embodiments, the target moiety is the GM-CSF receptor (heterodimer of CD116 and CD131 , UniProt-ID P15509 and P32927). In certain embodiments, the target moiety is IL-1 p (UniProt-ID P01584). In certain embodiments, the target moiety is the IL-1 receptor (IL-1 R, UniProt-ID P14778). In certain embodiments, the target moiety is CD121a (UniProt-ID P14778). In certain embodiments, the target moiety is CD121 b (UniProt-ID P27930). In certain embodiments, the target moiety is IL-1 RAcP (IL-1 receptor accessory protein, UniProt- ID P18510). In certain embodiments, the target moiety is the common beta chain receptor (CD131 , UniProt-ID P32927). In certain embodiments, the target moiety is the a1 subunit of the IL-22 receptor (UniProt-ID Q8N6P7). In certain embodiments, the target moiety is CDW210B (UniProt- ID Q08334). In certain embodiments, the target moiety is CD123 (UniProt-ID P26951). In certain embodiments, the target moiety is CD125 (UniProt-ID Q01344). In certain embodiments, the target moiety is CD116 (UniProt-ID P15509). In certain embodiments, the target moiety is the IL-22 binding protein (IL-22BP, UniProt-ID Q969J5).

In certain embodiments, the target moiety is selected from IL-22, IL-22 receptor, cd subunit of the IL-22 receptor, and common beta chain receptor. In certain embodiments, the target moiety is IL- 22. The ligand is administered to suppress the biological activity of IL-22 (e.g. neutralizing or shielding), to block the IL-22 receptor (antagonists, antibody binding, Ligand-receptor bindingcompetition), and/or to inhibit downstream signalling cascades of IL-22.

Anti-IL-22 antibodies are known in the art, as for example described in US10131709 B2 (Immunqure), US7638604 B2 (Genetics Institute), US2021230263 A1 (BioAtla), US8470993 B2 (Wyeth), and EP1986688 B1 (Wyeth), all of which are incorporated herein by reference in their entirety. In certain embodiments, the anti-IL-22 antibody is fezakinumab (CAS-NO: 1007106-86-6).

In certain embodiments, the target moiety is selected from IL-5, IL-5 receptor, CD125, and common beta chain receptor. In certain embodiments, the target moiety is IL-5. The ligand is administered to suppress the biological activity of IL-5 (e.g. neutralizing or shielding), to block the IL-5 receptor (antagonists, antibody binding, Ligand-receptor binding-competition), and/or to inhibit downstream signalling cascades of IL-5.

In certain embodiments, the ligand is selected from the group comprising an antibody, an antibody fragment, a soluble receptor construct, an aptamer, a non-immunoglobin scaffold, and an antibodylike molecule. In certain particular embodiments, the ligand is selected from an antibody and an antibody-fragment.

In other more particular embodiments, the ligand is an antibody.

In other more particular embodiments, the ligand is an antibody-fragment.

A soluble receptor construct is a receptor complex capable of binding the respective ligand whilst neutralizing the effect of this cytokine and therefore possessing antagonistic properties. It has the potential to regulate both local and systemic cytokine mediated events. The non-immunoglobin scaffolds possess a protein-protein-interaction and thus scaffold proteins are functional moderators by binding the respective cytokine. Non-immunoglobulin (non-lg) scaffolds are, in contrast to antibodies, small single-domain proteins that require no post- translational modification, often lack disulfide bonds, and can undergo straightforward multimerization. Among the 20 different types of non-lg scaffolds, Adhirons, Alphabodies, Centyrins, Pronectins, Repebodies, Affimers, and Obodies have been introduced in the past 4 years. 102 proteins have been specifically targeted by 139 different non-lg scaffold binders. The most frequent application of non-lg scaffolds is in the treatment and diagnosis of cancer and inflammatory diseases, and 10 non-lg scaffolds have already been tested in clinical trials. Recently, non-lg scaffolds have often been used in research as structure determination chaperones, for intracellular monitoring of post-translational modifications, and as antibody alternatives for microscopy, flow cytometry, and Western blotting.

In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody-fragment is the fragment of a monoclonal antibody.

In certain embodiments, the antibody is a humanized monoclonal antibody. In certain embodiments, the antibody is a humanized chimera. In certain embodiments, the antibodyfragment is a humanized antibody-fragment.

In certain embodiments, the ligand is a biopolymer ligand. In certain embodiments, the ligand is a ligand selected from the group comprising a polypeptide, a peptide, and a peptidomimetic. In certain embodiments, the ligand is a polypeptide.

In certain embodiments, the nucleic acid molecule is an siRNA capable of abrogating expression of its target mRNA. In certain particular embodiments, the nucleic acid molecule is an siRNA targeting IL-22. In certain particular embodiments, the nucleic acid molecule is an siRNA targeting the IL-22 receptor. In certain particular embodiments, the nucleic acid molecule is an siRNA targeting the a1 subunit of the IL-22 receptor.

In certain particular embodiments, the nucleic acid molecule is an siRNA targeting IL-5. In certain particular embodiments, the nucleic acid molecule is an siRNA targeting the IL-5 receptor. In certain particular embodiments, the nucleic acid molecule is an siRNA targeting CD125.

In certain embodiments, the nucleic acid molecule is an antisense oligonucleotide capable of forming a hybrid with an mRNA encoding the target moiety.

In certain embodiments, the antisense oligonucleotide comprises ribonucleotides and/or deoxyribonucleotides. In certain embodiments, the nucleic acid molecule is an RNA or a DNA molecule.

In certain embodiments, the antisense oligonucleotide comprises locked nucleic acid moieties and/or peptide nucleic acid moieties. In certain embodiments, the bone comprises a fracture and/or a surgically-induced disconnection. A surgically-induced disconnection of a bone occurs from sawing, drilling or intentionally breaking a bone during surgery.

A second aspect of the invention relates to a nucleic acid molecule encoding the agent according to the first aspect and its embodiments for use in treatment or prevention of non-fusion of a bone and/or delayed bone fracture healing.

A third aspect of the invention relates to a nucleic acid expression vector comprising the nucleic acid molecule of the second aspect for use in treatment or prevention of non-fusion of a bone and/or delayed bone fracture healing.

In certain embodiments, the patient shows an elevated pro-inflammatory reaction towards an injury associated with an increased immunological response of effector cells. In certain embodiments, the patient shows a frequency of CD28-CD8+ T cells is >30 % of all CD8+ T cells and/or a frequency of CD57+CD8+ T cells is >30 % of all CD8+ T cells. This frequency as a biomarker is discussed in EP2810065B1 , which is incorporated by reference herein.

In certain embodiments, the patient is of age >35. In certain embodiments, the patient is of age >55.

In certain embodiments, the ligand is administered by means of a local drug delivery system. A biomaterial scaffold-based local drug delivery system controls localization and sustained concentration of therapeutics. A biomaterial is any material of natural or synthetic origin which is placed within the human body, and may be degraded after a certain amount of time.

In certain embodiments, the pharmaceutical composition is formulated as an administration form for parenteral administration. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.

Medical treatment. Dosage Forms and Salts

Similarly, within the scope of the present invention is a method or treating impaired bone fracture healing and/or spine fusion in a patient in need thereof, comprising administering to the patient a non-agonist ligand or a nucleic acid molecule according to the above description.

In certain embodiments, the non-agonist ligand is an antibody, antibody fragment, an antibody-like molecule or a protein A domains derived polypeptide. In some embodiments, the non-agonist ligand is an immunoglobulin consisting of two heavy chains and two light chains. In some embodiments, the non-agonist polypeptide ligand is a single domain antibody, consisting of an isolated variable domain from a heavy or light chain. In some embodiments, the non-agonist polypeptide ligand is a heavy-chain antibody consisting of only heavy chains such as antibodies found in camelids.

In certain embodiments, the non-agonist ligand is an antibody fragment. In certain embodiments, the non-agonist ligand is a Fab fragment, i.e. the antigen-binding fragment of an antibody, or a single-chain variable fragment, i.e. a fusion protein of the variable region of heavy and the light chain of an antibody connected by a peptide linker.

Similarly, a dosage form for the prevention or treatment of impaired bone fracture healing and/or spine fusion is provided, comprising a non-agonist ligand or antisense molecule according to any of the above aspects or embodiments of the invention.

The skilled person is aware that any specifically mentioned drug compound mentioned herein may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.

Pharmaceutical Compositions and Administration

Another aspect of the invention relates to a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product. The pharmaceutical composition can be formulated for enteral administration, particularly oral administration or rectal administration. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).

The pharmaceutical composition can be formulated for parenteral administration, for example by i.v. infusion, intradermal, subcutaneous or intramuscular administration.

The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily. In certain embodiments, the compounds of the invention may be administered as a sustained release from a local drug delivery system.

In certain embodiments, the pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

Method of Manufacture and Method of Treatment according to the invention

The invention further encompasses, as an additional aspect, the use of a non-agonist ligand or a nucleic acid molecule as identified herein, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of impaired bone fracture healing and/or spine fusion. Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with a disease associated with impaired bone fracture healing and/or spine fusion. This method entails administering to the patient an effective amount of a non-agonist ligand or a nucleic acid molecule as identified herein, or its pharmaceutically acceptable salt, as specified in detail herein.

Wherever alternatives for single separable features such as, for example, a target moiety, a ligand type or a medical indication are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a target moiety may be combined with any of the alternative embodiments of a ligand type and these combinations may be combined with any medical indication mentioned herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures

Fig. 1 shows the analysis of the fracture gap from an osteotomy in 12 months old mice revealing a delayed fracture healing process in immunologically experienced mice (immunoaged). (A) Representative images of the newly formed bone (red) in the fracture gap 21 days post-surgery analysed via micro-computed x-ray tomography. (B) The callus volume (TV) was significantly decreased in the immunoaged group, whereas the bone volume (BV), bone mineral density (BMD), trabecular number (Tb.N) and trabecular thickness (Tb.Th) were not significantly different between the two groups. The diminished bone formation in the immunoaged group led to a less stable fracture callus as shown by a lower minimal polar moment of inertia (MMIp). N=4pergroup, Welch’s t-test

Fig. 2 shows the immune phenotype analysis of the local T cells at the site of injury in comparison to the bone marrow from untreated mice three days post-surgery revealing significant differences in T cell subtypes. The fracture gap including the hematoma and the adjacent bone marrow were analysed by flow cytometry three days post-surgery and compared to untreated bone marrow of the same groups. Sub populations for CD8+ (A) and CD4+ (B) T cells have been analysed. Initially the memory formation is more advanced in immunoaged mice, as seen in increased memory populations and decreased naive T cells. However, these differences were equalized three days post-surgery at the fracture site. Significant differences three days post-surgery could only be observed in short lived effector cells (SLEC) in the CD4+ T cell compartment around the fracture site. The introduction of an osteotomy significantly changed the memory populations in CD4+ T cells: The percentage of effector memory (EM) T cells were diminished, whereas the memory precursor cells (MPEC) are significantly increased compared to the untreated bone marrow. CD4+ regulatory T cells increased in the immunoaged group upon injury, also being less abundant in the intact bone marrow of untreated mice. CM central memory, EM, effector memory, MPEC memory

Fig. 3 shows the analysis of the local CD4+ T cells at the site of injury in comparison to the bone marrow from untreated mice (intact bone) three days post-surgery revealing significant differences in T cell activation states. Introducing a fracture increased the CXCR3 presentation on the cell surface in CD4+ T cells and on the other hand increased the expression of PD-1 accordingly. N=4 per group, Welch’s t-test, black * = between immunological experience, grey * = between timepoints

Fig. 4 shows the analysis of the local CD8+ T cells at the site of injury in comparison to the bone marrow from untreated mice (intact bone) three days post-surgery revealing significant differences in T cell activation states. CXCR3 expression on CD8+ T cells is highly regulated by the initial experience level, as aged mice downregulated the CXCR3 expression on CD8+ T cells three days post-surgery. Introducing a fracture activated CD8+ memory T cells, as showed by increased levels of CD107a on the cell surface. CD8+ T cells showed a significant higher activation state in immunoaged mice three days post-surgery. N=4 per group, Welch’s t-test, black * = between immunological experience, grey * = between timepoints

Fig. 5 shows the analysis of the local macrophage population at the site of injury three days postsurgery in comparison to the bone marrow from untreated mice revealing significant differences in macrophage polarization and activation. (A) Three days post-surgery significant more macrophages were present around the fracture gap compared to the untreated bone marrow. (B) The activation state of macrophages at the fracture site differed in their activation state, as significantly more M1 polarized macrophages could be found in the immunoaged group. Additionally, an increased level of M2a activated macrophages could be observed in the immunoaged group. This data showed an increased activation state in these mice with higher experience levels in the adaptive immune system and depicts a prolonged inflammatory phase.

Fig. 6 shows the cytokine pattern of the basal state and as response to an injury showing increased reactivity in immunoaged mice. Systemic cytokine levels were screened pre-surgery and three days post-surgery in the blood of experienced (immunoaged) and less-experienced (aged) 12 months old mice. The cytokine levels at basal state showed an inflammaged status in immunoaged mice: increased systemic levels of TNFoc, IL-6 and IL-10. Upon cell activation due to an injury, the cytokines were differentially contoured based on their experience level. Increased levels of IL-22, IL-5, GM-CSF and IL-1 b could be observed in immunoaged mice. Most importantly, IL-22 showed not only high basal expression levels in immunoaged compared to aged mice, IL-22 appeared to react specific in the respective experience phenotype with a significant downregulation upon injury in aged mice but an increase upon injury in immunoaged mice. N=4 per group, Welch’s t-test, black * = between immunological experience, grey * = between timepoints Fig. 7 shows IL-5, IL-22 and GM-CSF interfering with the regenerative potential of progenitor cells. All three cytokines were tested at different concentrations individually on the differentiating (A-C) and migratory/proliferative potential (F) of mesenchymal stromal cells as well as the angiogenic potential (D,E) of endothelial cells to form new tubes in vitro. IL-22 increased the mineralization of the extracellular matrix in the osteogenic differentiating assays, but higher concentrations of IL-22 decreased the migratory and proliferative potential as well as inhibiting the de novo formation of new vessels by lowering the tube number and branching points. IL-5 showed similar interference as IL-22. GM-CSF predominantly interfered with the migratory and proliferative behaviour of MSC, but did not inhibit the angiogenic potential of endothelial cells. N=5 different MSC donors per assay, endothelial cells (HUVEC) were pooled donors, tested in triplicate, unpaired t-test

Fig. 8 shows the substitution of IL-22 in less-experienced (aged) mice deteriorated the fracture healing process, whereas neutralization of IL-22 accelerated or ameliorated the healing process in experienced (immunoaged) mice. An osteotomy was introduced in long bones, the IL-22 levels were treated with either neutralization with antibodies or substitution with recombinant protein and the healing outcome was assessed 21 days post-surgery with pCT. (A, B) Less-experienced (aged) 12 months old mice, showing a lower level of IL-22 three days post-surgery, IL-22 was substituted during the first 7 days post-surgery, starting 24h post-surgery. The fracture healing was almost completely abolished 21 days post-surgery under IL-22 substitution treatment. Experienced 12 months old mice, showing a higher level of IL-22 three days post-surgery, IL-22 was neutralized during the first 7 days post-surgery, starting 24h post-surgery. The neutralization of IL-22 furthered the bone healing process, as an increased callus volume and bone volume could be observed 21 days post-surgery. The stability of the fracture gap is significantly increased (MMIp) and callus volume and stability reached the levels of less-experienced mice. N=4-6 per group, Welch’s t-test

Fig. 9 shows the substitution of IL-5 in less-experienced (aged) mice deteriorated the fracture healing process, whereas neutralization of IL-5 accelerated or ameliorated the healing process in experienced (immunoaged) mice. An osteotomy was introduced in long bones, the IL-5 levels were treated with either neutralization with antibodies or substitution with recombinant protein and the healing outcome was assessed 21 days post-surgery with pCT. (A, B) Less-experienced (aged) 12 months old mice, showing a lower level of IL-5 three days post-surgery, IL-5 was substituted during the first 7 days post-surgery, starting 24h post-surgery. The fracture healing was deteriorated 21 days post-surgery under IL-5 substitution treatment with a significant lower callus volume and bone vollume (TV and BV). Experienced 12 months old mice, showing a higher level of IL-5 three days post-surgery, IL-5 was neutralized during the first 7 days post-surgery, starting 24h post-surgery. The neutralization of IL-5 ameliorated the bone healing process, as an increased bone volume be observed 21 days post-surgery. N=6 per group, Welch’s t-test

Fig. 10 shows that IL-5 secretion is significantly increased in aged human donors, whereas IL-22 is not significantly increased. Isolated immune cells from peripheral blood (peripheral mononuclear cells, PBMC) from young healthy donors (aged 18-35) or aged healthy donors (aged 55-70) were activated via CD3 and CD28 antibody stimulation. The cytokine levels were measured after a three day activation scheme, revealing an increased IL5 production in aged donors compared to young donors. The IL-22 levels measured were relatively low and not significantly differing between the two groups. IL-22 might be secreted via a different activation scheme than CD3/CD28 stimulation. N=3 per group, unpaired t-test

Fig. 11 shows that the experience in the adaptive immune system, especially in T cells, is significantly increased under exposed housing conditions. Flow cytometry analysis of the immune phenotype systemically in the spleen and in the bone marrow. There were significant differences in immune cell composition between spleen and bone marrow, where the dominant populations in the bone marrow are part of the innate immune system, but no significant differences were seen between the immunoaged and aged group, except of dendritic cells, which were diminished in the immunoaged group. The proportion of naive T cells was significantly decreased in the immunoaged group. The dominant memory populations in the CD4+ T cells in the spleen were effector memory (EM) T cells, whereas in the CD8+ T cells, the dominant memory populations were central memory (CM) T cells. Effector cells, memory precursor effector cells (MPEC) and short-lived effector cells (SLEC), did not significantly differ in the spleen between the experience level in both the CD4+ and CD8+ T cell compartment, but significant difference were found in the bone marrow. The proportional contribution of the immune cell compartment is given in the table and p-values are given forthe comparison between the immunoaged and aged group. N=4 pergroup, Welch’s t-test

Example 1: Advanced pre-clinical model depicting different levels of immunological experience

This study was conducted in a murine pre-clinical model of aged mice (12 months old) with two groups of distinct immunological characteristics. After 12 months of aging under different housing conditions the immune system was analysed and phenotypical changes were monitored. Housing mice exposed to environmental microorganisms, by reducing the filtration of the air supply, allowed the formation of experience in the immune system. Individually ventilated cages however dampened the formation of experience and more naivety is kept in the immune system. The exposed housing conditions significantly shifted the sub-populations of lymphocytes such as the depletion of the naive T cell pools and subsequent accumulation of effector and memory T cells (Figure 11). As the two distinct groups of the same age (12 months) only differed in their immunological experience, the groups were termed henceforth as aged (less-experienced immune system) and immunoaged (experienced immune system). Systemic analysis of the immune cell compartment in the spleen and the bone marrow revealed no significant difference of the proportion of the broadest leukocyte classification between the two groups (granulocytes, monocytes, NK cells, B and T cells), except for dendritic cells, which could be found to be rarer in the bone marrow of the immunoaged group (Figure 11). However, significant differences could be found in the subclassification of lymphocytes in the adaptive immune compartment in the spleen and the bone marrow. Both naive immune cell pools of the CD4+ and CD8+ T cell compartment are significantly diminished in the immunoaged group. In parallel, a significant increased proportion of memory and effector T cells could be found in the immunoaged group. The CD4+ T cell compartment of the immunoaged group showed increased quantities of effector memory (EM) T cells and memory precursor effector (MPEC) T cells. The proportion of CD4+ T EM cells was significantly higher in the immunoaged group compared to the aged group (54.3%(+-4.6) and 37.3%(+- 1 .2), respectively). The central memory (CM) population of the CD4+ T cell pool and short-lived effector cells (SLEC) did not significantly differ between the two groups. The CD8+ T cell pool showed similar trends with increased memory formation and diminished naive T cells. The TCM pool was significantly increased (61 .1 %(+-1 .3) of all CD8+ T cells) in the spleen of the immunoaged group compared to the aged group with only 36.6%(+-2.4) CD8+ TCM (Figure 11). In the bone marrow some sub-populations of T cells were additionally stronger represented, as CD4+ SLEC and CD8+ MPEC were found in a higher proportion in the bone marrow of the immunoaged group (Figure 11). This data shows the basis of the study, as the inventors were able to reproduce immunologically experienced (immunoaged) and less-experienced (aged) individuals in a pre-clinical model of aged mice. This advanced model mimics the immunological diversity found in aged patients as the heterogeneity of immunological experience was shown to be increased with age.

Example 2: Fracture healinc is disturbed in the experienced croup

The healing outcome of a fracture in 12 months old mice was analysed 21 days post-surgery and revealed a less stable callus with experience in the adaptive immune system. An osteotomy was introduced in the left femur of mice with different experience levels of the immune system. Both groups were included at the age of 12 month and only differed in their immunological experience level. The analysis of the fracture healing by micro-computed x-ray tomography (pCT) revealed a more progressed and more stable callus in the immunologically less-experienced (aged) group. The callus of the immunoaged group showed a significant lower callus volume (TV) compared to the aged group and the minimal polar moment of inertia (MMIp), a computational analysis of torsional stability, depicted a less stable callus in the immunoaged group (Figure 1). Experience in the adaptive immune system significantly delayed the fracture healing process. As the immune system was the main diverging factor between the two groups, the systemic and local immunological events were monitored in order to understand the inferior healing outcome in the immunoaged group.

In order to understand the diverging healing outcome after bone injury, the local contribution of immune cells during the initial inflammatory reaction in the fracture healing process was monitored. The fracture gap including the hematoma and the adjacent bone marrow were analyzed by flow cytometry three days post-surgery. The hematoma consists of locally available immune cells as well as of attracted immune cells from the surrounding tissue and blood supply. Here, the inventors analyzed the local immune phenotype of T cells in the bone marrow from untreated mice in comparison with the hematoma phase in osteotomized mice. Immunoaged mice significantly differ in their immune cell composition in the bone marrow of intact bone compared to the untreated aged mice with less immunological experience. In intact bone marrow less naive CD8+ T cells were available superseded by memory cells, with a significant increased level of central memory CD8+ T cells in the immunoaged group (Figure 2 A). In the CD4+ T cell compartment a similar distribution could be observed, however, effector memory T cells were predominant. Especially, the level of effector cells differed between the experience level, as less MPEC could be observed in the immunoaged group, but the SLEC compartment was more abundant (Figure 2 B). The level of regulatory CD4+ T cells was significantly higher in the less-experienced (aged) group, showing a more pronounced attenuation and regulation of the adaptive immune system (Figure 2 C). Introducing an injury severely changed the local composition of immune cells. The aged group aligned to some extent to the immunoaged group with regard to less naivety locally at the fracture site. The immunoaged group, however significantly increased the local contribution of naive CD8+ T cells (Figure 3 A). In both groups more MPEC were present at the fracture site compared to the intact bone of untreated mice. The CD4+ T_reg population increased simultaneously in the immunoaged group. The CD4+ MPEC increased in both groups, showing an active contribution of effector cells during fracture healing. An interesting finding was that the CD4+ TEM cells reached similar levels in both groups at day three post-surgery. Significant alterations in the adaptive immune cell phenotype could consequently be observed at the injury site during the initial inflammatory phase, depicting a tight intercalation of adaptive immune cells and fracture healing.

Example 4: Immunological experience increased pro-inflammatorv response by stronger activation patterns and absent downregulation of CXCR3

The different subtypes of memory T cells were found to adjust to the same levels locally at the fracture site in both groups of different immunological experience levels. Analyzing the activation and inhibition patterns however showed impactful differences between the two groups. The chemokine receptor CXCR3 and the adhesion molecule CD43 are important surface proteins on memory T cells, depicting their efficiency towards a recall response. CXCR3-CD43- memory T cells show low secretion of effector cytokines upon activation, whereas CXCR3+CD43- memory T cells are prone for vast effector cytokine secretion, CD43+CXCR3int memory T cells are attributed to an intermediate effector function. CD8+ memory T cells locally in the fracture gap showed a significant downregulation in CXCR3 expression on the cell surface in the aged group, in CD4+ memory T cells both groups showed a decrease of the intermediate (CD43+CXCR3int) effector function (Figure 3 A and Figure 4 A). CD8+ memory T cells in the immunoaged group showed a more activated phenotype by an absence of downregulation of the CXCR3 and by the upregulated activation marker CD107a. CD137, however, was downregulated upon fracture but no difference between the two groups could be observed (Figure 4 B). CD107a expression correlates with cytokine and the CXCR3 and CD43 expression correlates with effector cytokine severity, depicting an overall more pronounced inflammatory phenotype on CD8+ memory T cells in the immunoaged group. Both groups showed in the initial hematoma phase, three days post-surgery, a slight increase of inhibitory marker PD-1 on CD8+ memory T cells (Figure 4 C). CD4+ memory T cells showed no significant difference in activation marker expression, but both groups showed a significant increase in PD-1 expression on the cell surface (Figure 3 B and C). This data showed that two systemically different experience levels in the adaptive immune system which somewhat merge locally within the early fracture hematoma, albeit a significant difference remained in cell activation and pro-inflammatory abilities of the cells in the early hematoma phase between the aged and immunoaged group. As cells of the adaptive and the innate immune system follow a close crosstalk during an immunological response upon injury the inventors further analyzed cells of the innate immune system locally at the fracture site.

Example 5: Innate immune cells in the fracture healing process showed a prolonged inflammatory phase with increased experience

Macrophages are key players in the healing process and are readily available upon injury and are referred as essential for successful healing. An osteotomy almost quadrupled the percentile macrophage population at the fracture site, and around 20 % of all leukocytes at the fracture site were allocated to the macrophage population (Figure 5 A). By analyzing the macrophage subtypes at the fracture site, it became apparent that in the immunoaged group the macrophages showed a prolonged activation phenotype and might be attributed to a prolonged inflammatory phase in the immunologically experienced (immunoaged) group. Macrophages are able to show two different polarization states, namely M1 (pro-inflammatory state) and M2 macrophages (alternatively activated state) and can be further divided into different activation subtypes, like M2a, M2b, M2c and M2d macrophages, all attributed to distinct cytokine secretion patterns/profiles and functionality. More macrophages seemed to be activated in the immunoaged group, as less MO (inactive macrophages) could be observed in the immunoaged group. However, significant more M1 activated macrophages, known for their pro-inflammatory properties, could be found at the fracture site in the immunoaged group compared to the aged group. Activation subtypes of the M2 polarized macrophages showed differences in their activation states and M2a activated macrophages could be found more abundant at the fracture site of the immunoaged group compared to the aged group three days post-surgery (Figure 5 B). This increased polarization and activation state of macrophages showed a heightened inflammatory state at the fracture site of the experienced group. Apart of the macrophage population, dendritic cells contribute to the inflammatory state by cross-presentation of molecules and are known for their direct stimulation and modulation of CD8+ T cells. Especially CD205+CD8+ dendritic cells directly guide and communicate with CD8+ T cells in mice. Different DC subsets exist in mice with distinct functionality and cytokine secretion patterns, here the inventors monitored three distinct DC populations, namely plasmacytoid dendritic cells (pDC), conventional DCs (eDC) and lymphoid tissue-resident classical DCs (IDC). This dendritic cell subtypes were affected by the experience in the adaptive immune system as more plasmacytoid dendritic cells (pDC) could be observed locally in the hematoma in the immunoaged group and lower levels of conventional DCs (eDC) and CD4+ lymphoid tissueresident classical DCs (IDC). The population of CD8+ IDCs is rather small and significant lower percentages could be found in the immunoaged group (Figure 5 C). However, in the immunoaged group more than 60% of all CD8+ DCs were CD205+, depicting a tighter communication between the innate and adaptive immune cells, as only around 25% were CD205+ in the aged group (Figure 5 D). CD8+ DCs in mice start type I cytotoxic immune responses and are known to be efficient for CD8+ T-cell activation, including antigen cross-presentation and cytokine production, hence playing a major role in the induction of a CD8+ T cell immune responses. DCs play a critical role in immunomodulation by balancing tolerance and immune responses and are able to orchestrate the adaptive immune response. Cells of the adaptive and the innate immune response orchestrate and steer inflammatory cascades, mostly by the secretion of cytokines communicating with the environment in autocrine and paracrine manners. The main cellular crosstalk is achieved via cytokines and therefore the inventors analyzed the diverging cytokine patterns induced by the distinct immunological experience level.

Example 6: Cytokine levels depict significant alterations in communication pattern in the immunoaqed group

Immune cell communication with adjacent cells is mostly done in a paracrine manner be secretion of cytokines. To analyze the altered communication by cytokines from experienced immune cells in vivo during the fracture healing process, the secreted cytokines were screened via multiplexed immunosorbent assay (Multiplex ELISA). The systemic cytokine levels were measured in untreated animals either being experienced (immunoaged) or less-experienced (aged) in the adaptive immune system, pre-surgery, three days post-surgery and 21 days post-surgery. The most striking cytokines measured pre-surgery and three days post-surgery are shown in Figure 6. The tumor necrosis factor a (TN Fa) level could be found to be systemically increased in 12 months old untreated mice with an experienced immune system (immunoaged group) compared to the same age mice with a less-experienced immune system (aged group), showing the inflammaged status in these mice with a low-grad pro-inflammatory milieu. Interestingly, interleukin (I L)-10 was likewise increased at the baseline state in the immunoaged group. Three days post-surgery, during the inflammatory phase of fracture healing, significant increased levels of cytokines could be observed in the immunoaged group. IL-1 b, IL-5, IL-22 and granulocyte-macrophage colony-stimulating factor (GM-CSF) showed significant increased systemic levels post-surgery in the immunoaged group. IL-6 seemed to be generally induced by an osteotomy in both groups, whereas IL-22 is significantly downregulated in the aged group significant higher levels could be observed in the immunoaged group. IL-22 is secreted by immune cells of the innate and the adaptive immune system and interestingly the IL-22 receptor can only be found on non-hematopoietic cells like endothelial and other stromal cells. GM-CSF can be secreted by varying immune cell populations, but also by endothelial and other stromal cells, like fibroblasts. GM-CSF can induce proliferation of macrophages but can also polarize macrophages towards the M1 lineage, not only higher GM-CSF levels could be observed but also an increased proportion of M1 macrophages at the fracture site (see Figure 5 B).

Example 7 IL-22, IL-5 and GM-CSF driving the regenerative process in vitro

The Cytokine milieu is significantly diverging between the experience levels in both 12 months old mice group. IL-5, IL-22 and GM-CSF were selected as potential cytokines interfering with the regenerative process during fracture healing. Therefore, all three cytokines were tested in vitro towards their interference during osteogenic, adipogenic and chondrogenic differentiation of MSC as well as migratory/proliferative potential of MSC in a wound healing assay. The cytokines were also tested in an endothelial cell tube formation assay, to state their interference with angiogenesis during fracture healing. Interestingly, all three cytokines induced the mineralization of the extracellular matrix of MSC during osteogenic differentiation at low concentrations, IL-5 and GM- CSF showed not difference in ECM mineralization at higher concentrations (Figure 7 B). The adipogenic differentiation and lipid storage in the cytoplasm was not affected by the cytokines, except for GM-CSF at low concentrations. Under the influence of GM-CSF at low concentration (1 ng/ml) significantly fewer fat vacuoles could be observed compared to the untreated control cells under adipogenic differentiation signals (Figure 7 A). The chondrogenic differentiation process was not altered under the influence of IL-5, IL-22 nor GM-CSF (Figure 7 C). However, the migratory properties were significantly deteriorated under the influence of all three cytokines, except for IL- 22 at low concentrations. The introduced wound by scratching took significant more time to heal compared to the untreated control (Figure 7 F). Not only the migratory properties of MSC were hampered also the tube formation from endothelial cells were affected by the cytokines. IL-22 and also IL-5 significantly reduced the ability to form new tubes by reducing the branching between neighboring tubes (branching points) and significant less new tubes were formed (Figure 7D and E). Cytokines released by immune cells showing elevated levels in the immunoaged group directly interfered with the regenerative process carried out by MSC and endothelial cells. Reducing the migratory potential of progenitor cells and delaying the revascularization in a healing scenario may delay bone fracture healing in spite of the increased mineralization of the ECM.

Example 8: Proof of concept: IL-22 neutralization or overexpression in vivo

IL-22 was shown to interfere with the major processes necessary for bone healing: migration of progenitor cells, osteogenic differentiation and angiogenesis. Therefore, in an in vivo proof of concept experiment IL-22 was either neutralized or added in 12 months old mice being either immunologically experienced or less-experienced (aged). In the aged group, showing decreased levels of IL-22 three days post-surgery, recombinant IL-22 was added/substituted during the initial seven days post-surgery; in the experienced group, showing elevated levels of IL-22, IL-22 was neutralized via an IL-22 neutralizing antibody. Analyzing the fracture healing outcome 21 days post- surgery, during the hard callus phase, the IL-22 treatment showed significant effects. The less- experienced (aged) 12 months old group receiving recombinant IL-22 showed significant inferior healing properties as seen by decreased callus volume (TV) and newly formed bone volume (BV). Neutralizing IL-22 in the experienced (immunoaged) group showed a healing progression comparable to the aged group. TV and BV were significantly increased and the minimal polar moment of inertia (MMIp), a computational analysis of torsional stability, showed an increased stability of the callus 21 days post-surgery (Figure 8 A and B). Neutralizing IL-22 during the healing process in immunologically experienced mice showed an accelerated healing process and showed a similar healing outcome as seen in the aged group. Substitution of IL-22 on the other hand almost abolished the healing process.

Example 9: Proof of conce t: IL-5 neutralization or overexpression in vivo

IL-5 was shown to interfere with the major processes necessary for bone healing: migration of progenitor cells, osteogenic differentiation and angiogenesis. Therefore, in an in vivo proof of concept experiment IL-5 was either neutralized or added in 6 months old mice being either immunologically experienced (immunoaged) or less-experienced (aged). In the aged group, showing decreased levels of IL-5 three days post-surgery, recombinant IL-5 was added/substituted during the initial seven days post-surgery; in the immunoaged group, showing elevated levels of IL-5, IL-5 was neutralized via an IL-5 neutralizing antibody. Analyzing the fracture healing outcome 21 days post-surgery, during the hard callus phase, the IL-5 treatment showed significant effects. The less-experienced (aged) group receiving recombinant IL-5 showed significant inferior healing properties as seen by decreased callus volume (TV) and newly formed bone volume (BV). Neutralizing IL-5 in the immunoaged group showed an elevated healing progression compared to the untreated group. The bone volume (BV) was significantly increased 21 days post-surgery (Figure 9 A and B). Neutralizing IL-5 during the healing process in immunologically experienced mice showed an accelerated healing process by increased bone formation. Substitution of IL-5 on the other hand decreased the regenerative capacity.

Example 10: Cytokine levels in young and aged individuals upon activation lnterleukin-5 and Interleukin-22 secretion in young and aged individuals was further monitored after stimulation of isolated immune cells from human donors. Isolated immune cells from peripheral blood (peripheral mononuclear cells, PBMC) from young healthy donors (aged 18-35) or aged healthy donors (aged 55-70) were activated via CD3 and CD28 antibody stimulation. The cytokine levels were measured after a three day activation scheme, revealing an increased IL5 production in aged donors compared to young donors. The IL-22 levels measured were relatively low and not significantly differing between the two groups. IL-22 might be secreted via a different activation scheme than CD3/CD28 stimulation. The increased IL-5 secretion from immune cells from aged human individuals depicts the translatability of the immunoaged setting with increased IL-5 levels in an aged preclinical model. Discussion

Altered cellular phenotype and functionality led to a distinct cytokine pattern which could be attributed to the level of immunological experience. Especially interleukin 22 and interleukin 5 could be identified as a hallmark of immunological experience associated diminished healing capacity in the aged. The increased experience in the adaptive immune system intensified and prolonged the inflammatory phase upon injury, significantly negatively affecting regeneration. Not only the response towards an injury was significantly amplified under immunological experience, but also at homeostatic level the immunological experience induced a slow-grad inflammatory state leading to immunoaged and inflammaged tissues. Patients with immunological experience induced delayed healing, which are more common in the elderly/aged population, are in need of new therapeutic approaches.

Aging is a multifaceted process and pre-clinical models mirroring the in-patient situation in the aged are highly warranted. Working on human aging, especially in the hematopoietic system, has important experimental limitations and for this reason adequate pre-clinical models mirroring aging characteristics and phenotypes needs to be taken into account. Here the inventors proposed a pre- clinical model of aged mice with discriminative levels of experience in the adaptive immune system. Aging per se is able to alter the immune cell composition, as a myeloid shift is seen in the aged population, hematopoietic stem cells favor the myeloid lineage over the lymphoid lineage in the aged (Pang et al. Proc Natl Acad Sci U S A (2011) 108:20012-20017. doi:10.1073/pnas.1116110108, Beerman et al. Curr Opin Immunol (2010) 22:500-506. doi:10.1016/j.coi.2010.06.007). Although, the number of myeloid cells is higher in the aged, the quality of myeloid cells seems compromised (Singh et al. Curr Stem Cell Reports (2018) 4:166- 181. doi:10.1007/s40778-018-0128-6, Chandel et al. Nat Cell Biol (2016) 18:823-832. doi:10.1038/ncb3385). However the exposure to the environment alters the immune composition in regard of memory formation individually. Understanding the mechanisms of immunological induced disturbed regeneration will provide the scientific community with new tools to improve the regenerative capacity. The hampered regenerative cascade in the elderly can often be attributed to an imbalanced immune reaction. The inventors and others showed the detrimental effect of immune cells during fracture healing (Baht et al. Springer (2018). doi: 10.1007/s11914-018-0423- 2, El-Jawhari et al. Injury (2016) 47:2399-2406. doi: 10.1016/j. injury.2016.10.008, Claes et al. Nat Rev Rheumatol (2012) 8:133-143. doi:10.1038/nrrheum.2012.1). Not only different cell types of the immune system impact the regenerative process, here the inventors could show that the increased experience in the adaptive immune system directly affected the healing process upon injury. Here the inventors showed that the exposure of mice to environmental microorganisms by reducing the ventilation shielding significantly promoted the formation of effector and memory cells in the adaptive immune system. In comparison to mice housed under specific pathogens free (SPF) conditions, this pre-clinical model allows to discriminate the influence of the experience in the adaptive immune system and aging during regeneration. Chronological age and the biological age (e.g. increased immunological experience) alters the behavior of cells and signalling cascades interdependently during the regenerative process. Previous studies already showed that aged mice exhibit a reduced capacity of regeneration (Bucher et al. Front Immunol (2019) 10:797. doi:10.3389/fimmu.2019.00797, Mehta et al. Bone (2010) 47:219-228. doi:10.1016/j. bone.2010.05.029, Hebb et al. J Orthop Res (2018) 36:149-158. doi: 10.1002/jor.23652, Clark et al. Curr Osteoporos Rep (2017) 15:601-608. doi: 10.1007/s11914- 017-0413-9), however only in this study the analysis of mineralization, biomechanical stability, systemic and local innate and adaptive immune cell composition, influencing factors currently discussed such as the microbiome and the analysis of the MSC subset composition have been brought together to form a more comprehensive picture of the underlying processes (data not shown). Most interestingly, the inventors found that the cytokine patterns were distinct and could be used to develop new treatment approaches. Here the inventors showed the aggravation of age- induced lack of regenerative capacity with immunological experience-induced deterioration of the process.

Analyzing the local inflammatory response upon injury revealed an amplification and prolongation of the initial inflammatory phase under immunological experienced conditions. Increased levels of CXCR3 on the surface of CD8+ T cells was found to correlate with increased effector functions in CD8+ T cells and with disease severity (Hikono et al. J Exp Med (2007) 204:1625-1636. doi:10.1084/jem.20070322, Kurachi et al. J Exp Med (2011) 208:1605-1620. doi:10.1084/jem.20102101 , Audemard et al. J Autoimmun (2019) 99:73-80. doi:10.1016/j.jaut.2019.01 .012, Zhou et al. Curr Neuropharmacol (2019) 17:142-150. doi:10.2174/1570159x15666171109161140). High levels of CXCR3 also facilitate the migratory behavior of CD8+ T cells (Hickman et al. Immunity (2015) 42:524-537. doi:10.1016/j.immuni.2015.02.009). Less-experienced mice showed a decreased level of CXCR3 on CD8+ T cells during the initial inflammatory process in bone healing, depicting a less invasive behavior and diminished effector function. As late stage effector CD8+ T cells could be attributed to delay healing after injury, a timely and local downregulation of CXCR3 favors the progression of healing. Further studies need to reveal the CXCR3 contribution during fracture healing and the time course of regulation of this chemokine receptor. The ligands for CXCR3 are known to be involved in the activity of diseases and tissue resident cells are main producers of these ligands, especially CXCL10 and CXCL9 are potent attractors and the release is induced by IFNy and TNFoc (Kuo et al. Front Med (2018) 5:271. doi:10.3389/fmed.2018.00271 , Van Raemdonck et al. Cytokine Growth Factor Rev (2015) 26:311-327. doi: 10.1016/j.cytogfr.2014.11 .009). Markers depicting an active state of T cells are CD107a and CD137, where CD107a is more known as an unspecific activation marker, whereas CD137 is an activation marker for the involvement of the T cell receptor (TCR) (Aktas et al. Cell Immunol (2009) 254:149-154. doi:10.1016/j.cellimm.2008.08.007, Wolfl et al. Blood (2007) 110:201-210. doi:10.1182/blood-2006-11-056168). Experienced mice showed increased levels of CD107a compared to the less-experienced mice in the initial inflammatory phase, whereas both groups showed slight downregulation of CD137 compared to the steady state pre-surgery, but the levels of CD137 in steady state neither showed active CD8+ T cells (<2% of CD8+ T cells were found CD137 positive). Markers showing inhibitory signalling in T cells are upregulated three days post-surgery especially in CD4+ T cells, whereas the PD-1 upregulation on CD8+ T cells only showed a slight trend. PD-1 limits the effector function of T cells in pathogenic situation as well as in cancer (Sun et al. Immunity (2018) 48:434-452. doi:10.1016/j.immuni.2018.03.014, Sharpe et al. Nat Rev Immunol (2018) 18:153-167. doi: 10.1038/nri.2017.108) and seems to limit the activity of T cells also in endogenous processes like fracture healing as shown in this study. Taken all together, this study showed an amplified and prolonged immune reaction of the adaptive immune system, especially CD8+ T cells, locally at the fracture site in immunological experienced mice by upregulated activation marker, increased chemokine receptor availability and lack of inhibitory signalling. Those alterations in phenotype and functionality might be an initiator of the observed delayed healing in immunologically experienced mice and interference with the aforementioned altered process might lead to new diagnostic and therapeutic approaches of patients in need with immunologically restricted healing scenarios.

The innate immune system is a pre-requisite of successful healing and depletion of macrophages (Schlundt et al. Bone (2018) 106:78-89. doi: 10.1016/j. bone.2015.10.019, Hozain et al. Bone (2020) 138: doi: 10.1016/j. bone.2020.115479), reduction of neutrophil granulocytes (Kovtun et al. Eur Cells Mater (2016) 32:152-162. doi:10.22203/eCM.v032a10) or deficient Mast cells (Kroner et al. J Bone Miner Res (2017) 32:2431-2444. doi:10.1002/jbmr.3234) hampers or even abolishes the fracture healing process. Macrophage show an incredible plasticity of their effector functions and are potent regulator of the regenerative process by initiating and steering the inflammatory cascade. The adaptive immune cells however are causer of amplified and prolonged or deregulated inflammatory process in the endogenous healing cascade. This is the first study to comprehensively analyse the macrophage plasticity locally at the fracture site by discriminating not only M1 and M2 polarized macrophages but also activated M2 subtypes namely M2a, M2b, M2c and M2d based on their distinct expression profiles. All subtypes show distinct differences in their secretome and can be attributed to divers functions: M2a is known as the classically alternatively activated macrophage subtype with a known anti-inflammatory secretome like IL-10 or TGFp, M2b macrophages, also known as type 2, are prone producers of IL-1 , IL-6, IL-10 and TNFoc, M2c are described as a more deactivated phenotype with a comparable secretome to M2a macrophages and M2d macrophages are relevant producers of VEGF and known for pro-angiogenic effector functions (Mantovani et al. Trends Immunol (2004) 25:677-86. doi: 10.1016/j.it.2004.09.015, Roszer et al. Mediators Inflamm (2015) 2015: doi: 10.1155/2015/816460). Immunologically experienced mice showed an alternative activation state of macrophages locally at the fracture site. M1 and M2a macrophages were increased and depict a more pronounced inflammatory state (pro- and anti-inflammatory). Due to the limitation of timely resolving of the local events, it remains unclear if the increased levels of M2a macrophages in the immunologically experienced group can be seen as a counteract to the M1 macrophages or if both activated macrophage subtypes are independently upregulated, however, increased amounts of activated macrophages (M1 and M2a) might result in increased cytokine levels and therefore increased cellular stress. The increased levels of GM-CSF, observed in the experienced group, could also be attributed to an increased M1 activation, as GM-CSF is the main driver of M1 polarization. Dendritic cells are a key player in the signal transmission towards T cells, especially CD8+CD205+ dendritic cells are known to tightly interact with CD8+ T cells and also CD4+ T cells. The CD205+ dendritic cell population is responsible for cross-presentation of apoptotic cell-derived antigens (Shrimpton et al. Mol Immunol (2009) 46:1229-1239. doi:10.1016/j.molimm.2008.11 .016). Introducing an injury significantly increased the CD205 expression on CD8+ DC locally at the fracture site as shown in this study.

All cellular changes in phenotype and function alters the local inflammatory milieu and also the systemic levels of cytokines. Secreted cytokines are difficult to measure locally, therefore most studies measure gene expression levels, and here the inventors wanted to analyse the systemic secreted levels of cytokines. Muliplexed ELISA offers the advantage of parallel measurement of cytokines, but the quality of the analysis is still not comparable to singleplex ELISA. Analysis of systemic cytokine levels at three different timepoint allows the temporal resolution of inflammatory events during bone regeneration. Increased systemic levels of TNFoc and IL-6 in the pre-surgery state of experienced mice displays the slow-grade inflammatory state which is known under the term inflammaging (Franceschi et al. Nat Rev Endocrinol (2018) 14:576-590. doi: 10.1038/s41574- 018-0059-4). IL-6 is widely acknowledged to play a major role during fracture healing with distinct function on osteoblastic as well as osteoclastic activity by exerting both pro-inflammatory and antiinflammatory effects (Blanchard et al. Cytokine Growth Factor Rev (2009) 20:19-28. doi:10.1016/j.cytogfr.2008.11 .004). IL-6 neutralization during the initial inflammatory phase or under knock-out conditions reduced the systemic inflammation and recruitment of immune cells to the fracture site, but thereafter significantly delayed the regenerative cascade (Prystaz et al. Am J Pathol (2018) 188:474-490. doi: 10.1016/j.ajpath.2017.10.011 , Yang et al. Bone (2007) 41 :928- 936. doi:10.1016/j.bone.2007.07.022, Wallace et al. J Orthop Res (2011) 29:1437-1442. doi: 10.1002/jor.21367). IL-6 on the other hand is responsible for a rapid induction of effector cytokine expression in CD8+ T cells with known negative effects on the fracture healing outcome (Reinke et al. Sci Transl Med (2013) 5:177ra36. doi: 10.1126/scitranslmed.3004754, Bbttcher et al. Cell Rep (2014) 8:1318-1327. doi: 10.1016/j.celrep.2014.07.008). IL-6 was found to be upregulated by both groups in this study post-surgery, but increased levels were observed in the homeostatic state of experienced mice. Less is known for the cytokines GM-CSF, IL-5 and IL-22 during fracture healing, therefor all three cytokines have been tested intensively in vitro in regeneration-related assays. GM-CSF application in vivo rapidly induces the proliferation of mononuclear cells in the bone marrow and GM-CSF is a key factor in macrophage and dendritic cell maturation, especially for the macrophage polarization towards M1 (Kim et al. Tissue Eng Regen Med (2019) 16:59-68. doi: 10.1007/s13770-018-0163-5, Hamilton J Exp Med (2020) 217: doi:10.1084/jem.20190945). Interestingly T cells produce GM-CSF after activation, recently depicted as a RORyt dependent activation pathway (Codarri et al. Nat Immunol (2011) 12:560-567. doi: 10.1038/ni.2027) and increased GM-CSF might prolong the inflammatory state during fracture healing by increasing the maturation and proliferation of dendritic cells and macrophage M1 polarization. IL-5 exerts pleiotropic activities on various targets mainly inducing proliferation or differentiation and is produced by both hematopoietic and non-hematopoietic cells including T cells and granulocytes. Little is known of IL-5 and regenerative processes, however one study revealed that systemic overexpression of IL-5 induced an ossification of the spleen and major changes in the cancellous and trabecular bone (Macias et al. J Clin Invest (2001) 107:949-959. doi: 10.1172/JC111232), also underlying the significant interplay of T cells and progenitor cells of the osteogenic lineage. IL-22 is produced during inflammatory conditions by activated T cells, mainly by T helper 22 (TH22), TH17 and also TH1 cells. Targets for IL-22 are predominantly cells of the non-hematopoietic lineage and induces inflammatory mediators in the target cells or potentiates the influence of TNFoc or IL-17 induced pro-inflammatory cytokine expression. IL-22 inhibits the differentiation and/or increases the proliferation of progenitor cells and protects them against damage. In psoriasis IL-22 signalling might also exert a severe pathogenic role and neutralization of IL-22 showed beneficiary effects (Sabat et al. Nat Rev Drug Discov (2014) 13:21-38. doi: 10.1038/nrd4176, Dudakov et al. Annu Rev Immunol (2015) 33:747-785. doi:10.1146/annurev-immunol-032414-112123). In bone regeneration the role of IL-22 is delusive, Monasterio et al and Diaz-Zuniga showed a correlation between IL-22 and alveolar bone resorption and osteoclast resorptive activity during periodontitis, on the other hand El-Zayadi showed the contribution of IL-22 to new bone formation spondyloarthropathies (Diaz-Zuniga et al. J Periodontal Res (2017) 52:893-902. doi:10.1111/jre.12461 , Monasterio et al. J Periodontal Res (2019) 54:513-524. doi:10.1111/jre.12654, El-Zayadi et al. Rheumatol (United Kingdom) (2017) 56:488-493. doi:10.1093/rheumatology/kew384). In the present study IL-5 and IL-22 both showed the involvement in regenerative process shown by the in vitro assays, namely reduced migratory or proliferative behavior of MSC as well as reduced tube number and branching of de novo formed blood vessels of endothelial cells. However, both cytokines induced mineralization of the extracellular matrix in vitro, but the osteogenic differentiation stimulation cocktail comprises dexamethasone, which may significantly interfere with the inflammatory response of MSC. El- Zayadi also showed the effect of pro-inflammatory stimulation of MSC and the subsequent increased effect of IL-22; in the present study the inventors did no pre-treat the MSC by pro- inflammatory stimuli. However, progenitor cells are exposed in vivo to an inflammatory milieu rather than single cytokine exposure and the potentiation or inhibition effects of the cytokines cannot directly be mirrored in vitro. Therefore, the inventors started a proof-of-concept study in vivo by either neutralization or overexpression of IL-22 during the fracture healing process. Neutralization of IL-22 in aged and experienced mice, which showed increased levels of IL-22 post-surgery, displayed a healing outcome comparable to less-experienced aged mice. Overexpression of IL-22, however, almost completely abolished the regenerative process. Some new bone formation could be observed directly at the fracture ends, but no dispersed callus formation in the fracture gap, speculative by inhibiting the de novo vessel formation and inhibited migration of progenitor cells. In conclusion the inventors were able to show the contribution and the complex consequences of the aging immune system on the course of regeneration. The inventors showed distinct immune cell phenotype changes and resulting altered cytokine pattern in an advanced animal model that can mimic the healing in inflammaged patients. Age-associated diminished capacity of healing is significantly affected by the immunological experience in the adaptive immune system, which altered cellular phenotype and functionality not only in innate immune cells, but also in mesenchymal stromal cells. The reduced healing capacity is linked to the age-associated challenges of fracture patient treatment. The experience in the adaptive immune system altered the cellular composition locally at the fracture site, as well as the phenotypic contribution and activation states of immune cells and progenitor cells. Novel players were identified with a perspective of future treatment of immunologically disturbed fracture patients. IL-22 and IL-5 were shown to be significantly increased post-surgery under immunologically experienced conditions and the neutralization of IL-22 and IL-5 inverted the diminished callus volume and stability. New treatment options are highly warranted for aged and immunologically disturbed fracture patients and future studies need to reveal the potential of immunomodulatory agents to revert and rejuvenate the regenerative process in the aged.

Materials and Methods

Animal housing and surgery

Female C57BL/6NCrl mice were purchased from Charles River France at the age of 6 weeks. The mice were housed under specific pathogen free (SPF) housing conditions or under exposed housing. Exposed housing was achieved by reducing the filtration of the air supply. The light/dark cycle was set as a 12h rhythm, the temperature was set and monitored at 20°C and water and food was offered ad libitum. After six or 12 months of aging under the different housing conditions one group each underwent a necropsy under deep anaesthesia and the organs and tissues were removed for further analysis. Another group each received an analgesic and antibiotics (Buprenorphine and Clindamycin, respectively) and were anesthetized continuously with an isoflurane/oxygen mixture. The mice were kept on a heating pad set at 37°C and the surgical area was shaved and sterilized. An incision along the axis of the left femur and blunt preparation of the adjacent muscles was performed by sparing the integrity of ligaments and muscles as possible. The femur was exposed with forceps by shielding the sciatic nerve from damage. An external rigid fixator (MouseExFix, RISystem, Davos, Switzerland) was mounted after drilling 0.4 mm holes orthogonal to the Femur length and fixation with 4 pins. An osteotomy was introduced between the inner pins of the installed fixator with a Gigli wire saw having a diameter of 0.66 mm to introduce a 0.7 mm osteotomy gap. Muscles and ligaments were repositioned at the respective places and the skin was closed with a non-resorbable suture and the wound was further locked with a spray adhesive. Post anaesthesia care was performed by subcutaneous injection of substitutive fluid for faster recovery from fluid depriving during surgery and heat supply by infrared lamps. Until three days post surgery further analgesic (T ramadol) was substituted via the drinking water and soaked food pellet offered on the cage bottom. All surgeries and animal handling were conducted according the FELASA and ARRIVE guidelines and by the local administration permission G0338/13, G0061/14, G0287/19, G0209/20 and T0114/19. The ARRIVE guidelines were followed with the exception of blinded randomization of the groups, which could not be followed due to the different housing conditions and different operation theatres. Three or 21 days post-surgery the osteotomized mice underwent necropsy under deep anaesthesia as mentioned above for the control groups and the organs and tissues were removed and prepared for further analysis.

Cell phenotype and functionality analysis

Spleen and bone marrow were brought to a single cell suspension by flushing the bone marrow and mincing the tissue through a nylon mesh filter. The hematoma was collected with the adjacent bone marrow around the fixation pins, which also resemble a hematoma due to the drilling of the bone, with fine forceps and mincing through a nylon mesh filter. The erythrocytes were lysed with an RBC Lysis buffer (BioLegend, San Diego, USA). A live dead discrimination was performed with the Fixable Live/Dead Blue staining kit for UV excitation (Invitrogen, Waltham, USA). Staining with antibodies was done in flow cytometry buffer (phosphate buffer saline (PBS) including bovine serum albumin and sodium acid to block the internalization of some surface marker) with constant cooling on ice. Unspecific binding of antibodies to fc receptors was blocked with the FcX blocking solution (BioLegend, San Diego, USA). Intracellular staining for cytoplasmic targets was done with a fixation and permeabilisation kit (BioLegend, San Diego, USA) and intranuclear staining was done with the T rueNuclear T ranscription Factor buffer set (BioLegend, San Diego, USA) according the manufacturers protocol. The stained cells were analysed on a LSR fortessa SORP system by applying a bead-based compensation matrix. Gating and analysation of the population was done with the FlowJo software (Tree Star, Ashland, OR, USA) using fluorescence minus one (FMO) controls to set the gates.

Cytokine secretion

Blood from all mice was collected by intracardial puncture under deep anaesthesia and storing of the blood in heparin coated tubes. The plasma was collected after centrifugation and stored at - 80°C until further use. The following multiplexed fluorescent-labelled immunosorbent assay kit were run according the manufactures instructions with filter plates: LegendPlex Mouse Cytokine Panel 2, LegendPlex Mouse T Helper Cytokine Panel and LegendPlex Mouse Proinflammatory Chemokine Panel (all BioLegend, San Diego, CA, USA). The multiplexed bead based immunoassays were measured on a CytoFlex LX system (BeckmanCoulter, Brea, CA, USA) and analysed with the LegendPlex Software (v8.0, BioLegend, San Diego, CA, USA).

Cell culture supernatants were collected and stored at -80°C until further use. The following singleplex immunosorbent assay kit were run according the manufactures instructions: IL-22 Human Uncoated ELISA Kit (ThermoFisher, Waltham, MA, USA) and IL-5 Human Uncoated ELISA Kit (ThermoFisher, Waltham, MA, USA). Signal intensity was measured with a Infinite 200 PRO plate reader (Tecan, Mannedorf, Switzerland).

Micro-computed x-ray tomography

The osteotomized bones were harvested post-necropsy and directly fixated in ice-cold 4% paraformaldehyde/PBS (Electron Microscopy Sciences, Hatfield, PA, USA) for 6h at 4°C with sequential shaking. The fixated bones were stabilized within a serological pipette and the external fixator was removed. The healing outcome was monitored via micro-computed tomography (pCT) with a SkyScan 1172 (Bruker, Kontich, Belgium). Voxel size was set at 8pm and a source energy of 70kV and 142pA was applied to scan the bones with beam filtering through a 0.5 mm aluminium filter. The shadow images were reconstruction using a modified Feldkamp algorithm implemented in the software suite NRecon (Bruker, Kontich, Belgium) with applying a ring artefact reduction and beam hardening corrections as specified by control scans. The reconstructed images underwent 2D and 3D analysis using the CTan software and 3D visualization was achieved with the CTvox software (both Bruker, Kontich, Belgium). The analysis of the fracture gap included a step for binarization, where a global threshold of 580mg HA/cm³ was set (standardized by measuring hydroxyapatite (HA) phantoms with known concentrations) and the trabecular structures of the fracture callus was binarized using an adaptive tresholding method. The newly formed bone was quantified by excluding the old cortical bone within the callus. This was achieved by using a morphological escalator, a new technique for automatic trabecular-cortical separation. Bone parameters are reported according the ASMBR guidelines for assessment of bone microstructure in rodents (Bouxsein et al. J Bone Miner Res (2010) 25:1468-1486. doi:10.1002/jbmr.141).

Progenitor cell differentiation assay

Bone marrow stromal/stem cells (MSC) were kindly provided by the Tissue Harvesting core unit of the BIH Center for Regenerative Therapies (BCRT) frozen in passage 2. All MSC were harvested from bone marrow aspirations from patients undergoing hip replacement with written consent. MSC were thawed and expanded in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher, Waltham, MA, USA) containing 1 g/l glucose and 110 mg/l sodium pyruvate, supplemented with 10% fetal bovine serum (FBS Superior, Biochrom, Berlin, Germany) and 1% penicillin-streptomycin (10000 U/ml, ThermoFisher, Waltham, MA, USA). Cells in passage 2+2 were used throughout the project and seeded in tissue-culture treated multiwall plate (Corning, Corning, NY, USA). Osteogenic differentiation was achieved culturing the MSC in StemXVivo Osteogenic/Adipogenic Base Media supplemented with StemXVivo Human Osteogenic Supplement (both R&D Systems, Minneapolis, MN, USA) according the manufacturers instruction for 14 days with media exchange twice a week. After culturing for 14 days the cells were fixated with 4% paraformaldehyde/PBS (Electron Microscopy Sciences, Hatfield, PA, USA), stained with 0.5% wt/vol Alizarin Red S (Sigma Aldrich, St. Louis, MO, USA) dissolved in dH2O and counterstained with 4',6-Diamidin-2- phenylindol (DAPI, Sigma Aldrich, St. Louis, MO, USA). DAPI fluorescence was measured with an Infinite 200 PRO plate reader(Tecan, Mannedorf, Switzerland) using multiple reads perwell (MRW) to ensure an unbiased signal measurement throughout the well area. A standard curve interpreting cell numbers was achieved by seeding defined cell numbers and directly staining with DAPI after cell attachment for normalization fluorescence and absorbance signals towards the cell number. Alizarin Red S absorbance was measured with an Infinite 200 PRO plate reader (Tecan, Mannedorf, Switzerland) after dissolving the bound dye from the culture plate with 10% wt/vol Cetylpyridinium chloride (Sigma Aldrich, St. Louis, MO, USA). Adipogenic differentiation was achieved culturing the MSC in StemXVivo Osteogenic/Adipogenic Base Media supplemented with StemXVivo Human Adipogenic Supplement (both R&D Systems, Minneapolis, MN, USA) according the manufacturers instruction for 14 days with media exchange twice a week. After culturing for 14 days the cells were fixated with 4% paraformaldehyde/PBS (Electron Microscopy Sciences, Hatfield, PA, USA), stained with 2pg/ml Nile Red (Sigma Aldrich, St. Louis, MO, USA) pre-dissolved in acetone and diluted in PBS and counterstained with DAPI. Both fluorescence signals were measured and the DAPI signal was interpreted according the osteogenic differentiation assay for normalization towards cell numbers. Chondrogenic differentiation was achieved culturing the MSC as a cell pellet in StemXVivo Chondrogenic Base Media supplemented with StemXVivo Human Chondrogenic Supplement (both R&D Systems, Minneapolis, MN, USA) according the manufacturers instruction for 21 days with media exchange twice a week. After culturing for 21 days the cell pellet was directly dissolved with 125 pg/ml papain from papaya latex (Sigma Aldrich, St. Louis, MO, USA) in buffered solution containing 5mM L-Cysteine (Sigma Aldrich, St. Louis, MO, USA) overnight at 60°C on a Thermomixer (VWR International, Radnor, PA, USA). Dissolved cell pellets were stored at -20°C until further use. The glycosaminoglycan (GAG) content was measured with 16 mg/l 1 ,9-Dimethyl-Methylene Blue (DMMB, Sigma Aldrich, St. Louis, MO, USA) in buffered solution and interpreted by dissolving known concentrations of chondroitin sulfate A from bovine trachea (Sigma Aldrich, St. Louis, MO, USA). To normalize the GAG content towards the cell number a DNA quantification assay was performed to achieve a ratio of GAG/DNA. DNA was quantified using the DNA Quantitation Kit, Fluorescence Assay (Sigma Aldrich, St. Louis, MO, USA) according the manufacturer’s instructions. Representative images for all assays were taken with a BZ-X810 microscope (Keyence, Osaka, Japan).

Wound healing assay

MSC were prepared as described above for the progenitor cell differentiation assay and used in passage 2+2. MSC were cultured in multiwell plates until reaching confluency in expansion medium consisting of DMEM (ThermoFisher, Waltham, MA, USA) containing 1 g/l glucose and 110 mg/l sodium pyruvate, supplemented with 10% fetal bovine serum (FBS Superior, Biochrom, Berlin, Germany), 1% GlutaMAX (ThermoFisher, Waltham, MA, USA) and 1% penicillin-streptomycin (10000 U/ml, ThermoFisher, Waltham, MA, USA). After reaching confluency the cells were checked for proliferation stop and a scratch was introduced with a 1 ml pipette tip, rinsed and replaced with fresh expansion medium. The wound closure was monitored after 6, 18, 24 and 48 hours post injury by taking phase contrast pictures (BZ-X810, Keyence, Osaka, Japan). Image analysis to assess the wound closure was done using an image J plugin, wound healing size tool (WHST), kindly provided by Suarez-Arnedo et al. (Suarez-Arnedo et al. PLoS One (2020) 15:e0232565. doi:10.1371/journaL pone.0232565) using the Fiji software (open source image processing package based on Imaged).

Tube formation assay

To assess the ability of cytokines to interfere with the angiogenic potential of endothelial cells, a tube formation assay was performed using human umbilical vein endothelial cells (HUVEC, pooled donor, Lonza, Basel, Switzerland). HUVECs were grown and expanded in endothelial cell growth medium (EGM-2, Lonza, Basel, Switzerland). Multiwell plates were pre-treated with a layer of extracellular matrix (Matrigel growth factor reduced, Corning, Corning, NY, USA) for 1 h a t 37°C prior seeding of 240,000 cells/cm² in a 24 well plate. The cells were imaged after 16 h with a BZ- X810 microscope (Keyence, Osaka, Japan) and tube formation was assessed with an image based analysis using Fiji software (open source image processing package based on Imaged).

IL-22 and IL-5 treatment

Mice were osteotomized as described above and the treatment was started 24h post-surgery to mimic the feasibility of this treatment under patient conditions. The substitution and neutralization of IL-22 was done by intraperitoneal injection of recombinant mouse IL-22 (PeproTech, Rocky Hill, NJ, USA) or rat anti-mouse functional grad IL-22 monoclonal antibody (clone IL22JOP, ThermoFisher, Waltham, MA, USA). IL-22 substitution was performed with 5 pg protein in 100 pl InVivoPure dilution buffer (BioXCell, Lebanon, NH, USA) every second day with the last injection timepoint one week post-surgery. The neutralizing IL-22 antibody was diluted at 100 pg in 100 pl InVivoPure dilution buffer with a treatment schedule likewise to the IL-22 substitution.

The substitution and neutralization of IL-5 was done by intraperitoneal injection of recombinant mouse IL-5 (PeproTech, Rocky Hill, NJ, USA) or InVivoMAb anti-mouse/human IL-5 (clone TRFK5, BioXCell, Lebanon, NH, USA). IL-5 substitution was performed with 5 pg protein in 100 pl InVivoPure dilution buffer (BioXCell, Lebanon, NH, USA) every day with the last injection timepoint one week post-surgery. The neutralizing IL-5 antibody was diluted at 100 pg in 100 pl InVivoPure dilution buffer with a treatment schedule likewise to the IL-5 substitution.

Immune cell activation in vitro

Peripheral blood mononuclear cells (PBMC) were isolated from blood drawn from healthy volunteers with written consent. PBMC were isolated by density gradient centrifugation using SepMate tubes and Lymphoprep medium (both Stemcell Technologies, Vancouver, Canada). The isolated cells were incubated with 10 pg/ml plate-bound CD3 antibody and 2pg/ml soluble CD28 antibody (BioLegend, San Diego, CA, USA) for 48h in RPMI1640 medium supplemented with 10% heat-inactivated FBS (ThermoFisher, Waltham, MA, USA). Supernatants were collected after centrifugation to remove cellular debris and frozen at -80°C until further use. Visualization and statistics

Statistical analysis and graphical visualization of the data was carried out with GraphPad Prism software (GraphPad, Software, San Diego, CA, USA). All values from in vitro assays are expressed as the Mean ± SEM and all values from animal experiments are depicted as Median ± Ranges (Whiskers Blot). Unpaired t-test was performed where appropriate including post-hoc tests and

Welch’s t-test was used in case of lack of Gaussian distribution (Kolmogorov-Smirnov test). Tukey’s post hoc test was used to exclude outliers. P <0.05 was considered as statistically significant. Power analysis was performed prior to animal tests by a statistician to determine the animal number (minimum 6 to get valuable results).

Claims

1 . An agent for use in treatment or prevention of delayed bone fracture healing and/or delayed non-union/fusion of a bone or spine segments, wherein the agent is selected from a. a non-agonist ligand of a target moiety; and b. a nucleic acid molecule capable of specifically suppressing expression of a target moiety; wherein the target moiety is selected from the group of: i. IL-22; ii. IL-22 receptor; iii. IL-3; iv. IL-3 receptor; v. IL-5; vi. IL-5 receptor; vii. GM-CSF; viii. GM-CSF receptor; ix. IL-1 P; x. IL-1 receptor; xi. CD121 a; xii. CD121 b; xiii. IL-1 RAcP; xiv. common beta chain receptor; xv. a1 subunit of the IL-22 receptor; xvi. CDW210B; xvii. CD123; xviii. CD125; xix. CD116; and xx. IL-22 binding protein.

2. The agent for use according to claim 1 , wherein the target moiety is selected from the group of IL-22, IL-22 receptor, a1 subunit of the IL-22 receptor, and common beta chain receptor, particularly wherein the target moiety is IL-22.

3. The agent for use according to claim 1 , wherein the target moiety is selected from the group of IL-5, IL-5 receptor, CD125, and common beta chain receptor, particularly wherein the target moiety is IL-5.

4. The agent for use according to any one of the preceding claims, wherein the ligand is selected from the group consisting of an antibody, an antibody fragment, a soluble receptor construct, an aptamer, a non-immunoglobin scaffold, and an antibody-like molecule, particularly wherein the ligand is an antibody or an antibody-fragment.

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5. The agent for use according to claim 4, wherein the antibody is a monoclonal antibody or the antibody-fragment of a monoclonal antibody.

6. The agent for use according to claim 5, wherein the antibody is a humanized monoclonal antibody or an antibody-fragment thereof.

7. The agent for use according to any one of the preceding claims 1 to 3, wherein the ligand is a biopolymer ligand, particularly a ligand selected from the group comprising a polypeptide, a peptide, and a peptidomimetic, more particularly wherein the ligand is a polypeptide.

8. The agent for use according to claim 1 to 3, wherein the nucleic acid molecule is an siRNA.

9. The agent for use according to claim 1 to 3, wherein the nucleic acid molecule is an antisense oligonucleotide, particularly the nucleic acid molecule is capable of forming a hybrid with an mRNA encoding the target moiety.

10. The agent for use according to claim 1 to 3 or 9, wherein the nucleic acid molecule is an antisense oligonucleotide that comprises ribonucleotides and/or deoxyribonucleotides, particularly wherein the antisense oligonucleotide molecule is an RNA or a DNA molecule.

11 . The agent for use according to claim 9 or 10, wherein the antisense oligonucleotide comprises locked nucleic acid moieties and/or peptide nucleic acid moieties.

12. The agent for use according to any one of the preceding claims, wherein said bone comprises a fracture and/or a bone fusion and/or a surgically-induced disconnection/osteotomy.

13. A nucleic acid molecule encoding the agent according to any one of the preceding claims for use in treatment or prevention of delayed bone fracture healing and/or non- union/fusion of a bone or spine segments.

14. A nucleic acid expression vector comprising the nucleic acid molecule of claim 13 for use in treatment or prevention of delayed bone fracture healing and/or non-union/fusion of a bone or spine segments.

15. The agent or the nucleic acid molecule or the nucleic acid expression vector for use according to any one of the preceding claims, wherein the patient shows an elevated pro- inflammatory reaction towards an injury associated with an increased immunological response of effector cells, particularly wherein a frequency of CD28-CD8+ T cells is >30 % of all CD8+ T cells and/or a frequency of CD57+CD8+ T cells is >30 % of all CD8+ T cells.

16. The agent or the nucleic acid molecule or the nucleic acid expression vector for use according to any one of the preceding claims in a patient of age over 35, particularly in a patient of age over 55.

38 The agent or the nucleic acid molecule or the nucleic acid expression vector for use according to any one of the preceding claims, wherein the ligand is administered by means of a local drug delivery system. A pharmaceutical composition comprising the agent according to any one of the preceding claims 1 - 12, the nucleic acid molecule according to claim 13, or the nucleic acid expression vector according to claim 14, and a pharmaceutically acceptable carrier, particularly formulated as an administration form for parenteral administration, more particularly for intravenous administration.