WO2024002914A1 - Prediction of, and composition to improve, tendon healing - Google Patents

Abstract

The present invention relates to a method for predicting a likelihood of compromised tendon healing, and to ligands, particularly antibodies, for treatment of compromised tendon healing, selected from selective ligands to a target comprised in the group of CD4, IL-17A, IL-23, the CCR6/CCL20 axis; CCR5, IL-6, CD11a, CD28, IL-1β, TNFα, IFN α/β.

Description

Prediction of, and Composition to Improve, Tendon Healing

This application claims the right of priority of European Patent Application EP22181354.6 filed 27 June 2022, which is incorporated by reference herein.

Field

The present invention relates to a method for predicting a likelihood of compromised tendon healing, and to ligands for treatment of compromised tendon healing.

Background of the Invention

Tendon healing is a complex process and often results in unfavorable healing. In case of acute rupture, the tendon fails to regenerate properly in approximately 30% of the patients. To date no clinically approved treatment option exists to support tendon healing after acute rupture or to prevent the development of chronic tendon pathologies. The early determination of patients at risk for compromised tendon healing will strengthen healing outcomes, due to the possibility of early interventions.

Cells of the immune system regulate healing processes all over the human body and lead to proper healing or, in case of disbalance, induce chronic tissue degeneration. The role of cells of the adaptive immune system, such as T cells, in tendon degeneration or regeneration is still unknown. These cells might act through mediation of inflammatory cytokines and thus modulation of the inflammatory tendon environment or through direct interplay with resident tenocytes.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to diagnose and treat compromised tendon 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. of the Invention

The inventors have surprisingly found that in acute Achilles tendon rupture, CD8+ T-cells and its subpopulations of CD28-, CD57+ and CD28-CD57+ memory T-cells in blood and hematoma aspirate at the time of surgery, positively correlated with the healing outcome 6 and 12 month after surgery, whereas CD4+ T-cells were associated with a negative healing potential. Therefore, CD4+/CD8+ T-cells can be used as diagnostic markers for the detection of patients developing pain or that have a risk for compromised healing after acute Achilles tendon rupture. A proof of mechanism in vitro study provided evidence that CD4+ T-cells negatively affect tenocytes and therefore Achilles tendon healing due to increasing the Collagen type III expression, IL17 receptor expression, and MMP levels, thus leading to a weakening of the extracellular tendon matrix. Inhibiting the CD4+ T-cell recruitment, activation, and/or proliferation at the rupture side can therefore be regarded as a promising tool to prevent inferior Achilles tendon healing.

The invention relates to findings that adaptive T cell populations serve as predictor for compromised Achilles tendon healing including tendon elongation and monitoring the course of treatment of a tendon rupture in a patient. In detail a higher CD4+/CD8+ ratio, likewise a higher CD4+ T cell percentage and lower CD8+ T cell percentage in peripheral blood at the time of Achilles tendon surgery is predicting compromised Achilles tendon healing. This leads to a further aspect of the invention relating to agents being capable of locally reducing CD4+ T cell recruitment, activation, proliferation, and/or locally reducing specific pro-inflammatory cytokines secreted by these T cells and/or locally inhibiting specific pro-inflammatory signalling pathways related to these T cells. This agent can be applied to patients that are identified to have a higher probability of compromised tendon healing by the diagnostic method of the present invention.

A first aspect of the invention relates to an agent for use in treatment of an Achilles tendon rupture, wherein the agent is capable of reducing the CD4⁺ T cell recruitment, CD4⁺ T cell activation, and/or CD4⁺ T cell proliferation and/or reducing specific pro-inflammatory cytokines and/or inhibiting specific pro-inflammatory signaling pathways in a patient. Agents that can be employed for this purpose according to the invention are specified in claim 1 .

A second aspect of the invention relates to a method for prediction of compromised tendon healing in a patient. An alternative of the second aspect of the invention relates to a method for prediction of tendon elongation in a patient. A further alternative of the second aspect of the invention relates to a monitoring the course of treatment of a tendon rupture in a patient. The method comprises the steps: a. providing at least one blood sample isolated from said patient; b. determining in said blood sample: i. a CD4⁺/CD8⁺ T cell ratio within CD3⁺ T cells; and/or ii. a CD4⁺ T cell frequency within CD3⁺ T cells; and/or iii. a CD8⁺ T cell frequency within CD3⁺ T cells; and/or iv. a frequency of CD11 a⁺⁺CD28' T cells within CD3⁺CD8⁺ T cells; and/or v. a frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells; and/or vi. a frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells; c. assigning a probability of compromised tendon healing to said patient; wherein i. a high CD4⁺/CD8⁺ T cell ratio within CD3⁺ T cells; and/or ii. a high CD4+ T cell frequency within CD3⁺ T cells; and/or iii. a low CD8+ T cell frequency within CD3⁺ T cells; and/or iv. a low frequency of CD11 a⁺⁺CD28^_ T cells within CD3⁺CD8⁺ T cells; and/or v. a low frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells; and/or vi. a low frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells; is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient.

A third aspect of the invention relates to the agent for use according to the first aspect, wherein a high probability of compromised tendon healing was assigned to said patient by the method according to the second aspect of the invention.

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 dictates 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.

Cell Biology, diagnostic method inventions: Markers, ligands

In the present specification, the term positive, when used in the context of expression of a marker, refers to expression of an antigen assayed by a fluorescently labelled antibody. Such expression of a marker is indicated by a superscript “plus” (⁺), following the name of the marker, e.g. CD4⁺, or by the addition of the plus sign after the marker name (“CD4+”). If the word “expression” is used herein in the context of “gene expression” or “expression of a marker or biomolecule” and no further qualification of “expression” is mentioned, this implies “positive expression” as defined above.

In the present specification, the term negative, when used in the context of expression of a marker, refers to expression of an antigen assayed by a fluorescently labelled antibody, wherein the median fluorescence intensity is less than 30% higher, particularly less than 15% higher, than the median fluorescence intensity of an isotype-matched antibody which does not specifically bind the same target. Such expression of a marker is indicated by a superscript minus ('), following the name of the marker, e.g. CD127', or by the addition of the minus sign after the marker name (“CD127-”).

High expression of a marker refers to the expression level of such marker in a clearly distinguishable cell population that is detected by FACS showing the highest fluorescence intensity per cell compared to the other populations characterized by a lower fluorescence intensity per cell. Cells designated double positive or “++” with respect to certain marker molecules means cells exhibiting a high expression of this certain marker molecule which can be separated as a distinct subpopulation by electronic gating. Double positive or “++” cells give a fluorescence signal significantly stronger than that given by cells at the lower end of the single positive “+” gate. “++” events can typically be distinguished as a distinct cluster. Cells “++” for a given marker are part of the “+” population for that marker.

Low expression of a marker, for example low expression of CD25, refers to the expression level of such marker in a clearly distinguishable cell population that is detected by FACS showing the lowest fluorescence intensity per cell compared to the other populations characterized by higher fluorescence intensity per cell. A low expression is indicated by superscript “low” or “Io” following the name of the marker, e.g. CD25^|OW. The term “is expressed lowly” refers to the same feature.

The expression of a marker may be assayed via techniques such as fluorescence microscopy, flow cytometry, ELISPOT, ELISA or multiplex analyses.

Surface molecule expression may also be assessed by adding detection antibodies to stimulation cultures e.g.: CD154 and CD137. In case CD154 is used, CD154 detection antibody may be added to culture at stimulation initiation or after stimulation. In the latter case, an antibody against CD40 may be added to facilitate CD154 detection. 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.

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.

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. compromised tendon 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.

In the context of the present specification, the term frequency refers to a quotient of the amount of a target population of cells and the amount of reference population of cells. For example, a CD4⁺ T cell frequency within CD3⁺ T cells is determined by counting the CD4⁺CD3⁺ T cell and dividing the count by the count of all CD3⁺ T cells. The frequency may be expressed as percentage.

In the context of the present specification, the term ratio refers to a quotient of two different frequencies. For example, a CD4⁺/CD8⁺ T cell ratio is determined by measuring the CD4⁺ T cell frequency and the CD8⁺ T cell frequency as described above, and then dividing the CD4⁺ T cell frequency by the CD8⁺ T cell frequency. The ratio is a non-unit number larger than 0.

In the context of the present specification, the term high frequency or high ratio refers to a frequency or ratio which is significantly above the frequency or ratio in a reference sample of a healthy subject.

In the context of the present specification, the term low frequency or low ratio refers to a frequency or ratio which is significantly below the frequency or ratio in a reference sample of a healthy subject.

In the context of the present specification, the term threshold refers to a value below or above which the respective patient has a high risk of developing compromised tendon healing. Detailed Description of the Invention

A first aspect of the invention relates to an agent for use in treatment of an Achilles tendon rupture. The agent is capable of reducing the CD4⁺ T cell recruitment associated with Achilles tendon injury. Likewise, application of the agent may interfere with CD4⁺ T cell activation, CD4⁺ T cell proliferation, and/or reduction of specific pro-inflammatory cytokines, and/or may inhibit specific pro- inflammatory signaling pathways. In certain embodiments, the agent is administered locally to a rupture side of the Achilles tendon. In certain embodiments, the agent is administered subcutaneously. In certain embodiments, the agent is administered systemically. In certain embodiments, the agent is administered intravenously.

In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to CD4. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to IL-17A. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to IL-23. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to the CCR6/CCL20 axis. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to CCR5. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to IL-6. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to CD11 a. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to CD28. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to IL-1 p. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to TNFa. In certain embodiments, the agent for use according to this first aspect of the invention is capable of binding to IFN a/p.

In certain embodiments, the agent for targeting CD4 is selected from the group comprising IT1208, Tregalizumab (BT-061), MAX.16H5 IgGi . In certain embodiments, the agent for targeting IL-17A is selected from the group comprising Sekunkinumab, Brodalumab, Ixekizumab. In certain embodiments, the agent for targeting IL-23 is selected from the group comprising Guselkumab, Risankizumab, Ustekinumab. In certain embodiments, the agent for targeting is selected from the group comprising CCR6/CCL20 axis: CCX9664, GSK3050002. In certain embodiments, the agent for targeting CCR5 is Ibalizumab. In certain embodiments, the agent for targeting IL-6 is Tocilizumab. In certain embodiments, the agent for targeting CD11a is Alefacept. In certain embodiments, the agent for targeting CD28 is Abatacept. In certain embodiments, the agent for targeting IL-1 p is selected from the group comprising Canakinumab, Anakinra. In certain embodiments, the agent for targeting TNFa is selected from the group comprising Adalimumab, Certolizumab, Etanercept, Golimumab, Infliximab. In certain embodiments, the agent for targeting IFN a/p is selected from the group comprising Anifrolumab, S95021 . IL-17 exists in different isoforms. IL-17A is the cytokine that is primarily involved in various chronic inflammatory diseases. Sekunkinumab and ixekizumab have IL-17A as a target. Brodalumab blocks the IL-17 receptor and thus the binding of IL-17A and F.

A second aspect of the invention relates to a method for prediction of compromised tendon healing in a patient. An alternative of the second aspect of the invention relates to a method for prediction of tendon elongation in a patient. A further alternative of the second aspect of the invention relates to a monitoring the course of treatment of a tendon rupture in a patient.

The method comprises the steps: a. providing at least one blood sample isolated from said patient having a tendon rupture; b. determining in said blood sample: i. a CD4⁺/CD8⁺ T cell ratio within a population of CD3⁺ T cells; and/or ii. a CD4⁺ T cell frequency within a population of CD3⁺ T cells; and/or iii. a CD8⁺ T cell frequency within a population of CD3⁺ T cells; and/or iv. a frequency of CD11 a⁺⁺CD28' T cells within a population of CD3⁺CD8⁺ T cells; and/or v. a frequency of CD11 a⁺⁺CD57⁺ T cells within a population of CD3⁺CD8⁺ T cells; and/or vi. a frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within a population of CD3⁺CD8⁺ T cells; c. assigning a probability of compromised tendon healing to said patient; wherein i. a high CD4⁺/CD8⁺ T cell ratio within a population of CD3⁺ T cells; and/or ii. a high CD4+ T cell frequency within a population of CD3⁺ T cells; and/or iii. a low CD8+ T cell frequency within a population of CD3⁺ T cells; and/or iv. a low frequency of CD11 a⁺⁺CD28' T cells within a population of CD3⁺CD8⁺ T cells; and/or v. a low frequency of CD11 a⁺⁺CD57⁺ T cells within a population of CD3⁺CD8⁺ T cells; and/or vi. a low frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within a population of CD3⁺CD8⁺ T cells; is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient.

In certain embodiments, a high CD4⁺/CD8⁺ T cell ratio within a population of CD3⁺ T cells is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient. In certain embodiments, high CD4+ T cell frequency within a population of CD3⁺ T cells is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient. In certain embodiments, a low CD8+ T cell frequency within a population of CD3⁺ T cells is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient. In certain embodiments, a low frequency of CD11 a⁺⁺CD28^_ T cells within a population of CD3⁺CD8⁺ T cells is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient. In certain embodiments, a low frequency of CD11a⁺⁺CD57⁺ T cells within a population of CD3⁺CD8⁺ T cells is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient. In certain embodiments, a low frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within a population of CD3⁺CD8⁺ T cells is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient.

In certain embodiments, a high probability of compromised tendon healing is assigned to said patient if criteria i, ii, and/or iii are met in a blood sample obtained at the time of surgery.

In certain embodiments, a high probability of tendon elongation is assigned to said patient if criteria iii, vi, v, and/or vi, are met in a blood sample obtained ~ six weeks after surgery.

In certain embodiments, a high probability of compromised tendon healing is assigned to the patient if the CD4⁺/CD8⁺ T cell ratio is above a threshold in a blood sample obtained at the time of surgery. In certain embodiments, the CD4⁺/CD8⁺ T cell ratio threshold is >1.7. In certain embodiments, the CD4⁺/CD8⁺ T cell ratio threshold is >2.3. In certain embodiments, the CD4⁺/CD8⁺ T cell ratio threshold is >2.6.

In certain embodiments, a high probability of compromised tendon healing is assigned to said patient if the CD4⁺ T cell frequency within all CD3⁺ cells is above a threshold in a blood sample obtained at the time of surgery. In certain embodiments, the CD4⁺ T cell frequency threshold is >57.2%. In certain embodiments, the CD4⁺ T cell frequency threshold is >59.2%. In certain embodiments, the CD4⁺ T cell frequency threshold is >66.2%.

In certain embodiments, a high probability of compromised tendon healing is assigned to said patient if the CD8⁺ T cell frequency within all CD3⁺ cells is below a threshold in a blood sample obtained at the time of surgery. In certain embodiments, the CD8⁺ T cell frequency threshold is <38.5%. In certain embodiments, the CD8⁺ T cell frequency threshold is <28.3%. In certain embodiments, the CD8⁺ T cell frequency threshold is <25.8%.

In certain embodiments, a high probability of tendon elongation is assigned to the patient if the frequency of CD11 a⁺⁺CD28^_ T cells within CD3⁺CD8⁺ T cells is below a threshold in a blood sample obtained ~ six weeks after surgery. In certain embodiments, the CD11 a⁺⁺CD28^_ T cell threshold is <29.6%. In certain embodiments, the CD11a⁺⁺CD28^_ T cell threshold is <13.8%. In certain embodiments, the CD11 a⁺⁺CD28^_ T cell threshold is <11 .8%. In certain embodiments, a high probability of tendon elongation is assigned to the patient if the frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells is below a threshold in a blood sample obtained ~ six weeks after surgery. In certain embodiments, the CD11 a⁺⁺CD57⁺ T cell threshold is <31.9%. In certain embodiments, the CD11a⁺⁺CD57⁺ T cell threshold is <19.7%. In certain embodiments, the CD11a⁺⁺CD57⁺ T cell threshold is <16.9%.

In certain embodiments, a high probability of tendon elongation is assigned to the patient if the frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells is below a threshold in a blood sample obtained ~ six weeks after surgery. In certain embodiments, the CD11 a⁺⁺CD28^_ CD57⁺ T cell threshold is <23.5%. In certain embodiments, the CD11a⁺⁺CD28'CD57⁺ T cell threshold is <12.1 %. In certain embodiments, the CD11a⁺⁺CD28'CD57⁺ T cell threshold is <9.6%.

In certain embodiments, a high probability of tendon elongation is assigned to the patient if the CD8⁺ T cell frequency within all CD3⁺ T cells is below a threshold in a blood sample obtained ~ six weeks after surgery. In certain embodiments, the CD8⁺ T cell threshold is <39.1 %. In certain embodiments, the CD8⁺ T cell threshold is <26.2%. In certain embodiments, the CD8⁺ T cell threshold is <23.2%.

In certain embodiments, the tendon is the Achilles tendon.

In certain embodiments, the blood sample is obtained from venous blood.

In certain embodiments, the blood sample is obtained from a hematoma or hematoma aspirate at a tendon rupture site.

A third aspect of the invention relates to the agent for use according to the first aspect, wherein a high probability of compromised tendon healing was assigned to said patient via the method of the second aspect.

A further aspect of the invention relates to a kit comprising a plurality of antibodies, wherein each antibody is covalently bound to a different detectable label, and wherein said plurality of antibodies consists of a. an antibody specifically binding to CD4; b. an antibody specifically binding to CD8; c. an antibody specifically binding to CD3; d. an antibody specifically binding to CD11a; e. an antibody specifically binding to CD28; and f. an antibody specifically binding to CD57.

In certain embodiments, said detectable label is a fluorescent label. A further aspect of the invention relates to a method for treatment of an Achilles tendon rupture in a patient in need thereof, wherein said method comprises the steps: a. providing at least one blood sample isolated from said patient; b. determining in said blood sample: i. a CD4⁺/CD8⁺ T cell ratio within CD3⁺ T cells; and/or ii. a CD4⁺ T cell frequency within CD3⁺ T cells; and/or iii. a CD8⁺ T cell frequency within CD3⁺ T cells; and/or iv. a frequency of CD11 a⁺⁺CD28^_ T cells within CD3⁺CD8⁺ T cells; and/or v. a frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells; and/or vi. a frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells; c. assigning a probability of compromised tendon healing to said patient; wherein i. a high CD4⁺/CD8⁺ T cell ratio within CD3⁺ T cells; and/or ii. a high CD4+ T cell frequency within CD3⁺ T cells; and/or iii. a low CD8+ T cell frequency within CD3⁺ T cells; and/or iv. a low frequency of CD11 a⁺⁺CD28^_ T cells within CD3⁺CD8⁺ T cells; and/or v. a low frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells; and/or vi. a low frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells; is indicative for a high probability of compromised tendon healing, d. assigning said patient to

- more frequent medical consultation; and/or

- more frequent magnetic resonance imaging; and/or

- more frequent functional rehabilitation; and/or

- prolonged wearing of a boot promoting plantar flexion; and/or

- wearing a boot with increased incline promoting plantar flexion; if said patient has a high probability of compromised tendon healing

Medical treatment

Similarly, within the scope of the present invention is a method for treating compromised tendon healing in a patient in need thereof, comprising administering to the patient a non-agonist compound or a non-agonist polypeptide (particularly an antibody) according to the above description.

In certain embodiments, the non-agonist polypeptide is an antibody, antibody fragment, an antibody-like molecule or a protein A domains derived polypeptide.

In some embodiments, the non-agonist polypeptide 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 polypeptide is an antibody fragment. In certain embodiments, the non-agonist polypeptide 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.

Pharmaceutical Compositions, Administration/Dosaqe Forms and Salts

According to one aspect of the compound of the invention, the compound is provided as a pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form.

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.

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.

Similarly, a dosage form forthe prevention ortreatment of compromised tendon healing is provided, comprising a non-agonist ligand according to any of the above aspects or embodiments of the invention.

The invention further encompasses 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.

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). Certain embodiments of the invention relate to a dosage form for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.

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 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 compound or a non-agonist polypeptide (particularly an antibody) 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 compromised tendon healing.

Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with a disease associated with compromised tendon healing. This method entails administering to the patient an effective amount of a non-agonist compound or a non-agonist polypeptide (particularly an antibody) as identified herein, or its pharmaceutically acceptable salt, as specified in detail herein. The invention further encompasses the use of labelled antibodies identified herein for use in the manufacture of a kit for the detection of compromised tendon healing.

The invention further encompasses a system configured to execute the method of the second aspect.

Wherever alternatives for single separable features such as, for example, an isotype protein or ligand type or 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 ligand may be combined with any medical indication or diagnostic method 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 correlations between the percentage of CD8+ and CD4+ T cells in pre-operative peripheral blood and the outcome of the patients 12 month after surgery: CD8+ cytotoxic T cells correlated in a negative fashion with VAS function (A) and in a positive fashion with the ATRS (B) and the total muscle volume (C). The CD4+ T-helper cells negatively correlated with the ATRS (B) and the total muscle volume (C) and positively with VAS function (A). Cell populations were measured by flow cytometry. r_s: Spearman correlation coefficient.

Fig. 2 shows correlations between the T cell activation status in peripheral blood 6 weeks after surgery and the outcome after 12 months: CD8+CD11 a++CD28- T cells (A), CD8+CD11 a++CD57+ T cells (B) and CD8+CD11a++CD28-CD57+ T cells (C) negatively correlated with the Matles test (tendon elongation), which is given as difference of flexion angle to the contralateral foot after 12 month. Cell populations were measured using flow cytometry. r_s: Spearman correlation coefficient.

Fig. 3 shows patient outcome scores after grouping in optimal healing and compromised healing patients according to the ATRS score (>90, <70). Subjective score: 0-6, with 6 being worst; VAS pain/function: 0-10, with 10 being worst; Hannover score: 0-100, with 100 being best; A heel rise repetitions: measured as difference to the contralateral side; total muscle volume: calculated as sum of the three muscles m. soleus, m. gastrocnemius lateralis/medialis and given in % to the pre-operative state; muscle volume of solely m. soleus given in % to pre-operative state; interval: time from rupture to surgery. Mann Whitney U test, p<0.05.

Fig. 4 shows CD4+ and CD8+ cell populations in the optimal healing and compromised healing group according to the ATRS score (>90, <70) after 12 month: Cell populations analyzed by flow cytometry in hematoma aspirate, as well as peripheral blood pre-operative, after 6 weeks, 6 month, and 12 month. N=8 optimal healer, n=5 compromised healer. Mann Whitney U test, p<0.05 (*) and p<0.01 (**).

Fig. 5 shows CD8+ T cell subsets in blood of patients with/without tendon elongation (Matles <4, >7) after 12 month: Cell populations analyzed by flow cytometry in hematoma fluid, as well as peripheral blood pre-operative, after 6 weeks, 6 month, and 12 month. N=15 non-elongation, n=7 elongation. Mann Whitney U test, p<0.05.

Fig. 6 shows ROC analysis with 95% confidence interval to evaluate the prognostic value of T cells for identifying patients with risk for compromised Achilles tendon healing. A) CD4+ of CD3+ T cells, B) CD8+ of CD3+ T cells and C) CD4+/CD8+ T cell ratio in pre-operative peripheral blood. At a cut-off at ATRS±70 n=5 compromised healers and n=21 healers were evaluated.

Fig. 7 shows ROC analysis with 95% confidence interval to evaluate the prognostic value of CD8+ memory T cells in identifying patients with risk for tendon elongation. A) CD11a++CD28- of CD8+ T cells, B) CD11 a++CD57+ of CD8+ T cells and C) CD11a++CD28-CD57+ of CD8+ T cells in peripheral blood 6 weeks after surgery. At a cut-off at Matles±7° n=7 tendon elongations and n=21 non-tendon elongations were evaluated.

Fig.8 Experimental set-up of proof of mechanism study.

Fig. 9 Gene expression and protein secretion of tenocytes kept in co-culture with unpolarized CD4+ or CD8+ T-cells in stimulation medium (! DMEM/HAMs + 10% FCS + 1% P/S, % X-Vivo,

unpolarized polarization medium). A) Gene expression calculated as normalized expression to the housekeeping gene HPRT and given as fold to untreated (culture medium: DMEM/HAMs F12 + 10% FCS + 1% P/S). B) Protein secretion given as fold to untreated. Statistics: Wilcoxon Test, p<0.05 (*). A dashed line indicates trends (<0.1).

Fig. 10 Gene expression, protein secretion and healing properties of tenocytes kept in coculture with IL17-polarized CD4+ or unpolarized CD8+ T-cells in the respective stimulation medium (% DMEM/HAMs + 10% FCS + 1% P/S, % X-Vivo, % IL17- polarized/unpolarized medium). A) Gene expression calculated as normalized expression to the housekeeping gene HPRT and given as fold to untreated (culture medium: DMEM/HAMs F12 + 10% FCS + 1% P/S). B) Protein secretion given as fold to untreated. C) Wound healing given as percentage of closure to the 0 h time point and contraction given as reduction of the size of the gel area in cm² compared to untreated. Statistics: Wilcoxon Test, p<0.05 (*). A dashed line indicates trends (<0.1).

Fig. 11 Gene expression, protein secretion and healing properties of tenocytes kept in coculture with IL17-polarized CD4+ or IFNy-polarized CD8+ T-cells in the respective stimulation medium (% DMEM/HAMs + 10% FCS + 1 % P/S, % X-Vivo, % IL17-/IFNy- polarized medium). A) Gene expression calculated as normalized expression to the housekeeping gene HPRT and given as fold to untreated (culture medium: DMEM/HAMs F12 + 10% FCS + 1% P/S). B) Protein secretion given as fold to untreated. C) Wound healing given as percentage of closure to the 0 h time point and contraction given as reduction of the size of the gel area in cm² compared to untreated. Statistics: Wilcoxon Test, p<0.05 (*). A dashed line indicate trends (<0.1).

Fig. 12: Proposed mechanism of CD4+ and CD8+ T-cells in compromised Achilles tendon healing.

Examples

Example 1: Clinical Study

Material and Methods: Clinical Study

Study design, tendon sampling and blood donation

A total of 31 patients (mean age: 39.4±10.0 years, mean BMI: 24.8±2.3, sex: 3 female, 28 male) with acute Achilles tendon rupture receiving minimal invasive Achilles tendon reconstruction 2 to 9 days (mean 4.9±1.7 days) after rupture were included in the study. Patients were mostly recreational sportsmen. Patients who received oral medication or local injections of systemic cortisone, antibiotics and anabolic steroids, which might influence tendon structure, were excluded. Peripheral blood was taken from the patients preoperatively, as well as at follow up after 6 weeks, 6 and 12 months. Additionally, hematoma aspirate was taken directly from the rupture side at the time of surgery. Leukocytes from peripheral blood as well as the hematoma aspirate were assessed for T cell surface markers including CD3, CD4, CD8, CD11a+ % T cells, CD57+ % T cells, and CD28- % T cells to evaluate the adaptive immunity in these patients. A clinical analysis of the subjective, functional and radiological status of the Achilles tendon healing outcome was performed at the three follow up time points to analyze the clinical relevance of the data. Five out of 31 patients did not complete the follow up and samples were excluded from the outcome correlation analysis. The study was approved by the local Institutional Review Board (IRB) of the Charite- Universitatsmedizin Berlin (EA2/074/14) and all patients gave their written informed consent prior to surgery.

Surgery and Rehabilitation

Patients with acute Achilles tendon rupture underwent minimal-invasive Achilles tendon repair using the Dresden instrument as inaugurated by Amlang et al. (Amlang et al., Unfallchirurg, 108: 529-36). In contrast to the original described technique, tendon ends were secured in an over- tightened manner and AT was shortened in maximum plantarflexion. Postoperatively, the foot was placed in a walker with 30° of plantarflexion for six weeks. Early functional rehabilitation was initiated with partial weight bearing and physiotherapy.

Physiotherapy program was supervised for twelve weeks and consisted of functional training of the muscles and proprioceptive as well as coordinative training. Plantarflexion exercises started after the third week and were limited to neutral position. After the sixth week, the heel height was reduced 10° per week, and weight bearing was increased as tolerated. Each patient received a detailed handout for physiotherapy after surgery.

Flow cytometric analysis

Human peripheral blood and hematoma aspirate of the Achilles tendon rupture patients were collected in EDTA sampling tubes at the time of surgery and 6 weeks, 6 month and 12 month postoperatively (blood only). White blood cells were isolated from 200pl of whole blood or all available volume of hematoma aspirate by incubation for 12 min with 4ml of lysis buffer (1x Red Blood Lysis (RBL) Buffer, eBioscience). After centrifugation the resulting cell pellet was washed and resuspended in FACS buffer (1 % FCS in PBS, Biochrom AG). The cells were stained for the T cell related surface markers CD3, CD4, CD8, CD11a, CD28, CD57, as well as CD19, CD45 and live/dead reagent in FACS-buffer for 25 minutes. Antibody details are listed in table 1. After staining, cells were fixed in 1% paraformaldehyde (PFA) solution and measured within one day using the BD Canto II System. Frequency minus one (FMO) controls were performed exemplarily for validation and gating strategy. The amount of cells is given as percentage of the parent population.

Table 1 : Antibodies for flow cytometric analysis

AK Conjugate Company Cat# Dilution

CD3 V500 BD Horizon 560772 50

CD4 APC Cy7 BD Bioscience 561839 50

CD8 PerCP Biolegend BDL-344708 50

CDlla PECy7 BD Bioscience 561387 50

CD19 PE BD Pharmingen 555413 10

CD28 FITC BD Pharmingen 555728 5

CD45 PE eBioscience 12-0459-41 100

CD57 APC BD Bioscience 560845 1000

L/D violet Invitrogen L34955 1000

PE: Phycoerythrin, APC: Allophycocyanin, PerCP: Peridinin Chlorophyll Protein, FITC: Fluorescein Isothiocyanat

Patient’s outcome analysis

To evaluate the subjective as well as functional healing outcome of the Achilles tendons, the patients were recruited 6 weeks, 6 month as well as 12 month after surgery to the outpatient clinic. According to a standardized protocol the following parameters were evaluated: subjective score for patient satisfaction (1-6, 6=bad), visual analog scale (VAS) for pain (1-10, 10=bad), VAS for function (1-10, 10=bad), Matles Test for Achilles tendon lengthening (Achilles tendon resting angle in plantarflexion relative to contralateral side, high=bad), Tegner activity score (high=good), Achilles tendon total rupture score (ATRS, 1-100, 100=best), Hannover score (1-100, 100=best), heel rise test (heights and repetitions relative to contralateral side, high=bad), and the maximum calf circumference (MCC, relative to pre-operative state in cm, high=bad). For measuring maximum heel-rise height, patients were asked to stand in a maximum one-legged tiptoeing position. Maximum heel rise height was measured bilaterally and defined as the distance between the plantar side of the heel and the floor. To test endurance of the calf muscles, maximum single-leg heel-rise repetitions were counted for each leg separately. The patient was asked to perform as many single-leg heel-rises as possible in one minute. The test ended either after one minute or was interrupted when the patient was unable to perform a proper heel-rise. If the test was interrupted, the performing time was noted and test time for testing maximum heel rise repetitions on the unaffected leg was adjusted accordingly. Using magnetic resonance imaging (MRI) the tendon length, the muscle volume (MV) of the Achilles tendon related muscles (musculus soleus, m. gastrocnemius lateralis/medialis), as well as the fatty degeneration of the m. soleus, m. gastrocnemius lateralis/medialis were evaluated. For the purpose of this study each patient underwent in total 4 MRI examinations of both lower legs. The first MRI was done pre- operatively to record the muscle volume, fatty degeneration and the Achilles tendon length of the contralateral side. This data served as internal control. The measured tendon length is given in % to the pre-operative contralateral side of the Achilles tendon and the muscle volume in % to the pre-operative muscle volume of the affected leg. The measured percentage of fatty degeneration of the muscles is given as fold to the pre-operative stage of the affected side. The muscle volume and the fatty degeneration were calculated as a sum of all three muscles (MV total) as well as for the m. soleus only, which is the most affected muscle regarding Achilles tendon pathologies. All MRI examinations were carried out with a 1 .5 Tesla imaging system (Magnetom Aera; Siemens Healthcare).

Statistics

Statistical analysis was performed using GraphPad Prism Version 7.0 (GraphPad Software, San Diego, CA, USA). To evaluate a relationship between the T cell composition in hematoma aspirate and peripheral blood with the patient outcome after 6 and 12 months, a Spearman correlation analysis was performed. Additionally, the patients were grouped according to ATRS in “optimal healer” (ATRS > 90 points) and “compromised healer” (ATRS < 70). Patients with ATRS scores between 89 and 71 were not considered in the healer analysis. The Mann-Whitney-U Test was performed to analyze significant differences between the groups and p<0.05 was considered as statistically significant. ROC analysis with 95% confidence interval was performed to evaluate the prognostic value of T cells with a cut-off for healers and non-healers at ATRS of 70 points and tendon elongation versus non-elongation at Matles test of 7°. Results: Clinical Study

Distribution of cell populations in hematoma aspirate and peripheral blood

The mean amount of analyzed cell populations within the whole patient cohort in hematoma aspirate, as well as in peripheral blood pre-operatively and after 6 weeks, 6 months and 12 months is listed in table 2. The percentage of lymphocytes was lowest in the hematoma aspirate and peripheral blood at the time of surgery and increased with the time after surgery, however without reaching significant differences. The percentage of T cells (CD3+) was significantly lower in hematoma aspirate from the tendon rupture side compared to all peripheral blood groups (preoperative: p<0.0001 , 6 weeks: p=0.011 , 6 month: p=0.027, 12 month: p=0.005, as indicted by * in T able 2). The amount of the other cell population were comparable between the groups.

Table 2: Distribution of cell populations in hematoma aspirate and peripheral blood:

*) Significant difference to hematoma aspirate group

Correlation between blood and hematoma status and the clinical outcome of the patients

The percentage of analyzed cell populations in hematoma aspirate, as well as peripheral blood was correlated to the clinical outcome scores after 6 and 12 months post-surgery. The correlations were more pronounced at the 12-month-follow-up (data from the 6-month clinical follow-up are not presented). A list of all correlations between the cell populations and the clinical follow up after 12 month is listed in table 3. The amount of T cells (CD3+) marginally negatively correlated with the Matles test (table 3), which indicates a positive contribution of this cell type for the tendon healing outcome.

The percentage of CD8+ T cells correlated with a positive healing outcome for the patient showing negative correlations to the subjective score, VAS pain, VAS function, the Matles test and the fatty degeneration of the m. soleus, as well as positive correlations to the ATRS, the maximum calf circumference, the total muscle volume, and the muscle volume of the m. soleus (Table 3 and Figure 1 A-C). This indicates a positive contribution of higher CD8+ T cell percentages in Achilles tendon healing.

In contrast, the percentage of CD4+ T cells showed a negative relationship to the healing outcome, indicated by a negative correlation of T-helper cells with the Tegner activity score, the ATRS, the maximum calf circumference, the total muscle volume, and the muscle volume of the m. soleus. Positive correlations occurred between the percentage of T-helper cells and the subjective score, the VAS pain and function, and the Matles test (Table 3 and Figure 1 A-C). Altogether, this shows a negative contribution of higher percentages of CD4+ T cells in Achilles tendon healing.

At the 6 and 12 month follow-up time points, CD4+ and CD8+ T cells in peripheral blood showed comparable results compared to the preoperative time points, but fewer and less pronounces significances to the subjective score, VAS pain/function, Matles and ATRS. This indicates the high potential of the CD4+/CD8+ T-cells ratio as an early prognostic marker for compromised Achilles tendon healing. A positive influence on the healing outcome was also seen for T cell subpopulations, but at later follow-up time points. This was indicated by a negative correlation of the Matles test (tendon elongation) and also the VAS pain and function with CD11a++, CD28-, CD57+ and CD28-CD57+ memory T cells (CD8+CD11a++) in peripheral blood from patients 6 weeks, 6 month and 12 month after surgery (Table 3 and Figure 2 A-C).

Table 3: Correlations between cell populations analyzed by flow cytometry and clinical outcome scores after 12 months

Heal rise reps: Heal rise repetitions; MV: Muscle volume

Clinical evaluation of optimal and compromised healing patients

Patients that displayed a compromised healing outcome (ATRS < 70) showed an increased subjective score (p=0.002), VAS pain score (p=0.016), VAS function score (p=0.01 1 ), and higher difference in heel rise repetitions (p=0.048) compared to the patients that reported optimal outcome results (ATRS > 90) (Figure 3 A-C, E)). Additionally, the Hannover score, as well as the total muscle volume and muscle volume of the m. soleus was significantly lower in the compromised healing group compared to the optimal healing group (p=0.030, p=0.006, p=0.024, respectively) (Figure 3 D, F, G). Altogether, this shows that the ATRS score is a suitable parameter for differentiating between optimal and compromised Achilles tendon healing. Interestingly, also the interval between rupture and surgery influenced the healing outcome with earlier time points of surgery leading to better clinical outcome results (p=0.024) (Figure 3 H). The age and BMI of the patients did not influence the outcome (data not shown). Factors indicating an Achilles tendon elongation (Matles test and tendon length measured by MRI) as well as the fatty degeneration of the corresponding muscles did not differ between the compromised and optimal healers grouped according to the ATRS score (data not shown).

Cell populations analyzed in optimal and compromised healing patients

When grouping the Achilles tendon patients according to the healing outcome in an “optimal healing” and “compromised healing” group utilizing the ATRS with a cut-off at 90 and 70 points, CD4+ T-helper cells were significantly increased in the compromised healing group compared to the optimal healing group in hematoma aspirate (p=0.030), pre-operative peripheral blood (p=0.006), peripheral blood 6 weeks and 6 months after surgery (p=0.002, p=0.030 respectively) (Figure 4). In contrast, CD8+ cytotoxic T cells were found in significantly higher amounts in the optimal healing group compared to the compromised healing group in hematoma aspirate (p=0.002), pre-operative peripheral blood (p=0.002) as well as blood 6 weeks and 12 months after surgery (p=0.031 , p=0.030 respectively) (Figure 4). Other cell populations did not differ between the healing groups according to the ATRS. In summary, this group analysis showed that a higher CD4+ to CD8+ T cell ratio is an indicator for compromised Achilles tendon healing.

CD8+ subpopulations analyzed in patients with and without tendon elongation

Patients with and without tendon elongation 12 month after surgery were identified by Matles Test (<4, >7) and evaluated regarding the impact of CD8+ T cell subpopulations. No significant differences occurred for CD8+ T cell subsets in pre-operative hematoma aspirate and peripheral blood. The analysis revealed that lower percentages of CD28-, CD57+ as well as CD28-CD57+ memory T cells (CD8+CD11 a++) in peripheral blood taken 6 weeks after surgery contributed to tendon elongation after 12 month (p=0.014, p=0.032, p=0.011 , respectively). In blood taken 6 month after surgery no significant differences occurred, but after 12 month CD28- memory T cells are decreased in patients with tendon elongation (p=0.032)(Figure 5).

CD4+ and CD8+ T cells as prognostic markers for healing outcome after Achilles tendon reconstruction:

Receiver operating characteristic (ROC) analysis was performed to investigate the prognostic value of CD4+ and CD8+ T cell percentages in pre-operative peripheral blood to identify patients with a risk to develop compromised tendon healing (ATRS <70). T cells in pre-operative blood showed the strongest relationship to the healing outcome and would present an optimal analysis medium to predict compromised healers during surgery in the future. In pre-operative peripheral blood, a CD4+ T cell percentage of >59.2% from CD3+ T cells was identified as cut-off for compromised Achilles tendon healing (ATRS <70) with a sensitivity of 100% and a specificity of 76.2% (AUC=0.848, p=0.018). For CD8+ T cells a cut-off value of <28.3% from CD3+ T cells was found for compromised healers, which produced 100% sensitivity and 85.7% specificity (AUC=0.867, p=0.012). The CD4+/CD8+ T cell ratio identified compromised healers at a cut-off at >2.3 with a sensitivity of 100% and a specificity of 81 % (AUC=0.848, p=0.018)(Figure 6).

CD8+CD11 a++CD28-CD57+ T cells as prognostic markers for tendon elongation:

The investigation of CD8+ memory T cell subsets in peripheral blood taken 6 weeks after surgery by ROC analysis revealed its prognostic value regarding tendon elongation. A CD11a++CD28- T cell percentage of <13.8% derived from CD8+ T cells was found as the cut-off value for Achilles tendon elongation (Matles >7), with a sensitivity of 71 ,4% and a specificity of 94,7% (AUC=0.842, p=0.009). For CD11 a++CD57+ T cells a percentage of <19.7% derived from CD8+ T cells was the cut-off, which produced 71.4% sensitivity and 84.2% specificity (AUC=0.782, p=0.030). Also CD11a++CD28-CD57+ T cells from CD8+ T cells with <12.1 % define patients with an Achilles tendon elongation (Matles >7), with a sensitivity of 71 .4% and a specificity of 89.5% (AUC=0.835, p=0.010)(Figure 7).

Conclusion

The data of the correlation analysis and healing group analysis suggest that a higher CD4 + 1 CD8 + T cell ratio in hematoma aspirate and peripheral blood at the time of surgery is associated with a worse clinical outcome with regard to pain and function 12 months after surgery. Thereby, the ATRS score is an optimal parameter for dividing patients into optimal and compromised healers. In addition to CD4+ and CD8+ T cells, an increased percentage of CD11 ++CD28-, CD11 ++CD57+, CD11 ++CD28-CD57 + from CD8+ T cells after 6 weeks, 6 months and 12 months in the peripheral blood is primarily associated with less tendon elongation. Tendon elongation does not seem to be a typical healing parameter (no correlation to ATRS), where other cell populations (memory T cells) are involved. In summary, the CD4 + 1 CD8 + T cell ratio at the time of surgery can be used as predictive diagnostic marker to identify patients with an increased risk of compromised Achilles tendon healing.

Example 2: Proof of Mechanism in vitro study

Material and Methods: Proof of Mechanism

For understanding the regulatory mechanism behind the negative contribution of a higher CD4+/CD8+ T-cell ratio on acute Achilles tendon healing, an in vitro study was performed. Here, we investigated the impact of direct co-culture of tenocytes with autologous unpolarized CD4+ or CD8+ T-cells. IFNy-polarized CD8+ T-cells and IL17-polarized CD4+ T-cells (Th17 cells) represent the physiologically most abundant cell populations and were additionally used for comparison, due to their high clinical relevance.

Human Achilles tendon samples and cell isolation:

Six male patients with a mean age of 36 years (Range: 24-50 years) and a mean BMI of 26.2 (Range: 22.7-31.1) with an acute Achilles tendon rupture receiving minimal invasive Achilles tendon reconstruction were included in the cell culture experiments. Patients with severe illness (HIV, Hepatitis C) or receiving medication (cortisone, anabolic steroids) were excluded. The study was approved by the local Institutional Review Board (IRB, EA2/074/14) and all patients gave their written informed consent prior to surgery. Peripheral blood was taken pre-operatively from the Achilles tendon rupture patients and collected in Heparin sampling tubes. PBMCs were directly isolated using SepMate™-50 tubes and Lymphoprep™ medium (both STEMCELL Technologies Inc.) by density gradient centrifugation. Isolated PBMCs were stored in RPMI 1640 (PAN Biotech) supplemented with 60% fetal calf serum (FCS, Sigma-Aldrich) and 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich) at-180°C until further use. Tenocytes of the respective donor were isolated by 0.3% collagenase digestion as described previously (Pauly et al. 2010, Eur Cell Mater, 20: 84-97). Cells were cultured with tenocyte medium (DMEM/Ham’s F12 (1 :1 , Sigma-Aldrich) with 10% FCS and 1% penicillin/streptomycin (Sigma-Aldrich)) at 37°C, 95% humidity and 5% CO2. After expansion, cells were stored at -180°C until further use.

Polarization and T-cell sorting

For polarization, round bottom 96-well plates (Greiner-Bio-One) were coated with anti-human CD3 antibody (BD Bioscience) in PBS and incubated at 4°C overnight. Next day, PBMCs were thawed and seeded into coated 96- Well plates with X-Vivo™ medium (Lonza) supplemented with antihuman CD28 antibody (BD Bioscience) and the respective agents to polarize T-cells either into IFNy, or IL17 producers, or left them unpolarized for four days. Polarizations were performed according to a modified protocol of Deiens et al. for IL17 production using 25 ng/mL rhulL6, 6.25 ng/mL rhuTGFpi , 12.5 ng/mL rhulLI (3 (all Peprotech), 25 ng/mL rhulL-23 (R&D), and 500 ng/mL anti-human IFNy antibody (BD Bioscience)(Delens et al. 2019, Biol Blood Marrow Transplant, 25: 204-15). IFNy polarization was induced with 20 ng/mL rhulL12p70 (Peprotech), 5 ng/ml rhulL2 (Novartis), and 500 ng/ml anti-human IL4 antibody (Biolegend). Afterwards, polarization medium (Pol-M) of the respective groups were collected and 1*10⁶ cells incubated with Live/Dead Fixable (L/D, Invitrogen) in 100 pl PBS in the dark at 4°C for 30 minutes. Subsequently 1*10⁶ cells were stained with antibodies against CD45, CD3, CD4, CD8 and TCRyS (table 2) diluted in PBS with 2% FCS and incubated at 4°C for 15 min. After staining, cells were sorted into CD4+ or CD8+ T-cells using the cell sorter BD FACS Aria II by the BIH Cytometry Core Facility (Berlin, Germany) and directly used for co-culture with autologous tenocytes. Wound healing assay

Tenocytes were seeded with 2.5*10⁴ cells/ml Tenocyte medium into 24-well plates and cultured at 37°C until reached 100% confluence. A wound (scratch) was induced using a 100 pl pipette tip and wells were scanned with a multiplate reader (Tecan SPARK®, Tecan). Afterwards, 2.5*10⁴ of the sorted CD4+ or CD8+ T-cells were added to the tenocytes in 1 ml of the respective experimental medium (Exp-M: % DMEM/Ham’s F12 + 10% FCS + 1% P/S, ¹/4 X-Vivo™, % Pol-M IFNy, IL-17 or Unpol). Tenocytes in culture medium (DMEM/Ham’s F12 + 10% FCS + 1 % P/S) without T-cells served as untreated controls. Cultivation was done at 37°C for 45 h. At time points 0, 15, 25, and 45 h the wound healing was documented with the Tecan SPARK®. After 45 h, supernatants were taken, cells were lyzed immediately for RNA isolation and supernatants stored at -20°C for further analysis. The wound closure was measured using Imaged (version 1 .530).

Cell contraction assay

A cell suspension with 7*10⁴ tenocytes and 3.5*10⁴ sorted CD4+ or CD8+ T-cells was prepared in the respective Exp-M IFNy, IL-17 or Unpol. Tenocytes in culture medium served as untreated controls. Gel suspension was prepared on ice with 1 ,5x PBS (1 Ox Biochrom), 6.4 mM sodium hydroxide (Sigma-Aldrich), 1.54 mg/ml Collagen Typ I (rat tail tendon, Corning) ad 250 pl ddH2O per gel. A total of 250pl Gel suspension was added to cell suspension and transferred into selfmanufactured silicone rings (1 cm inner diameter) placed in a 12-well plate (BD Falcon). After 30 minutes incubation at 37°C, 1 ml of the respective Exp-M or culture medium (untreated) was added to each well and silicone rings removed carefully to release the polymerized gels containing the tenocyte/T-cell co-culture. The gels were incubated for 65 h and gel contraction documented at time points 0, 15, 25, 45 and 65 hours. Reduction of gel size was analyzed using Imaged.

Gene expression analysis

RNA was isolated from the cell migration experiment by using the NucleoSpin® RNA isolation mini kit (Macherey Nagel) according to the manufacturer instructions. The RNA was quantified with the NanoDrop™ 1000 spectrophotometer (PeqLab Biotechnologie) and afterwards stored at -80°C. A total of 100 ng RNA was transcribed into cDNA with the qScript™ cDNA synthesis SuperMix (Quanta Biosciences). Quantitative Real-Time PCR (qRT-PCR) was performed with the PerfeCTa® SYBR® Green SuperMix (Quanta Biosciences) according to the manufacturer and using the Light Cycler 480 System (Roche). Primer sequences specific for targets: HPRT (NM_000194); Col-1 (NM_000088.3); Col-3 (NM_000090.3); MMP1 NM_002421.3; MMP2 (NM_004530); MMP3 (NM_002422.3); TIMP1 (NM_003254.2); IL6 (NM_000600) and IL1 (NM_000576) were designed using Primer 3 software (Freeware; available online: http://frodo.wi.mit.edu/primer3) and produced by Tib Molbiol. All primers were tested for amplification efficiency and an efficiency correct equation was used to calculate the normalized gene expression to the reference gene hypoxanthine phosphoribosyl transferase (HPRT) which was tested to be the most constant housekeeping gene. Protein analysis

Protein concentrations of IFNy and IL17 was measured for supernatants of the PBMC polarization to verify that IFNy and IL17 polarization has worked. MMP1 , MMP2, and MMP3 concentrations were quantified in supernatants from the migration assay (45 h time point). All proteins were quantified by DuoSet® ELISA Kits (R&D Systems) for human IFNy, human IL17, human MMP1 , 2, and 3. To achieve sample concentrations which fit in the range of the standard curve, samples were diluted according to the used ELISA kit with 1x Reagent Diluent (DuoSet® Kit). The assays were performed in accordance to the instructions of the manufacturer and absorbance measured with a the Tecan Infinite Pro® multiplate reader (Tecan).

Statistics:

For the in vitro study, statistical differences were analyzed using Wilcoxon Test for paired nonparametric samples (GraphPad Prism Version 7.0). P<0.05 was considered as statistically significant and p<0.1 indicate trends.

Results: Proof of Mechanism:

CD4+ T-cells increase expression of Col III , IL17 receptors and IL17 downstream targets: Tenocytes kept in co-culture with CD4+ T-cells that derived from unpolarized PBMCs showed an increased expression of Col III compared to CD8+ T-cells (p=0.031), resulting in a decreased Col l/Col III ratio (p=0.031). Expression of the IL17 receptors A and C was significantly higher in tenocytes co-cultured with CD4+ T-cells compared to CD8+ T-cells (both p=0.031)(Fig. 9A). Furthermore, the downstream targets of IL17 signaling, the MMPs, were higher in co-culture with CD4+ T-cells compared to CD8+ T-cells, as shown on gene expression (MMP1 : p=0.031 , MMP2: p=0.063, MMP3: p=0.063) as well as by trend on protein level (MMP1 and 2: p=0.094)(Fig. 9A,B).

IL17 polarization amplifies the negative effect of unpolarized CD4+ T-cells on tenocytes: IL17-polarized CD4+ T-cells, which are highly abundant in physiological conditions, significantly increased the Col III (p=0.031) and by trend the Col I expression in tenocytes, leading to a reduced Col l/Col III ratio compared to co-culture with unpolarized CD8+-T-cells (both p=0.063). The expression of IL1 b and IL17RC was significantly increased in tenocytes co-cultured with IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells (both p=0.031). The same trend was observed for IL6 expression (p=0.063). Tenocytes’ MMP1 , 2, and 3 levels were significantly higher in co-culture with IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells (all p=0.031 )(Fig. 10A,B). The wound healing capacity of tenocytes was by trend reduced in the presence of IL17-polarized CD4+ T-cells compared to CD8+ T-cells after 25h (p=0.063)(Fig. 10C). Comparison to the IFNy-polarized CD8+ T-cells underlines the negative effect of IL17-polarized CD4+ T-cells on tendon healing properties:

IFNy-producing CD8+ T-cells and IL17-producing CD4+ T-cells are the physiologically most abundant T-cell populations. Compared to IFNy-polarized CD8+ T-cells, the IL17-polarized CD4+ T-cells by trend increased the Col I, Col III, MMP1 , 2, 3 expression and MMP2 secretion (p=0.063). Furthermore, the wound healing capacity of tenocytes was reduced and the matrix contraction increased in the presence of IL17-polarized CD4+ T-cells compared to IFN-polarized CD8+ T-cells (p=0.063)(Fig. 11). The data are less significant as for the comparison to the unpolarized CD8+ group, due to a lower n-number (5 instead of 6).

Conclusion:

The proof of mechanism in vitro study revealed that CD4+ T-cells in unpolarized condition, and even more in IL17-polarized condition, lead to an increase in IL17 receptor expression of tenocytes compared to CD8+ T-cells. This increases the susceptibility of tenocytes to IL17. Therefore, the IL17 downstream mediators MMP1 , 2, and 3 are increased in tenocytes kept in coculture with CD4+ T-cells compared to CD8+ T-cells, which in turn increases the weakening of the extracellular matrix and thus impairs tendon healing. On the other hand, CD4+ T-cells increase the expression of Col III and by that decrease the Col l/Col III ratio, which is associated with less biomechanical competence of tendon tissue. IFNy-polarized CD8+ T-cells and IL17- polarized CD4+ T-cells are the physiologically most abundant T-cell populations and have a high clinical relevance. Here, next to similar trends in gene expression and protein secretion, the IL17- polarized CD4+ T-cells by trend decreased the wound healing capacity and increased the ECM contraction compared to IFNy-polarized CD8+ T-cells. A too strong matrix contraction in the early tendon healing phase is hypothesized to hinder a proper healing and growth of tendon stumps into each other. In summary, the proof of mechanism study supports that a reduction of CD4+ T- cells or the CD4+/CD8+ T-cell ratio would be beneficial for acute Achilles tendon healing due to decreasing the susceptibility of tenocytes towards IL17 and the weakening of the extracellular tendon matrix. The mechanism can be summarized as follows: CD4+ T-cells as strong producers of IL17 lead to an increased IL17 receptor expression, resulting in increased MMP levels and thus a weakening of the ECM. Biomechanical competence of the ECM is furthermore impaired by increased Col III expression. Taken together, this mechanism would lead to impaired tendon healing in vivo.

Claims

Claims

1 . An agent being capable of reducing the CD4⁺ T cell recruitment, CD4⁺ T cell activation, CD4⁺ T cell proliferation, and/or reducing specific pro-inflammatory cytokines and/or inhibiting specific pro-inflammatory signaling pathways in a patient for use in treatment of an Achilles tendon rupture, and wherein the agent is capable of selectively binding to a target selected from the group comprising: a. CD4; b. IL-17A: c. IL-23; d. CCR6/CCL20 axis; e. CCR5; f. IL-6; g. CD11a; h. CD28; i. IL-1 P; j. TNFa; k. IFN a/p;

2. The agent for use according to claim 1 , wherein the agent is selected from the group comprising: a. for targeting CD4: IT1208, Tregalizumab (BT-061), MAX.16H5 IgGi, b. for targeting IL-17A: Sekunkinumab, Brodalumab, Ixekizumab, c. for targeting IL-23: Guselkumab, Risankizumab, Ustekinumab, d. for targeting CCR6/CCL20 axis: CCX9664, GSK3050002, e. for targeting CCR5: Ibalizumab, f. for targeting IL-6: Tocilizumab, g. for targeting CD11a: Alefacept, h. for targeting CD28: Abatacept, i. for targeting IL-1 p: Canakinumab, Anakinra, j. for targeting TNFa: Adalimumab, Certolizumab, Etanercept, Golimumab, Infliximab, k. for targeting IFN a/p: Anifrolumab, S95021 ,

3. The agent for use according to claim 1 or 2, wherein said agent is administered locally to a rupture side of the Achilles tendon.

4. The agent for use according to claim 1 or 2, wherein said agent is administered subcutaneously. The agent for use according to claim 1 or 2, wherein said agent is administered systemically, particularly wherein said agent is administered intravenously. A method for

I. prediction of compromised tendon healing in a patient; and/or

II. prediction of tendon elongation in a patient; and/or

III. monitoring the course of treatment of a tendon rupture in a patient; said method comprising the steps: a. providing at least one blood sample isolated from said patient; b. determining in said blood sample: i. a CD4⁺/CD8⁺ T cell ratio within CD3⁺ T cells; and/or ii. a CD4⁺ T cell frequency within CD3⁺ T cells; and/or iii. a CD8⁺ T cell frequency within CD3⁺ T cells; and/or iv. a frequency of CD11 a⁺⁺CD28^_ T cells within CD3⁺CD8⁺ T cells; and/or v. a frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells; and/or vi. a frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells; c. assigning a probability of compromised tendon healing to said patient; wherein i. a high CD4⁺/CD8⁺ T cell ratio within CD3⁺ T cells; and/or ii. a high CD4+ T cell frequency within CD3⁺ T cells; and/or iii. a low CD8+ T cell frequency within CD3⁺ T cells; and/or iv. a low frequency of CD11 a⁺⁺CD28' T cells within CD3⁺CD8⁺ T cells; and/or v. a low frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells; and/or vi. a low frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells; is indicative for a high probability of compromised tendon healing, and/or for the need to apply a treatment to said patient. The method according to claim 6, wherein a high probability of compromised tendon healing is assigned to said patient if criteria i, ii, and/or iii are met in a blood sample obtained at the time of surgery. The method according to claim 6, wherein a high probability of tendon elongation is assigned to said patient if criteria iii, vi, v, and/or vi, are met in a blood sample obtained ~ six weeks after surgery. The method according to claim 6, wherein a high probability of compromised tendon healing is assigned to said patient if the CD4⁺/CD8⁺ T cell ratio is above a threshold in a blood sample obtained at the time of surgery, wherein said threshold is >1.7, particularly >2.3, more particularly >2.6. The method according to claim 6, wherein a high probability of compromised tendon healing is assigned to said patient if the CD4⁺ T cell frequency within all CD3⁺ cells is above a threshold in a blood sample obtained at the time of surgery, wherein said threshold is >57.2%, particularly >59.2%, more particularly >66.2%. The method according to claim 6, wherein a high probability of compromised tendon healing is assigned to said patient if the CD8⁺ T cell frequency within all CD3⁺ cells is below a threshold in a blood sample obtained at the time of surgery, wherein said threshold is <38.5%, particularly <28.3%, more particularly <25.8%. The method according to claim 6, wherein a high probability of tendon elongation is assigned to said patient if the frequency of CD11 a⁺⁺CD28^_ T cells within CD3⁺CD8⁺ T cells is below a threshold in a blood sample obtained ~ six weeks after surgery, wherein said threshold is <29.6%, particularly <13.8%, more particularly <11.8%. The method according to claim 6, wherein a high probability of tendon elongation is assigned to said patient if the frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells is below a threshold in a blood sample obtained ~ six weeks after surgery, wherein said threshold is <31.9%, particularly <19.7%, more particularly <16.9%. The method according to claim 6, wherein a high probability of tendon elongation is assigned to said patient if the frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells is below a threshold in a blood sample obtained ~ six weeks after surgery, wherein said threshold is <23.5%, particularly <12.1%, more particularly <9.6%. The method according to claim 6, wherein a high probability of tendon elongation is assigned to said patient if the CD8⁺ T cell frequency within all CD3⁺ T cells is below a threshold in a blood sample obtained ~ six weeks after surgery, wherein said threshold is <39.1%, particularly <26.2%, more particularly <23.2%. The method according to any one of the preceding claims 6 to 15, wherein said blood sample is obtained from venous blood. The method according to any one of the preceding claims 6 to 15, wherein said blood sample is obtained from a hematoma or hematoma aspirate at a tendon rupture site. The agent for use according to any one of claims 1 to 5, wherein a high probability of compromised tendon healing was assigned to said patient via the method of any one of claims 6 to 17. A kit comprising a plurality of antibodies, wherein each antibody is covalently bound to a different detectable label, and wherein said plurality of antibodies consists of a. an antibody specifically binding to CD4; b. an antibody specifically binding to CD8; c. an antibody specifically binding to CD3; d. an antibody specifically binding to CD11a; e. an antibody specifically binding to CD28; and f. an antibody specifically binding to CD57. The kit according to claim 19, wherein said detectable label is a fluorescent label. A method for treatment of an Achilles tendon rupture in a patient in need thereof, wherein said method comprises the steps: a. providing at least one blood sample isolated from said patient; b. determining in said blood sample: i. a CD4⁺/CD8⁺ T cell ratio within CD3⁺ T cells; and/or ii. a CD4⁺ T cell frequency within CD3⁺ T cells; and/or iii. a CD8⁺ T cell frequency within CD3⁺ T cells; and/or iv. a frequency of CD11 a⁺⁺CD28^_ T cells within CD3⁺CD8⁺ T cells; and/or v. a frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells; and/or vi. a frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells; c. assigning a probability of compromised tendon healing to said patient; wherein i. a high CD4⁺/CD8⁺ T cell ratio within CD3⁺ T cells; and/or ii. a high CD4+ T cell frequency within CD3⁺ T cells; and/or iii. a low CD8+ T cell frequency within CD3⁺ T cells; and/or iv. a low frequency of CD11 a⁺⁺CD28' T cells within CD3⁺CD8⁺ T cells; and/or v. a low frequency of CD11 a⁺⁺CD57⁺ T cells within CD3⁺CD8⁺ T cells; and/or vi. a low frequency of CD11 a⁺⁺CD28'CD57⁺ T cells within CD3⁺CD8⁺ T cells; is indicative for a high probability of compromised tendon healing, d. assigning said patient to

- more frequent medical consultation; and/or

- more frequent magnetic resonance imaging; and/or

- more frequent functional rehabilitation; and/or

- prolonged wearing of a boot promoting plantar flexion; and/or

- wearing a boot with increased incline of plantar flexion; if said patient has a high probability of compromised tendon healing.