DPP3 binders for the treatment of endothelial dysfunction

Abstract

Subject of the invention are DPP3 binders for use in therapy or prevention of endothelial dysfunction in a patient wherein the level of Pro-Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient is above a threshold, as well as methods of therapy guidance or therapy stratification or therapy monitoring of a treatment of a patient with a DPP3 binder, the methods comprising: providing a sample of bodily fluid of said patient; determining the level of Pro-Adrenomedullin or a fragment thereof in said sample; and comparing said level of Pro-Adrenomedullin or a fragment thereof to a threshold; wherein the level of Pro-Adrenomedullin or a fragment thereof in said sample being above said threshold is indicative that said patient requires treatment with a DPP3 binder, or wherein if said level of Pro-Adrenomedullin or a fragment thereof in said sample is above said threshold, said patient is treated with a DPP3 binder.

Description

[0001] T75369WO BOEHMERT & BOEHMERT

[0002] DPP3 binders for the treatment of endothelial dysfunction

[0003] Subject of the invention are DPP3 binders for use in therapy or prevention of endothelial dysfunction in a patient wherein the level of Pro-Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient is above a threshold, as well as methods of therapy guidance or therapy stratification or therapy monitoring of a treatment of a patient with a DPP3 binder, the methods comprising: providing a sample of bodily fluid of said patient; determining the level of Pro-Adrenomedullin or a fragment thereof in said sample; and comparing said level of Pro- Adrenomedullin or a fragment thereof to a threshold; wherein the level of Pro-Adrenomedullin or a fragment thereof in said sample being above said threshold is indicative that said patient requires treatment with a DPP3 binder, or wherein if said level of Pro-Adrenomedullin or a fragment thereof in said sample is above said threshold, said patient is treated with a DPP3 binder.

[0004] State of the art

[0005] DPP3

[0006] Dipeptidyl peptidase 3 - also known as Dipeptidyl aminopeptidase III, Dipeptidyl arylamidase III, Dipeptidyl peptidase III, Enkephalinase B or red cell angiotensinase; short name: DPP3, DPPIII - is a metallopeptidase that removes dipeptides from physiologically active peptides, such as enkephalins and angiotensins. DPP3 was identified and its activity measured in extracts of purified bovine anterior pituitary (Ellis and Nuenke 1967). The enzyme, which is listed as EC 3.4.14.4, has a molecular mass of about 83 kDa and is highly conserved in procaryotes and eucaryotes (Prajapati and Chauhan 2011). The amino acid sequence of the human variant is depicted in SEQ ID NO: 1. DPP3 is a mainly cytosolic peptidase which is ubiquitously expressed. Despite lacking a signal sequence, a few studies reported membranous activity (C. Lee and Snyder 1982).

[0007] DPP3 is a zinc-depending exo-peptidase belonging to the peptidase family M49. It has a broad substrate specificity for oligopeptides from three / four to ten amino acids of various compositions and is also capable of cleaving after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, including angiotensin II, III and IV; Leu- and Met-enkephalin; endomorphin 1 and 2. The metallopeptidase DPP3 has its activity optimum at pH 8.0-9.0 and can be activated by addition of divalent metal ions, such as Co2+and Mg2+. T75369WO BOEHMERT & BOEHMERT

[0008] Structural analysis of DPP3 revealed the catalytic motifs HELLGH (SEQ ID NO: 30; human DPP3 [hDPP3] 450-455) and EECRAE (SEQ ID NO: 31; hDPP3 507-512), as well as following amino acids, that are important for substrate binding and hydrolysis: Glu316, Tyr, 318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666 and Arg669 (Prajapati and Chauhan 2011) (Kumar et al. 2016); numbering refers to the sequence of human DPP3, see SEQ ID NO: 1. Considering all known amino acids or sequence regions that are involved in substrate binding and hydrolysis, the active site of human DPP3 can be defined as the area between amino acids 316 and 669.

[0009] The most prominent substrate of DPP3 is angiotensin II (Ang II), the main effector of the reninangiotensin system (RAS). The RAS is activated in cardiovascular diseases (Dostal et al. 1997) (Roks et al. 1997), sepsis, and septic shock (Correa, Takala, and Jakob 2015). Ang II, in particular, has been shown to modulate many cardiovascular functions including the control of blood pressure and cardiac remodeling.

[0010] Recently, two assays were developed, characterized, and validated to specifically detect DPP3 in human bodily fluids (e.g., blood, plasma, serum): a luminescence immunoassay (LIA) to detect DPP3 protein concentration and an enzyme capture activity assay (ECA) to detect specific DPP3 activity (Rehfeld et al. 2019). A washing step removes all interfering substances before the actual detection of DPP3 activity is performed. Both methods are highly specific and allow the reproducible detection of DPP3 in blood samples.

[0011] Circulating DPP3 (cDPP3) levels were shown to be increased in septic, cardiogenic and vasodilatory shock patients (Rehfeld et al. 2019). Moreover, it was associated with an increased risk of short-term mortality and severe organ dysfunction in patients with cardiogenic shock (Deniau et al. 2020). Moreover, in patients with severe sepsis or septic shock showed that the higher the initial cDPP3 was, the greater the need for organ support and vasopressors upon admission and the longer the need for vasopressor(s), mechanical ventilation or renal replacement therapy (RRT) and the higher the need for fluid load (Blet et al. 2021) (wherein in Blet et al, the term „severe sepsis“ was based on the Sepsis-2 ..main inclusion criteria was patients admitted to the ICU for severe sepsis or septic shock based on the former sepsis definition^).

[0012] Thus, efforts have been made to pharmacologically inhibit supranormal concentrations of cDPP3.

[0013] DPP3 binders T75369WO BOEHMERT & BOEHMERT

[0014] Numerous binders that inhibit DPP3 have been described (Abramic and Agic 2022): Except for the polypeptide aprotinin, all others are small molecules and include flavonoids, coumarin and benzimidazole derivatives. Presented are current strategies for the discovery or development of DPP3 inhibitors, and mechanisms of inhibitory actions. The most potent inhibitors yet reported (propioxatin A and B, Tyr-Phe- and Phe-Phe-NHOH, and JMV-390) are active in low nanomolar range and contain hydroxamic acid moiety. High inhibitory potential possesses oligopeptides from the hemorphin group, valorphin and tynorphin, which are poor substrates of DPP3.

[0015] Procizumab, a humanized monoclonal IgGl antibody specifically binding circulating DPP3, targets and modulates the activity DPP3. Its mode of action is relevant in acute diseases that are associated with massive cell death and uncontrolled release of intracellular DPP3 into the bloodstream. Translocated DPP3 remains active in the circulation where it cleaves bioactive peptides in an uncontrolled manner.

[0016] Procizumab is able to inhibit circulating DPP3 thereby inhibiting bioactive peptide degradation in the bloodstream. This inhibition results in stabilization of cardiovascular and renal function and reduction of short-term mortality. Preclinical studies of Procizumab in animal models of cardiovascular failure showed impressive and instant efficacy. In several preclinical cardiovascular failure models, Procizumab has shown to normalize ejection fraction and kidney function and reduces mortality (Malovan et al. 2023).

[0017] Adrenomedullin

[0018] Adrenomedullin (ADM) is one of the best studied peptide hormone, which plays a role in a vast range of physiological and pathophysiological processes, including inter alia vasodilation, angiogenesis, and hormone regulation. ADM is also involved in bronchodilatation, renal function, cell growth, differentiation, neurotransmission, and modulation of the immune response. ADM belonging to the ADM / calcitonin gene-related peptide (CGRP) superfamily of peptides and is known to be produced in various human organs and tissues, including the heart, adrenal endothelial cells, lungs, kidneys, adipose tissue, and vascular endothelium, which contribute to ADM blood levels.

[0019] ADM mRNA encodes a preprohormone of 185 amino acids (SEQ ID NO: 32), the pre-pro- Adrenomedullin that is enzymatically converted into Proadrenomedullin by cleavage of the N- terminal signal peptide. Proadrenomedullin (SEQ ID NO: 33) is then further process by several prohormone convertases to result in four peptides, namely T75369WO BOEHMERT & BOEHMERT

[0020] PAMP-Gly (SEQ ID NO: 35): Proadrenomedullin N-terminal 20 peptide with a C- terminal glycine residue, inactive precursor of biologically active PAMP (PAMP-NH2 (SEQ ID NO: 34)),

[0021] MR-proADM (SEQ ID NO: 38): Mid-regional Proadrenomedullin, a stable and inert peptide,

[0022] ADM-Gly (SEQ ID NO: 37): C-terminally glycine extended, inactive precursor of biologically active ADM (mature ADM / ADM-NH2 (SEQ ID NO: 375-20) and CT-proADM (SEQ ID NO: 39 C-terminal Proadrenomedullin or Adrenotensin.

[0023] ADM-Gly is the direct, inactive biosynthetic precursor of the fully activated ADM form (mature ADM), often referred to as the intermediate form of ADM, and represents the dominating circulating form of ADM in humans. To gain its biological activity, ADM-Gly is activated by the Vitamin C dependent enzyme peptidylglycine-alpha amidating monooxygenase (PAM). PAM recognizes the C-terminal glycine and catalyses a sequential two-step reaction also referred to as amidation or C-terminal amidation.

[0024] Mature ADM has several physiological effects, such as vasodilation, angiogenesis, cardioprotection, nephroprotection, anti-oxidation, anti-apoptosis and tissue repair and regeneration. Mature ADM is involved in blood pressure regulation, bronchodilatation, renal function, hormone secretion, cell growth, differentiation, neurotransmission, and modulation of the immune response. Moreover, ADM plays a crucial role as autocrine factor during proliferation and regeneration of endothelial cells.

[0025] Additionally, mature ADM promotes angiogenesis, arteriogenesis, prevents cognitive decline after chronic cerebral hypoperfusion and is therefore considered as therapeutic agent in vascular dementia (reviewed in (Garcia et al. 2006) and (Balint et al. 2023).

[0026] Endothelial dysfunction

[0027] Endothelial (barrier) dysfunction has been described for a number of diseases and is considered to contribute to the pathogenesis of these diseases. These include a severe infectious disease, inflammation, sepsis, shock, pulmonary diseases including ARDS, cardiovascular diseases including acute myocardial infarction, acute or chronic heart failure, organ dysfunction, hepatic diseases, renal diseases, autoimmune disease including ulcerative colitis, arthritis other chronic inflammatory diseases, age-related macular degeneration, diabetic macular edema, psychiatric and neurodegenerative diseases, cancer, (Claesson- Welsh, Dejana, and McDonald 2021) (Rodrigues and Granger 2015) (O Karpinich et al. 2011) (Soussi et al. 2023) (Opal and Van T75369WO BOEHMERT & BOEHMERT

[0028] Der Poll 2015), (W. L. Lee and Slutsky 2010), (Chistiakov, Orekhov, and Bobryshev 2015) (Claesson-Welsh 2015).

[0029] Among cardiovascular diseases, heart failure has been extensively described in the literature to be associated with endothelial dysfunction (Arrigo et al. 2020). Clinically, this manifests as congestion, in particular as peripheral congestion. Congestion has been recognized as a driver in progression of disease (Mocan et al. 2024). Patients upon decompensation frequently need to be hospitalized, and have an elevated mortality risk. Peripheral congestion becomes symptomatic by the development of edema, and is associated with increased levels of circulating plasma bio- ADM (Voors et al. 2019).

[0030] In patients with sepsis, typically progressive subcutaneous and body-cavity edema develops, suggesting widespread increases in vascular permeability. Tissue edema is not benign: the accumulation of parenchymal and interstitial fluid could impair organ function by increasing the distance required for the diffusion of oxygen and by compromising microvascular perfusion because of increased interstitial pressure. A well-recognized feature of recovery from septic shock is a spontaneous diuresis with reduction in edema, consistent with restoration of vascular integrity. Given that all blood vessels are lined with endothelial cells, vascular leak and tissue edema in sepsis are due to endothelial dysfunction (W. L. Lee and Slutsky 2010). The barrier function of the endothelial surface is almost uniformly impaired in septic shock, and it is considered to strongly contribute to adverse outcomes (Opal and Van Der Poll 2015).

[0031] Endothelial dysfunction and Adrenomedullin

[0032] Elevation of plasma ADM is widely accepted as a surrogate marker for disturbed endothelial barrier function. High ADM levels likely represent a failing compensatory response, aimed at restoring endothelial barrier function (D. van Lier, Kox, and Pickkers 2020). Elevated plasma concentrations have been described in most of the diseases, which are associated with endothelial dysfunction (see above).

[0033] Endothelial dysfunction and DPP3

[0034] Nothing in the art suggests that DPP3 is linked to endothelial dysfunction, neither as an epiphenomenon nor causally. Conversely, it has been reported that elevated bio-ADM, the biomarker indicating endothelial dysfunction, and elevated cDPP3 are predicting mortality risk in sepsis independently from each other (results from the AdrenOSS-1 study (D. van Lier, Kox, and Pickkers 2020)). Consequently, a pharmaceutical approach to improve survival of septic T75369WO BOEHMERT & BOEHMERT shock patients is through restoration of the endothelial barrier function by enhancing the plasma bio-ADM activity using an anti-N-terminal ADM antibody yielded promising results, when patients with supranormal concentrations of cDPP3 were excluded (Dirk van Lier et al. 2022).

[0035] Now, surprisingly, it has been found by the present inventors that endothelial dysfunction, which is in particular represented by pathological elevation of circulating ADM (and / or other peptides related to pro-ADM as detailed herein), is a condition, where application of a DPP3 binder is beneficial. The present Examples demonstrate that not only pre-existing endothelial dysfunction can be restored by treatment with a DPP3 binder, but also the development of endothelial dysfunction can be prevented by such treatment.

[0036] Immune effector cell therapy

[0037] Over the past decades, an increasing number of ways to treat cancer have been developed (Link 2019). These include chemotherapy, hormone therapy, hyperthermia, photodynamic therapy, radiation therapy, stem cell transplant, surgery, targeted therapy, and immunotherapy. Recently, Immune Effector Cell Therapies (IECT) have emerged as the most promising therapy in oncology (Forero-Forero et al. 2021). Under the term IECT several types of cell therapies are summarized, which include Chimeric Antigen Receptor T-cell (CAR-T) therapy, natural killer (NK) or chimeric antigen receptor natural killer cell (CAR-NK) therapy, T cell receptor- engineered T cell (TCR T) therapy, tumor-infiltrating T cell (TIT)), and cytokine-induced killer cell (CIK) therapy, of which CAR T-cell therapy has been studied and used the most (Forero- Forero et al. 2021). CAR- T-cell therapy has turned out to be particularly effective in the treatment of patients with B cell malignancies (Sun et al. 2024). Currently, CAR T-cell therapy is approved for the treatment of B cell relapsed or refractory leukemia and lymphoma, and most recently, multiple myeloma. In these different diseases, it has led to excellent complete and overall response rates depending on the patient population and therapy. The field of CAR T-cell therapy continues to expand rapidly. IECTS other than CAR-T, including CAR-NK, TIL, CIK are being explored in hematologic malignancies and solid malignancies such as germ cell tumor, sarcoma, neuro-blastoma, and melanoma (Kanate et al. 2023).

[0038] Despite promising efficacy, CAR T-cell therapy as well as the other IECTs mentioned above are frequently associated with significant side effects (Lee et al. 2019). The two most notable toxicities are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) (Chohan, Siegler, and Kenderian 2023). T75369WO BOEHMERT & BOEHMERT

[0039] CRS has been defined as “a supraphysiologic response following any immune therapy that results in the activation or engagement of endogenous or infused T cells and / or other immune effector cells. Symptoms can be progressive, must include fever at the onset, and may include hypotension, capillary leak (hypoxia) and end organ dysfunction. (Lee et al. 2019)”.

[0040] ICANS has been defined as “a disorder characterized by a pathologic process involving the central nervous system following any immune therapy that results in the activation or engagement of endogenous or infused T cells and / or other immune effector cells. Symptoms or signs can be progressive and may include aphasia, altered level of consciousness, impairment of cognitive skills, motor weakness, seizures, and cerebral edema (Lee et al. 2019)”. Similar to CRS, ICANS should be applied to any immune effector cell engaging therapy, not just CAR T- cells.

[0041] CRS is one of the most common side effects associated with CAR T-cell therapy with an incidence of 20-50% and manifests as fever and difficulty of breathing, low blood pressure, nausea and vomiting, and poses a notable safety challenge and can lead to life-threatening conditions, such as hypotension, respiratory distress and organ insufficiency (Sun et al. 2024).

[0042] Regimes have been developed to treat CAR T-induced toxicity. These include supportive care, the use of tocilizumab, an IL-6 receptor antibody, and steroids, if patients are refractory to tocilizumab. Even with the use of tocilizumab, which is FDA approved to treat severe CRS, severe CRS and death still occur.

[0043] For the treatment of neurotoxicity, tocilizumab is not effective, and steroids are used as first line of treatment there. The pathophysiology and mechanisms underlying neurotoxicity are only barely understood (Neelapu et al. 2018).

[0044] Other agents which have been investigated and are generally reserved for refractory CRS include include anti-IL-6 antibody (siltuximab), anti-TNF antibody (etanercept, infliximab) and anti-IL-1 antibody (anakinra). They all aim at downregulating the overactivation of the immune system.

[0045] Due to the side-effects of lECTs and the so far limited possibilities to treat these, experts have called for novel toxicity-directed therapies and low-toxicity constructs which do not compromise treatment efficacy, which are desperately needed (Chohan, Siegler, and Kenderian 2023).

[0046] The immune system emerges as a key player not only mediating cytokine responses but potentially contributing to endothelial injury in CAR T-cell toxicity (Gavriilaki et al. 2020). T75369WO BOEHMERT & BOEHMERT

[0047] Thus, endothelial dysfunction has been investigated in CAR T-cell toxicity (Sumransub et al.

[0048] 2022). Following CAR T-cell therapy, endothelial dysfunction characterized by increased pro- inflammatory signaling (e.g., IL-6, IL-8, MCP-1) loss of barrier function has been observed, and CAR T-cells have been shown to directly induce endothelial dysfunction (Rosen et al.

[0049] 2023).

[0050] The administration of CAR T-cells is preceded by a chemotherapy to achieve lymphodepletion, which is applied typically for three days, and after a rest of two days the CAR T-cells are infused. One study has been published, in which the influence of chemotherapy on the levels of Adrenomedullin was investigated (Giiler et al. 2006). The study was restricted to pediatric patients and did not comprise a following CAR T-cell therapy. The authors reported slightly higher levels of Adrenomedullin after chemotherapy compared to baseline (30.3 pmol / mL vs 25.1 pmol / mL).

[0051] At this point, there are no publications describing biomarkers existent prior to immune effector cell administration (in particular CAR T-cell administration) in the frame of an immune effector cell therapy (in particular in the frame of a CAR T-cell therapy) or shortly after administration of immune effector cells (in particular CAR T-cells) in the frame of an immune effector cell therapy (in particular in the frame of a CAR T-cell therapy), which would predict an increased risk for endothelial dysfunction, which would develop as a side effect of immune effector cell therapy (in particular CAR T-cell therapy), nor how to prevent such side-effect.

[0052] The present invention found that inhibition of DPP3 is beneficial in therapy or prevention of endothelial dysfunction, depending on the patient’s level of Pro- Adrenomedullin or a fragment thereof and that this may be applied beneficially in therapy guidance, stratification monitoring.

[0053] Brief description of the Figures

[0054] Figure 1: Change of bio-ADM-, ADM-Gly- and MR-proADM concentrations after treatment with humanized anti-hDPP3 antibody. Shown are mean / SD values obtained from three patients treated with humanized anti-hDPP3 antibody.

[0055] Figure. 2: Change of norepinephrine dosing after treatment with humanized anti-hDPP3 antibody. Shown are mean / SD values obtained from three patients treated with humanized anti-hDPP3 antibody.

[0056] Figure 3: Development of bio-ADM increase after treatment.

[0057] Figure 4: Schematic representation of the experimental procedure. T75369WO BOEHMERT & BOEHMERT

[0058] Figure 5: Baseline bio-ADM and ADM-Gly concentrations of patients treated with CAR

[0059] T-cell therapy. Shown are box and whiskers plots (with median, 25th and 75th percentiles and min and max data indicated).

[0060] Figure 6: Baseline bio-ADM- (Fig. 6 A) and ADM-Gly (Fig. 6 B) concentrations of patients treated with CAR T-cell therapy depending on their Cytokine Release Syndrome (CRS) Grading <2 or >2. Shown are box and whiskers plots (with median, 25th and 75th percentiles and min and max data indicated).

[0061] Figure 7: Time course of bio-ADM (Fig. 7 A) and ADM-Gly (Fig. 7 B) after start of T-cell administration in the frame of CAR T-cell therapy dependent on the Cytokine Release Syndrome (CRS) Grading <2 or >2 following the CAR T-cell therapy. As blood draws were not taken at the exact same time points after start of CAR T-cell therapy for all patients, bio-ADM- and ADM-Gly measurements were grouped in time intervals of four days, e.g. > 0 and <4 days, > 4 and <8 days, > 8 and <12 days, > 12 and <16 days, > 16 days, after start of CAR T-cell therapy. Displayed are bars for median with interquartile range.

[0062] Figure 8: Inhibition curve of native DPP3 from blood cells with murine anti-hDPP3 antibody. Inhibition of DPP3 by a specific antibody is concentration dependent, with an IC50 at ~15 ng / ml when analysed against 15 ng / ml DPP3.

[0063] Figure 9: Association and dissociation curve of the murine anti-hDPP3 antibody-DPP3 binding analysis using Octet. Murine anti-hDPP3 antibody loaded biosensors were dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM) and association and dissociation monitored.

[0064] Figure 10: Western Blot of dilutions of blood cell lysate and detection of dilutions of blood cell lysate and recombinant human DPP3 with murine anti-hDPP3 antibody as primary antibody.

[0065] Figure 11: Representative calibration curve of the bio-ADM assay (in the presence or absence of 100 pg / mL antibody NT-H).

[0066] Figure 12: Representative calibration curve of the ADM-Gly Assay with synthetic human 1-53 ADM-Gly.

[0067] Figure 13: Frequency distribution (histogram) of ADM-Gly (pg / mL) in self-reported healthy individuals (n=157). T75369WO BOEHMERT & BOEHMERT

[0068] Figure 14: Schematic representation of the development and selection of anti-DPP3 antibodies using the SELMA technology.

[0069] Figure 14 A: Myeloma cells are used for fusion with B-cells. The myeloma cells are stable transfected with a construct enabling the expression of a surface marker containing the biotin acceptor peptide sequence for site-specific biotinylation by biotin ligase (BirA).

[0070] Figure 14 B: After fusion of these myeloma cells with the B-cells derived from an immunized mouse, the pool of hybridoma cells are biotinylated in vitro at their surface via biotin ligase.

[0071] Figure 14 C: A separately prepared DPP3 -streptavidin conjugate is added to the pool of cells, where it binds to the biotin-residues carried by the hybridoma cells.

[0072] Figure 14 D: During a subsequent incubation, the hybridoma cells secrete their monoclonal antibodies (mAbs). Those mAbs directed against the target protein DPP3 immediately after secretion bind to the DPP3 -streptavidin conjugate nearby, whereas other mAbs from other hybridoma cells diffuse into the culture medium.

[0073] Figure 14 E: A fluorescence-labeled anti-mouse IgG is added and binds to mAbs. “Positive” hybridoma cells now carry a fluorescence label (complex formed by biotin covalently attached at the cell surface* Streptavidin conjugated DPP3*anti-DPP3 mAb* fluorescence-labeled anti-mouse IgG); “negative” hybridoma cells do not. By fluorescence-activated cell sorting (FACS), “positive” hybridoma cells can be easily isolated.

[0074] Figure 15: Schematic representation of designs for assessment of DPP3 binding and influence on DPP3 enzymatic activity for anti-DPP3 antibodies.

[0075] Subject Matter of the Invention

[0076] Throughout this application, the term “anti-DPP3 binder” and “DPP3 binder” are used interchangeably.

[0077] Subject matter of the present invention is a DPP3 binder for use in therapy or prevention, in particular therapy, of endothelial dysfunction in a patient wherein the level of Pro- Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient is above a threshold.

[0078] The embodiments of the present invention likewise read on a DPP3 binder for use in therapy or prevention, in particular therapy, of endothelial dysfunction in a patient wherein said patient has a level of Pro-Adrenomedullin or a fragment thereof above certain threshold, in particular in a sample of bodily fluid of said patient. T75369WO BOEHMERT & BOEHMERT

[0079] The embodiments of the present invention likewise read on a DPP3 binder for use in therapy or prevention, in particular therapy, of endothelial dysfunction in a patient, comprising determining the level of Pro- Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient, and administering said DPP3 binder if the level of Pro- Adrenomedullin or a fragment thereof is above certain threshold.

[0080] The embodiments of the present invention likewise read on a DPP3 binder for use in therapy or prevention, in particular therapy, of endothelial dysfunction in a patient, comprising determining the level of Pro-Adrenomedullin or a fragment thereof of said patient, and administering said DPP3 binder if the level of Pro-Adrenomedullin or a fragment thereof is above certain threshold.

[0081] In particular embodiments of the invention relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said Pro-Adrenomedullin fragment is a fragment of at least 10 amino acids in length.

[0082] In the present invention, said level of Pro-Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient, is in particular the level of circulating Pro- Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient.

[0083] In the present invention, where said level of Pro-Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient being above a threshold, said patient is characterized by having said level of Pro-Adrenomedullin or a fragment thereof above said threshold, and thus in particular said patient is characterized by having said level of circulating Pro-Adrenomedullin or a fragment thereof above said threshold.

[0084] In particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said Pro-Adrenomedullin or a fragment thereof is selected from the group comprising Pro-Adrenomedullin (SEQ ID No. 33), PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT-proADM (SEQ ID NO: 39).

[0085] In particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, the level of a fragment of Pro- Adrenomedullin in a sample of bodily fluid obtained from said patient is above a threshold, wherein in particular the fragments are mature PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID T75369WO BOEHMERT & BOEHMERT

[0086] NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT-proADM (SEQ ID NO: 39).

[0087] In particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, the level of Pro-Adrenomedullin or a fragment thereof is determined in a sample of bodily fluid obtained from said patient and is above a threshold, wherein in particular the fragments are mature PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT-proADM (SEQ ID NO: 39).

[0088] In particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, the total level (in other words the combined level) of mature PAMP (SEQ ID NO: 34) and PAMP-Gly (SEQ ID NO: 35) in a sample of bodily fluid obtained from said patient is determined and is above a threshold.

[0089] In particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, the total level (in other words the combined level) of ADM-Gly (SEQ ID NO: 37) and mature ADM (SEQ ID NO: 36) in a sample of bodily fluid obtained from said patient is determined and is above a threshold.

[0090] In the aforementioned embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, wherein the total / combined level of PAMP (SEQ ID NO: 34) and PAMP-Gly (SEQ ID NO: 35) or ADM-Gly (SEQ ID NO: 37) and mature ADM (SEQ ID NO: 36), respectively, is determined, the determination may be made with a single assay, e.g. involving a binder binding to both PAMP (SEQ ID NO: 34) and PAMP- Gly (SEQ ID NO: 35) or both ADM-Gly (SEQ ID NO: 37) and mature ADM (SEQ ID NO: 36), respectively. This is possible due to the large sequence overlap between PAMP (SEQ ID NO: 34) and PAMP-Gly (SEQ ID NO: 35), and ADM-Gly (SEQ ID NO: 37) and mature ADM (SEQ ID NO: 36), respectively.

[0091] In the embodiments of the invention, said threshold is in particular a predetermined threshold.

[0092] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is MR-proADM and the threshold of the level of MR-proADM is between 0.5 and 2 nmol / L, more preferred between 0.6 and 1.5 nmol / L, even more preferred between 0.7 and 1 nmol / L, most preferred said threshold is 0.8 nmol / L. T75369WO BOEHMERT & BOEHMERT

[0093] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is MR-proADM and the threshold of the level of MR-proADM is an x-fold of the median level of MR-proADM in a healthy population, particularly in the range between 1.2-fold and 4.9-fold, more preferred between 1.5-fold and 3.7-fold, even more preferred between 1.7-fold and 2.4- fold, most preferred said threshold is 2.0-fold of the median of the level of MR-proADM in a healthy population.

[0094] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is mature PAMP and the threshold of the level of mature PAMP is between 0.7 and 1.2 pmol / L, more preferred between 0.8 and 1.0 pmol / L, most preferred said threshold is 0.9 pmol / L.

[0095] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is mature PAMP and said threshold level of mature PAMP is an x-fold of the mean level of mature PAMP in a healthy population, particularly in the range between the 1.4-fold and 2.4-fold, more particular in the range between 1.6-fold and 2.0-fold, most particular said threshold is the 1.8- fold of the mean of the level of mature PAMP in a healthy population. In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro- Adrenomedullin is PAMP-Gly and the threshold of the level of PAMP-Gly is between 1.5 and 2.4 pmol / L, even more preferred between 1.8 and 2.2 pmol / L, most preferred said threshold is 2 pmol / L and 2.4 pmol / L, even more preferred between 1.8 and 2.2 pmol / L, most preferred said threshold is 2 pmol / L.

[0096] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is PAMP-Gly and said threshold level of PAMP-Gly is an x-fold of the mean level of PAMP-Gly in a healthy population, particularly in the range between the 1.3-fold and 2.1-fold, more particular in the range between 1.6-fold and 1.9-fold, most particular said threshold is the 1.7- fold of the mean of the level of PAMP-Gly in a healthy population.

[0097] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is ADM-Gly and the threshold of the level of ADM-Gly is between 25 and 125 pg / ml, more T75369WO BOEHMERT & BOEHMERT preferred between 30 and 100 pg / ml, even more preferred between 35 and 75 pg / ml, most preferred said threshold is 40 pg / ml.

[0098] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly in the range between the 0.9-fold and 4.6-fold, more particular in the range between the 1.1 -fold and 3.7-fold, more particular in the range between 1.3-fold and 2.8-fold, most particular said threshold is the 1.5-fold of the median of the level of ADM-Gly in a healthy population.

[0099] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is mature ADM and the threshold of the level of mature ADM is between 35 and 125 pg / ml, more preferred between 40 and 100 pg / ml, even more preferred between 50 and 90 pg / ml, most preferred said threshold is 70 pg / ml.

[0100] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is mature ADM and said threshold level of mature ADM is an x-fold of the median level of mature ADM in a healthy population. In a specific embodiment of the invention the threshold is an x- fold of the median level of ADM in a healthy population, particularly in the range between the2.6-fold and 9.1-fold, more particular in the range between the 2.9-fold and 7.3-fold, more particular in the range between 3.6-fold and 6.6-fold, most particular said threshold is the 5.1- fold of the median of the level of mature ADM in a healthy population.

[0101] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is CT-proADM and the threshold of the level of CT-proADM is between 75 and 350 pmol / L, more preferred between 100 and 250 pmol / L, even more preferred between 125 and 200 pmol / L, most preferred said threshold is 150 pmol / L.

[0102] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said fragment of Pro-Adrenomedullin is CT-proADM and said threshold level of CT-proADM, and / or the threshold of the level of CT- proADM is an x-fold of the median level of CT-proADM in a healthy population, particularly T75369WO BOEHMERT & BOEHMERT in the range between the 1.0-fold and 4.5-fold, more particular in the range between the 1.3- fold and 3.2-fold, more particular in the range between 1.6-fold and 2.6-fold, most particular said threshold is the 1.9-fold of the median of the level of CT-proADM in a healthy population.

[0103] In further more particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro- Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly, and the threshold of the level of ADM-Gly is between 40 and 175 pg / ml, more preferred between 40 and 125 pg / ml, most preferred said threshold is 40 pg / ml.

[0104] In certain more particular embodiments, said patient is a cancer patient, suffering from B cell relapsed or refractory leukemia or lymphoma, or multiple myeloma.

[0105] In further more particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro- Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly in the range between 1.5-fold and 6.5- fold, more preferred between 1.5-fold and 4.6-fold, most preferred said threshold is 1.5-fold of the median of the level of ADM-Gly in a healthy population.

[0106] In further more particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro- Adrenomedullin is mature ADM and said threshold level of mature ADM is between 25 and 150 pg / ml, more preferred between 30 and 125 pg / ml, most preferred said threshold is 30 pg / ml.

[0107] In further more particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro- Adrenomedullin is mature ADM and said threshold level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, particularly in the range between 1.8-fold and 10.9-fold, more preferred between 2.2-fold and 9.1-fold, most preferred said threshold is 2.2-fold of the median of the level of mature ADM in a healthy population. T75369WO BOEHMERT & BOEHMERT

[0108] In particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said sample is a bodily fluid sample selected from the group comprising whole blood, plasma and serum.

[0109] In particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said patient is a patient suffering from a severe infectious disease, inflammation, sepsis, shock, pulmonary diseases including ARDS, cardiovascular diseases including acute myocardial infarction, acute or chronic heart failure, organ dysfunction, a hepatic disease, a renal disease, an infectious disease, an autoimmune disease including ulcerative colitis, arthritis other chronic inflammatory diseases, other chronic inflammatory diseases, age-related macular degeneration, diabetic macular edema, a psychiatric disease, a neurodegenerative disease, or cancer, in particular wherein the patient is a cancer patient receiving immune effector cell therapy (IECT).

[0110] In particular embodiments of the invention, said patient is not suffering from shock, more particularly, said patient is not suffering from refractory shock,

[0111] In particular embodiments of the invention, said patient is suffering from a severe infectious disease, inflammation, sepsis, pulmonary diseases including ARDS, cardiovascular diseases including acute myocardial infarction, acute or chronic heart failure, organ dysfunction, a hepatic disease, a renal disease, an infectious disease, an autoimmune disease including ulcerative colitis, arthritis other chronic inflammatory diseases, other chronic inflammatory diseases, age-related macular degeneration, diabetic macular edema, a psychiatric disease, a neurodegenerative disease, or cancer, in particular wherein the patient is a cancer patient receiving immune effector cell therapy (IECT).

[0112] In even more particular embodiments of the present invention, said immune effector cell therapy (IECT) is selected from the group comprising Chimeric Antigen Receptor T-cell (CAR- T) therapy, natural killer cell (NK) therapy, chimeric antigen receptor natural killer cell (CAR- NK) therapy, T cell receptor-engineered T cell (TCR T) therapy, tumor-infiltrating T cell (TIT)), and cytokine-induced killer cell (CIK) therapy.

[0113] In a particular embodiment of the invention, said patient is a cancer patient receiving or about to receive chimeric antigen receptor T-cell therapy (CAR T-cell therapy).

[0114] In embodiments of the present invention relating to immune effector cell therapy (IECT), the DPP3 binder is administered in particular in the time span between lymphodepletion and T75369WO BOEHMERT & BOEHMERT administration of the immune effector cells, even more particularly within 14 days, more preferred within 7 days, most preferred within 2 days before said immune effector cells are administered to said patient.

[0115] In other particular embodiments of the present invention relating to immune effector cell therapy (IECT), the DPP3 binder is administered within 48 hours, more preferred within 24 hours, more preferred within 12 hours, more preferred within 6 hours before said immune effector cell are administered to said patient.

[0116] The DPP3 binder in particular embodiments is administered, in particular prophylactically, prior to immune effector cell administration at a timepoint selected according to the half-life time of the DPP3 binder to ensure that the circulating concentration of the DPP3 binder within the first 24 hours after the administration of the immune effector cells is sufficiently high to effectively bind circulating DPP3.

[0117] Dosage and Formulation

[0118] Dosage regimens are determined and adjusted to provide the optimum desired response (e.g., a therapeutic response).

[0119] In one embodiment of the invention said anti-DPP3 Binder is administered at a dose between 1 to 30 mg / kg body weight, preferably 2 to 20 mg / kg body weight, preferably 3 to 15 mg / kg body weight, preferably 5 to 12 mg / kg body weight, more preferably 7 to 10 mg / kg body weight, and most preferably 10 mg / kg body weight. In a further embodiment of the invention said anti-DPP3 Binder is administered intravenously at a dose of 10 mg / kg bodyweight, after which it is titrated up to no greater than 30 mg / kg body weight or titrated down to no less than 1 mg / kg body weight. In one embodiment of the invention said anti-DPP3 Binder is administered at a dose of 5 to 12 mg / kg bodyweight, wherein said anti-DPP3 Binder is subsequently administered in a plurality of additional doses and wherein said additional doses are no greater than 30 mg / kg body weight and no less than 1 mg / kg bodyweight.

[0120] The terms “Binder” and “binder” are used interchangeably herein.

[0121] In one embodiment of the invention said anti-DPP3 Binder is applied as infusion over 1 to 3 hours, preferably 1.5 to 2.5 hours and most preferred 2 hours.

[0122] In one embodiment of the invention said anti-DPP3 Binder is administered at a dose of 10 mg / kg bodyweight and applied as infusion over 1 to 3 hours, preferably 1.5 to 2.5 hours and T75369WO BOEHMERT & BOEHMERT most preferred 2 hours. In another embodiment the anti-DPP3 Binder is administered intravenously at a dose no greater than 30 mg / kg body weight to no less than 1 mg / kg body weight for a period of up to 3 hours. In one embodiment of the invention said anti-DPP3 Binder is administered intravenously at a dose of 10 mg / kg bodyweight, after which said dose is titrated up to no greater than 30 mg / kg bodyweight or titrated down to no less than 1 mg / kg bodyweight.

[0123] The term “pharmaceutical formulation” or “dosage form” means a pharmaceutical ingredient in combination with at least one pharmaceutically acceptable excipient, which is in such form as to permit the biological activity of a pharmaceutical ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The term “pharmaceutical ingredient” means a therapeutic component (e.g., an anti-DPP3 binder or an anti-DPP3 binder fragment or an anti-DPP3 non-Ig scaffold) which can be combined with pharmaceutically acceptable excipients to provide a pharmaceutical formulation or dosage form.

[0124] In one embodiment the pharmaceutical formulation suitable for infusion or injection of the anti- DPP3 binder is an aqueous solution comprising a buffer. In another embodiment such solution comprises a sugar, wherein the sugar is sucrose, trehalose, or mannitol. In a further embodiment such solution comprises an amino acid, wherein the amino acid is lysine, arginine, or histidine. In one embodiment the pharmaceutical formulation suitable for injection is a dispersion comprising glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof. Pharmaceutical formulation solutions are sterilized prior to administration to a patient.In one embodiment the pharmaceutical formulation is in a freeze-dried state. Such a freeze-dried state may be a lyophilized cake that can be reconstituted to create a concentrated solution or a solution suitable for infusion or injection as described above.

[0125] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said patient suffers from organ organ dysfunction, in particular kidney dysfunction, lung dysfunction, heart dysfunction, or liver dysfunction.

[0126] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said patient suffers from shock, in particular selected from the group comprising cardiogenic shock, septic shock and anaphylactic shock. T75369WO BOEHMERT & BOEHMERT

[0127] Particular embodiments of the invention include a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, the DPP3 binder is selected from the group comprising small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non-Ig scaffolds.

[0128] Since a patient is particularly a human patient, references to DPP3 are in particular to hDPP3 (human DPP3), unless otherwise stated.

[0129] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said DPP3 binder is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID No. 1 :

[0130] MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYI YALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFV PNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFS GNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEV TSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQ GSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERL ASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKN VSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQ DEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLH PQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILR VLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVA GGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASA AGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA.

[0131] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said DPP3 binder is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in

[0132] SEQ ID No. 2: CETVINPETGEQIQSWYRSGE, particularly SEQ ID NO: 3: INPETG.

[0133] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said antibody is a monoclonal antibody or monoclonal antibody fragment. T75369WO BOEHMERT & BOEHMERT

[0134] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity determining regions (CDRs) in the heavy chain comprise the sequences:

[0135] CDR1 : SEQ ID NO: 6: GFSLSTSGMS

[0136] CDR2: SEQ ID NO: 7: IWWNDNK

[0137] CDR3: SEQ ID NO: 8: ARNYSYDY and wherein the complementarity determining regions (CDRs) in the light chain comprise the sequences:

[0138] CDR1: SEQ ID NO: 9: RSLVHSIGSTY

[0139] CDR2: KVS

[0140] CDR3: SEQ ID NO: 10: SQSTHVPWT.

[0141] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said monoclonal antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.

[0142] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence: SEQ ID NO: 11 :

[0143] MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSV GWIRQPPGI<ALEWLAHIWWNDNI<SYNPALI<SRLTITRDTSI<NQVVLTMTNMDPVDT GTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

[0144] HEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI< VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG, and wherein the light chain comprises the sequence: SEQ ID NO: 12:

[0145] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLY

[0146] WYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQST T75369WO BOEHMERT & BOEHMERT

[0147] HVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

[0148] VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC.

[0149] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence: SEQ ID NO: 68:

[0150] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDN KSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTGTYYCARNYSYDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

[0151] PREEQ YNSTYRVVS VET VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, and wherein the light chain comprises the sequence: SEQ ID NO: 69:

[0152] DIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0153] In further particular embodiments of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said DPP3 binder is a small molecule and is selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, or a derivative or analog of any of the aforementioned compounds, and benzimidazol derivatives (i.e. compounds having a benzimidazole scaffold).

[0154] In the context of the present invention, prevention of endothelial dysfunction includes prevention of worsening of endothelial dysfunction. For instance, a patient having a level of Pro-Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient that is above the threshold but to a limited extent, e.g. up to about 5 % or up to about 10 % above the threshold, may have early signs of endothelial dysfunction. The administration of the T75369WO BOEHMERT & BOEHMERT

[0155] DPP3 binder may then prevent further escalation of the endothelial dysfunction / further worsening of endothelial dysfunction.

[0156] Subject matter of the present invention is furthermore a method of therapy guidance or therapy stratification or therapy monitoring of a treatment of a patient with a DPP3 binder, the method comprising:

[0157] • providing a sample of bodily fluid of said patient,

[0158] • determining the level of Pro-Adrenomedullin or a fragment thereof in said sample, and

[0159] • comparing said level of Pro-Adrenomedullin or a fragment thereof to a threshold, wherein the level of Pro-Adrenomedullin or a fragment thereof in said sample being above said threshold is indicative that said patient requires treatment with a DPP3 binder, or wherein if said level of Pro-Adrenomedullin or a fragment thereof in said sample is above said threshold, said patient is treated with a DPP3 binder.

[0160] In particular embodiments of the method of the invention, said Pro-Adrenomedullin or a fragment thereof is selected from the group comprising Pro-Adrenomedullin (SEQ ID NO: 33), mature PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT-proADM (SEQ ID NO: 39).

[0161] In particular embodiments of the method of the invention, the level of a fragment of Pro- Adrenomedullin in a sample of bodily fluid obtained from said patient is above a threshold, wherein in particular the fragments are mature PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT-proADM (SEQ ID NO: 39).

[0162] In particular embodiments of the method of the invention, the level of Pro- Adrenomedullin or a fragment thereof is determined in a sample of bodily fluid obtained from said patient and is above a threshold, wherein in particular the fragments are mature PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT-proADM (SEQ ID NO: 39).

[0163] In particular embodiments of the method of the invention, the total level (in other words the combined level) of mature PAMP (SEQ ID NO: 34) and PAMP-Gly (SEQ ID NO: 35) in a sample of bodily fluid obtained from said patient is determined and is above a threshold. T75369WO BOEHMERT & BOEHMERT

[0164] In particular embodiments of the method of the invention, the total level (in other words the combined level) of ADM-Gly (SEQ ID NO: 37) and mature ADM (SEQ ID NO: 36) in a sample of bodily fluid obtained from said patient is determined and is above a threshold.

[0165] In the aforementioned embodiments of the method of the invention wherein the total / combined level of PAMP (SEQ ID NO: 34) and PAMP-Gly (SEQ ID NO: 35) or ADM-Gly (SEQ ID NO: 37) and mature ADM (SEQ ID NO: 36), respectively, is determined, the determination may be made with a single assay, e.g. involving a binder binding to both PAMP (SEQ ID NO: 34) and PAMP-Gly (SEQ ID NO: 35) or both ADM-Gly (SEQ ID NO: 37) and mature ADM (SEQ ID NO: 36), respectively. This is possible due to the large sequence overlap between PAMP (SEQ ID NO: 34) and PAMP-Gly (SEQ ID NO: 35), and ADM-Gly (SEQ ID NO: 37) and mature ADM (SEQ ID NO: 36), respectively.

[0166] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is MR-proADM and the threshold of the level of MR-proADM is between 0.5 and 2 nmol / L, more preferred between 0.6 and 1.5 nmol / L, even more preferred between 0.7 and 1 nmol / L, most preferred said threshold is 0.8 nmol / L.

[0167] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is MR-proADM and the threshold of the level of MR-proADM is an x-fold of the median level of MR-proADM in a healthy population. In a specific embodiment of the invention the threshold level of MR-proADM is in the range between 1.2-fold and 4.9-fold, more preferred between 1.5-fold and 3.7-fold, even more preferred between 1.7-fold and 2.4- fold, most preferred said threshold is 2.0-fold of the median of the level of MR-proADM in a healthy population.

[0168] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is mature PAMP and the threshold of the level of mature PAMP is between 0.7 and 1.2 pmol / L, more preferred between 0.8 and 1.0 pmol / L, most preferred said threshold is 0.9 pmol / L.

[0169] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is mature PAMP and said threshold level of mature PAMP is an x-fold of the mean level of mature PAMP in a healthy population. In a specific embodiment of the invention the threshold level of mature PAMP is in the range between the 1.4-fold and 2.4-fold, more T75369WO BOEHMERT & BOEHMERT particular in the range between 1.6-fold and 2.0-fold, most particular said threshold is the 1.8- fold of the mean of the level of mature PAMP in a healthy population.

[0170] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is PAMP-Gly and the threshold of the level of PAMP-Gly is between 1.5 and 2.4 pmol / L, even more preferred between 1.8 and 2.2 pmol / L, most preferred said threshold is 2 pmol / L.

[0171] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is PAMP-Gly and said threshold level of PAMP-Gly is an x-fold of the mean level of PAMP-Gly in a healthy population. In a specific embodiment of the invention the threshold level of PAMP-Gly is in the range between the 1.3-fold and 2. 1-fold, more particular in the range between 1.6-fold and 1.9-fold, most particular said threshold is the 1.7-fold of the mean of the level of PAMP-Gly in a healthy population.

[0172] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is ADM-Gly and the threshold of the level of ADM-Gly is between 25 and 125 pg / ml, more preferred between 30 and 100 pg / ml, even more preferred between 35 and 75 pg / ml, most preferred said threshold is 40 pg / ml.

[0173] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly in the range between the 0.9-fold and 4.6-fold, more particular in the range between the 1.1-fold and 3.7-fold, more particular in the range between 1.3-fold and 2.8-fold, most particular said threshold is the 1.5-fold of the median of the level of ADM-Gly in a healthy population.

[0174] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is mature ADM and the threshold of the level of mature ADM is between 35 and 125 pg / ml, more preferred between 40 and 100 pg / ml, even more preferred between 50 and 90 pg / ml, most preferred said threshold is 70 pg / ml.

[0175] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is mature ADM and said threshold level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, particularly in the range between the 2.6- fold and 9.1 -fold, more particular in the range between the 2.9-fold and 7.3-fold, more particular T75369WO BOEHMERT & BOEHMERT in the range between 3.6-fold and 6.6-fold, most particular said threshold is the 5.1-fold of the median of the level of mature ADM in a healthy population.

[0176] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is CT-proADM and the threshold of the level of CT-proADM is between 75 and 350 pmol / L, more preferred between 100 and 250 pmol / L, even more preferred between 125 and 200 pmol / L, most preferred said threshold is 150 pmol / L.

[0177] In further particular embodiments of the method of the invention, said fragment of Pro- Adrenomedullin is CT-proADM and said threshold level of CT-proADM, and / or the threshold of the level of CT-proADM is an x-fold of the median level of CT-proADM in a healthy population. In a specific embodiment of the invention the threshold level of CT-proADM is in the range between the 1.0-fold and 4.5-fold, more particular in the range between the 1.3-fold and 3.2-fold, more particular in the range between 1.6-fold and 2.6-fold, most particular said threshold is the 1.9-fold of the median of the level of CT-proADM in a healthy population.

[0178] In further more particular embodiments of the method of the invention, said patient is a cancer patient receiving or about to receive IETC, in particular CAR T-cell therapy, said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly, and the threshold of the level of ADM-Gly is between 40 and 175 pg / ml, more preferred between 40 and 125 pg / ml, most preferred said threshold is 40 pg / ml.

[0179] In further more particular embodiments of the method of the invention, relating to a DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient, said patient is a cancer patient receiving or about to receive IETC, in particular CAR T-cell therapy, said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly is an x- fold of the median level of ADM-Gly in a healthy population, particularly in the range between 1.5-fold and 6.5-fold, more preferred between 1.5-fold and 4.6-fold, most preferred said threshold is 1.5-fold of the median of the level of ADM-Gly in a healthy population.

[0180] In further particular embodiments of the method of the invention, said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro- Adrenomedullin is mature ADM and said threshold level of mature ADM, and the threshold of the level of ADM is between 25 and 150 pg / ml, more preferred between 30 and 125 pg / ml, most preferred said threshold is 30 pg / ml. T75369WO BOEHMERT & BOEHMERT

[0181] In further particular embodiments of the method of the invention, said patient is a cancer patient receiving or about to receive IETC, in particular CAR T-cell therapy, said fragment of Pro- Adrenomedullin is mature ADM and said threshold level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, particularly in the range between 1.8-fold and 10.9-fold, more preferred between 2.2-fold and 9.1-fold, most preferred said threshold is 2.2-fold of the median of the level of mature ADM in a healthy population

[0182] In particular embodiments of the method of the invention, said sample is a bodily fluid sample selected from the group comprising whole blood, plasma and serum.

[0183] In particular embodiments of the method of the invention, said patient is a patient suffering from a severe infectious disease, inflammation, sepsis, shock, pulmonary diseases including ARDS, cardiovascular diseases including acute myocardial infarction, acute or chronic heart failure, organ dysfunction, a hepatic disease, a renal disease, an infectious disease, an autoimmune disease including ulcerative colitis, arthritis other chronic inflammatory diseases, other chronic inflammatory diseases, age-related macular degeneration, diabetic macular edema, a psychiatric disease, a neurodegenerative disease, or cancer, in particular wherein the patient is a cancer patient receiving immune effector cell therapy (IECT).

[0184] In a specific embodiment said immune effector cell therapy is chimeric antigen receptor T-cell therapy (CAR T-cell therapy).

[0185] In further particular embodiments of the method of the invention, said patient suffers from organ organ dysfunction, in particular kidney dysfunction, lung dysfunction, heart dysfunction, or liver dysfunction.

[0186] In further particular embodiments of the method of the invention, said patient suffers from shock, in particular selected from the group comprising cardiogenic shock, septic shock and anaphylactic shock.

[0187] In particular embodiments of the method of the invention, the DPP3 binder is selected from the group comprising small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non-Ig scaffolds.

[0188] In further particular embodiments of the method of the invention, said DPP3 binder is an anti- DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID No. 1 : T75369WO BOEHMERT & BOEHMERT

[0189] MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYI YALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFV PNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFS GNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEV TSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQ GSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERL VASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKN VSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQ DEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLH PQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILR VLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVA GGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASA AGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA.

[0190] In further particular embodiments of the method of the invention, said DPP3 binder is an anti- DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in

[0191] SEQ ID No. 2: CETVINPETGEQIQSWYRSGE, particularly SEQ ID NO: 3: INPETG.

[0192] In further particular embodiments of the method of the invention, said antibody is a monoclonal antibody or monoclonal antibody fragment.

[0193] In further particular embodiments of the method of the invention, said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity determining regions (CDRs) in the heavy chain comprise the sequences:

[0194] CDR1 : SEQ ID NO: 6: GFSLSTSGMS

[0195] CDR2: SEQ ID NO: 7: IWWNDNK

[0196] CDR3: SEQ ID NO: 8: ARNYSYDY and wherein the complementarity determining regions (CDRs) in the light chain comprise the sequences:

[0197] CDR1: SEQ ID NO: 9: RSLVHSIGSTY

[0198] CDR2: KVS

[0199] CDR3: SEQ ID NO: 10: SQSTHVPWT. T75369WO BOEHMERT & BOEHMERT

[0200] In further particular embodiments of the method of the invention, said monoclonal antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.

[0201] In further particular embodiments of the method of the invention, said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence: SEQ ID NO: 11:

[0202] MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSV GWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDT GTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

[0203] HEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI< VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG, and wherein the light chain comprises the sequence: SEQ ID NO: 12:

[0204] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLY WYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQST HVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC.

[0205] In further particular embodiments of the method of the invention, said DPP3 binder is a small molecule and is selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, or a derivative or analog of any of the aforementioned compounds, and benzimidazol derivatives (i.e. compounds having a benzimidazole scaffold).

[0206] General Definitions

[0207] Throughout the specification the term “ADM-Gly” includes all splice variants of ADM-Gly if not stated otherwise. Throughout the specification the term “mature ADM” includes all splice variants of mature ADM if not stated otherwise.

[0208] As used herein, the terms "comprising" and "including" or grammatical variants thereof are to be taken as specifying at least the stated features, integers, steps or components but do not T75369WO BOEHMERT & BOEHMERT preclude the addition of one or more additional features, integers, steps, components or groups thereof. These terms encompass the terms "consisting of’ and "consisting essentially of’ that are understood to specify only the stated feature, integers, steps or components to the exclusion of any additional features.

[0209] As used herein “concentration” particularly refers to mass per volume, e.g. mass of DPP3 in a volume of sample, e.g., ng / mL.

[0210] The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

[0211] The term "patient" as used herein refers to a living human or non-human organism that is receiving medical care or that should receive medical care due to a disease. This includes persons with no defined illness who are being investigated for signs of pathology. Thus, the methods and assays described herein are applicable to human disease.

[0212] The term "pharmaceutical composition" means a pharmaceutical ingredient in combination with at least one pharmaceutically acceptable excipient, which is in such form as to permit the biological activity of a pharmaceutical ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. The term "pharmaceutical ingredient" means a therapeutic composition which can be optionally combined with pharmaceutically acceptable excipients to provide a pharmaceutical composition or dosage form.

[0213] DPP3 Binders

[0214] In particular embodiments of the invention, the DPP3 binder inhibits the activity of DPP3. In certain embodiments, the DPP3 binder is an inhibitor of DPP3 activity. Inhibitors are molecules that preferably significantly inhibit DPP3 activity. Those molecules can be peptides and small molecules, antibodies, antibody fragments or non-Ig scaffolds.

[0215] Significantly inhibiting means in particular inhibiting the activity of DPP3 at least 10%, preferably at least 20%, more preferably at least 30%, even more preferred at least 40%, even more preferred at least 50%, even more preferred at least 60%, even more preferably at least 70%, even more preferably at least 80 %, even more preferably at least 90 %, even more preferably about or actually 100% inhibition. T75369WO BOEHMERT & BOEHMERT

[0216] For the avoidance of doubt, this means that less than 90%, preferably less than 80%, more preferably less than 70%, even more preferred less than 60%, even more preferred less than 50%, even more preferred less than 40%, even more preferred less than 30%, even more preferably less than 20 %, even preferably less than 10 %, even more preferably about or actually 0% of DPP3 activity remains.

[0217] In other particular embodiments of the invention, the DPP3 binder inhibits the activity of DPP3 by at least 20 %. In other particular embodiments of the invention, the DPP3 binder inhibits the activity of DPP3 by at least 60 %.

[0218] The activity of DPP3 can be bound unspecifically by different general protease binders (e.g., PMSF, TPCK), sulfhydryl reagents (e.g., pHMB, DTNB) and metal chelators (EDTA, o-phenantroline) (Abramic etal. 2000. Biological Chemistry, 381: 1233 1243; EP 2949332).

[0219] DPP3 activity can be further inhibited specifically by different kinds of compounds: an endogenous DPP3-binder is the peptide spinorphin. Several synthetic derivatives of spinorphin, e.g., tynorphin, have been produced and shown to inhibit DPP3 activity to varying extents (Yamamoto et al. 2000. Life sciences 62 (19): 1767-1773). Other published peptide binders of DPP3 are propioxatin A and B (US 4804676) and propioxatin A analogues (Inaoka et al. 1988. J. Biochem 104 (5): 706-711).

[0220] A “derivative or analogue” is a chemical compound that is derived from a parent compound by a chemical reaction with the replacement of one atom or substitution of a group of atoms by a functional group. Parent and derivative compounds have similar chemical structures.

[0221] DPP3 can also be bound by small molecules such as fluostatins and benzimidazol derivatives. Fluostatins A and B are antibiotics produced in Streptomyces sp. TA-3391 that are non-toxic and strongly inhibit DPP3 activity. So far, 20 different derivatives of benzimidazol have been synthesized and published (Agic etal. 2007. Bioorganic Chemistry 35 (2): 153 169; Rastija et al. 2015. Acta Chimica Slovenica 62: 867-878), of which the two compounds 1’ and 4’ show the strongest inhibitory effect (Agic etal. 2007. Bioorganic Chemistry 35 (2): 153 169). Several dipeptidyl hydroxamic acids have been shown to inhibit DPP3 activity as well (Cvitesic et al., 2016. J Enzyme Inhib Med Chem 31(sup2):40-45).

[0222] A “small molecule” is in particular a low molecular weight (more particularly < 1000 daltons) organic compound. Such small molecules may in particular regulate a biological process, e.g. bind a specific biological macromolecule, in the present invention in particular DPP3, and act as an effector, in particular an inhibitor, altering the activity or function of the biological macromolecule. T75369WO BOEHMERT & BOEHMERT

[0223] Particular examples of small molecule and peptide binders of DPP3 are shown in the following Table 1. Compounds 1 ’ and 4’ show the strongest inhibitory effect (Agic et al. 2007, see above).

[0224] Table 1: Peptide and small molecule binders of DPP3.

[0225] T75369WO BOEHMERT & BOEHMERT T75369WO BOEHMERT & BOEHMERT T75369WO BOEHMERT & BOEHMERT

[0226] The spinorphin and spinorphin derivatives in the above table have (insofar as comprising 4 amino acids or more) SEQ ID NO: 44 through SEQ ID NO: 66 (see herein below).

[0227] In particular embodiments of the invention, therapy or prevention of endothelial dysfunction does not involve the administration of a binder to Adrenomedullin (ADM) or a fragment thereof, in particular an anti-ADM antibody or a fragment thereof.

[0228] In certain embodiments of the invention relating to a DPP3 binder for for use in the treatment of a patient in need thereof, the DPP3 binder is selected from the group comprising small molecules, anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold.

[0229] In one embodiment of the invention the DPP3 binder is a small molecule selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, enzimidazole or derivatives or analogues thereof.

[0230] In another embodiment of the invention said binder is an anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold.

[0231] In one embodiment said anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non- Ig scaffold exhibits a minimum binding affinity to full-length DPP3 of equal or less than 10'7M.

[0232] In one embodiment said anti-DPP3 antibody is a monoclonal antibody or monoclonal antibody fragment.

[0233] Throughout the specification the “antibodies”, or “antibody fragments” or “non-Ig scaffolds” in accordance with the invention that are capable to bind DPP3, and thus are directed against DPP3, can be referred to as “anti-DPP3 antibodies”, “anti-DPP3 antibody fragments”, or “anti- DPP3 non-Ig scaffolds”. T75369WO BOEHMERT & BOEHMERT

[0234] Likewise, throughout the specification the “antibodies”, or “antibody fragments”, or “non-Ig scaffolds” in accordance with the invention that are capable to bind Pro-Adrenomedullin or a fragment thereof, and thus are directed against Pro-Adrenomedullin or a fragment, can be referred to as “anti-Pro-Adrenomedullin or anti-Pro-Adrenomedullin-fragment antibodies”, “anti-Pro-Adrenomedullin or anti-Pro-Adrenomedullin-fragment antibody fragments”, or “anti-Pro-Adrenomedullin or anti-Pro-Adrenomedullin-fragment non-Ig scaffolds”, wherein the term “Pro-Adrenomedullin fragment” can be replaced with the specific Pro- Adrenomedullin fragment as detailed herein, where applicable.

[0235] The term “antibody” generally comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single-chain-antibodies” (Bird, R. E., et al. (1988) Science, 242(4877), 423- 426.), chimeric, humanized, in particular CDR-grafted antibodies, and dia- or tetrabodies Also comprised are immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample. In this context the term “specific binding” refers to antibodies raised against the molecule of interest or a fragment thereof. An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least preferably 50-fold higher, more preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to make antibodies and to select antibodies with a given specificity.

[0236] In one embodiment of the invention the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold is monospecific.

[0237] Monospecific means that said antibody or antibody fragment or non-Ig scaffold binds to one specific region encompassing at least 4 amino acids within the target DPP3. Monospecific antibodies or fragments or non-Ig scaffolds according to the invention are antibodies or fragments or non-Ig scaffolds that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies may also be produced by other means than producing them from a common germ cell.

[0238] In particular, monospecific anti-DPP3 antibody or monospecific anti-DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold means that said antibody or antibody fragment or non-Ig scaffold binds to one specific region encompassing at least 5 amino acids within the target DPP3 (SEQ ID NO: 1). Monospecific anti-DPP3 antibody or monospecific anti-DPP3 T75369WO BOEHMERT & BOEHMERT antibody fragment or monospecific anti-DPP3 non-Ig scaffold are anti-DPP3 antibodies or anti- DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies may also be produced by other means than producing them from a common germ cell.

[0239] In specific embodiments said anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold is an inhibiting antibody, fragment or non-Ig scaffold. In certain specific embodiments, said anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold inhibits the activity of DPP3 more than 10%, preferably more than 20%, more preferably more than 30%, even more preferred more than 40%, even more preferred more than 50%, even more preferred more than 60%, even more preferably more than 70%, even more preferably more than 80 %, even more preferably more than 90 %, even more preferred more than 95%, even more preferably almost or actually 100%. For the avoidance of doubt, this means that less than 90%, preferably less than 80%, more preferably less than 70%, even more preferred less than 60%, even more preferred less than 50%, even more preferred less than 40%, even more preferred less than 30%, even more preferably less than 20 %, even preferably less than 10 %, even more preferably about or actually 0% of DPP3 activity remains.

[0240] An antibody or fragment according to the present invention is a protein including one or more polypeptides substantially encoded by immunoglobulin genes that specifically binds an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgGi, IgG2, IgG?, IgG4), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length.

[0241] Full-length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acids in length. Light chains are encoded by a variable region gene at the NH2-terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus. Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0242] The basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., T75369WO BOEHMERT & BOEHMERT

[0243] Lanzavecchia, A., & Scheidegger, D. (1987) European Journal of Immunology, 17(1), 105— 111.; Huston, J. S., et al. (1988) Proceedings of the National Academy of Sciences, 85(16), 5879-5883; Bird, R. E., et al. (1988) Science, 242(4877), 423-426; Hood et al. 1984, Immunology, Benjamin, N.Y.,2nd ed.; Hunkapiller and Hood 1986. Nature 323: 15-16}. An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al. 1983, U.S. Department of Health and Human Services}. As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. An immune complex is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody or human antibody, or functional antibody fragment, specifically bound to the antigen.

[0244] Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a nonhuman (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor" and the human immunoglobulin providing the framework is termed an "acceptor". In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.

[0245] A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other T75369WO BOEHMERT & BOEHMERT monoclonal antibodies can have additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions.

[0246] Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr.

[0247] Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Patent No. 5,585,089). Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (for example, see WO 93 / 12227; WO 91 / 10741).

[0248] A human antibody is an antibody wherein the light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art.

[0249] Antibodies can be produced by immortalizing a (e.g. mammalian) B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by Epstein-Barr Virus (EBV) infection of a B-cell or by fusing a B cell with a myeloma to make a hybridoma or fusing a B cell to a hybridoma cell to produce a trioma cell. Antibodies can also be produced by phage display methods (see, e.g., WO 91 / 17271; WO 92 / 001047; WO 92 / 20791) or selected from a combinatorial monoclonal antibody library (see the Morphosys website).

[0250] In a certain further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by isolating the antibody from a transgenic plant or transgenic animal or a secretion or product of a transgenic plant or transgenic animal having a nucleotide sequence encoding for the antibody.

[0251] Said animal may e.g., be selected from a chicken, a mouse, a rat, a rabbit, a cow, a goat, a sheep, a pig; said secretion or product may e.g. be milk or an egg. Said plant may e.g. be selected from tobacco (N. tabacum or N. benlhamianct). duckweed (Lemna minor), Chlamydomonas reinhardlii, rice, Arabidopsis thaliana, alfalfa (Medicago sativa), lettuce, maize.

[0252] The antibodies can in certain embodiments be isolated by physicochemical fractionation, e.g. size exclusion chromatography, precipitation, e.g. using ammonium sulphate, ion exchange chromatography, immobilized metal chelate chromatography gel filtration, zone electrophoresis; based on their classification e.g. binding to bacterial proteins A, G, or L, jacalin; antigen-specific affinity purification via immobilized ligands / antigens; if necessary, low molecular weight components can be removed by methods like dialysis, desalting, and diafiltration. T75369WO BOEHMERT & BOEHMERT

[0253] In some embodiments the antibody is encoded by a nucleotide sequence where the nucleotide sequence is a reverse transcription of an amino acid sequence from an antibody produced by one of the processes described herein.

[0254] Thus, the anti -DPP3 -fragment antibody may have the formats known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR- grafted antibodies. In a preferred embodiment antibodies according to the present invention are recombinantly produced antibodies as e.g. IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F-variable domain of heavy and / or light chain as e.g. chemically coupled antibodies (fragment antigen binding) including but not limited to Fab- fragments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-V5Sx2; bivalent Fab (mini -antibody) dimerized with the CH3 domain; bivalent Fab or multivalent Fab, e.g. formed via multimerization with the aid of a heterologous domain, e.g. via dimerization of dHLX domains, e.g. Fab-dHLX-FSx2; F(ab‘ ^-fragments, scFv-fragments, multimerized multivalent or / and multi-specific scFv-fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T-cell engager), trifunctional antibodies, polyvalent antibodies, e.g. from a different class than G; single-domain antibodies, e.g. nanobodies derived from camelid or fish immunoglobulins and numerous others.

[0255] In certain embodiments the anti-DPP3 or anti -DPP3 -fragment antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment and scFv-Fc Fusion protein. In another preferred embodiment the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFv fragment and bioavailability optimized conjugates thereof, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0256] Non-Ig scaffolds may be protein scaffolds and may be used as antibody mimics as they are capable to bind to ligands or antigens. In one embodiment non-Ig scaffolds may be selected from the group comprising tetranectin-based non-Ig scaffolds (e.g. described in US 2010 / 0028995), fibronectin scaffolds (e.g. described in EP 1 266025 lipocalin-based scaffolds (e.g. described in WO 2011 154420}: ubiquitin scaffolds (e.g. described in WO 2011 / 073214), transferrin scaffolds (e.g. described in US 2004 / 0023334), protein A scaffolds (e.g. described in EP 2 231 860), ankyrin repeat based scaffolds (e.g. described in WO 2010 / 060748), microproteins preferably microproteins forming a cysteine knot) scaffolds (e.g. described in EP 2314308), Fyn SH3 domain based scaffolds (e.g. described in WO 2011 / 023685) EGFR-A- T75369WO BOEHMERT & BOEHMERT domain based scaffolds (e.g. described in WO 2005 / 040229} and Kunitz domain based scaffolds (e.g. described in EP 1 941 867}.

[0257] In addition to antibodies other biopolymer scaffolds are well known in the art to complex a target molecule and have been used for the generation of highly target specific biopolymers. Examples are aptamers, spiegelmers, anticalins, and conotoxins.

[0258] In one embodiment of the invention anti-DPP3 antibodies may be produced as outlined in Example 4 by synthesizing fragments of DPP3 as antigens or full-length DPP3. Thereafter, binder to said fragments are identified using the below described methods or other methods as known in the art.

[0259] The fragments as DPP3 antigen are a peptide, wherein the peptide is 15 to 55 amino acids long; is 15, 20, 25, 30, 35, 40, 45, 50, or 55 amino acids long; or is about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, or about 55 amino acids long, wherein the term “about” in this context of amino acid length for the peptide can be plus or minus 2 amino acids, e.g., about 20 amino acids means the range of 23-27 amino acids long.

[0260] For the antigen comprises the peptide, the peptide comprises the catalytic site from 316 to 669 amino acids, preferably include the catalytic motifs SEQ ID NO: 30 or SEQ ID NO: 31, preferably at least one or more of the important amino acids important for substrate binding and hydrolysis chosen from the list consisting of Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669, or more preferably SEQ ID NO: 2.

[0261] Humanization of murine antibodies may be conducted according to the following procedure:

[0262] For humanization of an antibody of murine origin the antibody sequence is analyzed for the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modelling. (Almagro 2008) The use of molecular modeling to achieve a humanized antibodies may involve additional conservative amino acid substitutions at the junction of the CDR-FR. Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr. Additionally, back mutations may T75369WO BOEHMERT & BOEHMERT be implemented, where selection of residues outside of the region defining the specificity and thus target for back mutation (to mimic the source sequence, i.e., murine) to restore or improve the affinity of the humanized antibody. (Almagro 2008). When available, the three-dimensional models of the antibodies may be used to guide the humanization process.

[0263] Antibody fragment humanization may also be done by a CDR-grafting method, such as described in Jones et al. 1986. Nature 321, 522-525. Particular steps may be: Extract total RNA from hybridomas; First round of RT-PCR with primer sets specific for the antibody heavy and light chains, with multiple heavy chain and light chain RT-PCR reactions for each RNA sample, using degenerate forward primer mixtures covering the leader sequences of variable regions; reverse primers are to be located in the constant regions of heavy and light chains, no restriction sites need to be engineered into the primers. Further amplification of the RT-PCR products from the first-round reactions in a second-round PCR, using semi-nested primer sets specific for antibody variable regions. Detecting DNA fragments amplified in the PCR reaction samples, e.g. with gel, such as agarose gel methods. Sequencing cloned DNA fragments amplified by nested RT-PCR and determining suitable and correctly cloned sequences. Protein sequence alignment and CDR analysis may be used to identify heavy chain and light chains. Optionally, amino acids in the variable heavy and / or light chains that are critical to the binding properties may be reverted to correspond the (e.g. murine) original sequence.

[0264] The humanized anti-DPP3 antibody may recombinantly be produced with a CHO DG44 cell line. In a particular embodiment, the creation of a production cell, e.g., the CHO cell line encoding the DPP3 Binder, is used for producing the DPP3 Binder. The antibody binds to its target molecule DPP3, inhibiting its enzymatic activity as part of its mode of action. The humanized anti-DPP3 antibody is directed against a linear epitope SEQ ID NO: 3 (INPETG within the DPP3 sequence). The in silico derived molecular weights of the light and heavy chain of PCZ are 23,943 Dalton (Da) and 48,950 Da, respectively.

[0265] A unit dose in mg / kg refers to mg of antibody per kg of patient is based on a certain molecular weight (“MW”) of the administered antibody. All references to mg / kg in this application refer to the full-length anti-DPP3 antibody having a MW of 145,734 Da determined in silico. For any other MW of antibodies and fragments thereof the appropriate doses may be calculated accordingly. T75369WO BOEHMERT & BOEHMERT

[0266] Corresponding molar doses of antibodies or antibody fragments having other molecular weights can in particular be calculated according to the following explanation:

[0267] A unit of dose in mg of antibody or fragment thereof per kg of patient is based on a specific molecular weight of the administered antibody or fragment thereof. If the molecular weight of the antibody would be for instance 145,734 g / mol (as is the case for the humanized anti-DPP3 antibody AK1967 (Procizumab; PCZ)), a dose of about 18 to 22 mg / kg would correspond to about 0.12 to 0.15 pmol / kg. If, in contrast, the molecular weight of the antibody would be for instance 165,000 g / mol, the same molar doses of about 0.12 to 0.15 pmol / kg would correspond to about 20 to 25 mg / kg.

[0268] The method of how the mass of PCZ was determined was as follows: The PCZ concentration attributed to the drug substance and drug product as well as the doses described in mass per weight (mg / kg) were based on measurement of the absorption of the PCZ at 280 nm measured in a spectrophotometer with a path length of 1 cm. Conversion of A280 nm measurement into mol / L was done by employing a theoretically calculated extinction coefficient Smoiar using the formula: molar Concentration=A280 nrn / smoiar. The extinction coefficient for the PCZ was calculated as follows: The amino acid sequences of the heavy and light chain of the PCZ, as shown below, were combined, duplicated, and then pasted in a calculator program (Expasy - ProtParam; https: / / web expasy.org / protparam / ; Protein Identification and Analysis Tools on the Expasy Server; Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A.; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607 Full text - Copyright Humana Press.):

[0269] PCZ heavy chain amino acid sequence (SEP ID No. 68):

[0270] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWN DNI<SYNPALI<SRLTITRDTSI<NQVVLTMTNMDPVDTGTYYCARNYSYDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG T75369WO BOEHMERT & BOEHMERT

[0271] PCZ light chain amino acid sequence (SEP ID No. 69):

[0272] DIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0273] The resulting calculation was:

[0274] Molar extinction coefficient Smoiar (at 280nm in H2O) = 228,440 M-I*cm_|

[0275] The value was calculated under the assumption that all cysteine residues form cystines. The molar concentration of PCZ can be determined by dividing the measured absorption at 280 by the molar extinction coefficient Smoiar.

[0276] For the conversion from mol into mass of PCZ a molecular weight of 145,734 g / mol was used. This molecular weight was calculated by amino acid sequence-based in silico prediction using the same calculator program as mentioned above (Expasy - ProtParam; https: / / web expasy.org / protparam / ): First, the amino acid sequence SEQ ID No. 68 of the heavy chain was predicted as having a molecular weight of 48,950 g / mol. Secondly, the amino acid sequence SEQ ID No. 69 of the light chain was predicted as having a molecular weight of 23,943 g / mol. The molecular weight for the full-length PCZ was then calculated as 2 x 48,950 Da + 2 x 23,943 Da = 145,734 g / mol (1 Da = 1 g / mol).

[0277] By using the method described above for the definition of the concentration of the PCZ, all concentrations and doses of the PCZ mentioned in the current invention are defined by the amino acid composition of the antibody only and do not take into consideration post- translational modifications such as glycosylation.

[0278] In another preferred embodiment, the anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold is a full-length antibody, antibody fragment, or non-Ig scaffold.

[0279] In certain embodiments of the invention, the DPP3 binder is an anti-DPP3 antibody or anti- DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID NO: 1. In one embodiment said binder is an anti- DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an T75369WO BOEHMERT & BOEHMERT epitope of at least 4 amino acids in length comprised in SEQ ID NO: 1. In another embodiment said binder is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 5 amino acids in length comprised in SEQ ID NO: 1.

[0280] In certain embodiments of the invention, the DPP3 binder is an anti-DPP3 antibody or anti- DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID NO: 2, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO: 1.

[0281] In certain embodiments of the invention, the DPP3 binder is an anti-DPP3 antibody or anti- DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 amino acids in length comprised in SEQ ID NO: 2, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO: 1. In another embodiment said binder is an anti-DPP3 antibody or anti- DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 5 amino acids in length comprised in SEQ ID NO: 2, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO: 1.

[0282] In certain embodiments of the invention, the DPP3 binder is an anti-DPP3 antibody or anti- DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID NO: 3, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO: 1. In one embodiment said binder is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 amino acids in length comprised in SEQ ID NO: 3, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO: 1. In another embodiment said binder is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 5 amino acids in length comprised in SEQ ID NO: 3, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO: 1.

[0283] An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies. For example, the epitope is the specific piece of the antigen to which an antibody binds. The part of an antibody that binds to the epitope is called a paratope. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the involved epitope residues and the shape or tertiary structure of other segments of the antigen. T75369WO BOEHMERT & BOEHMERT

[0284] A conformational epitope is formed by the 3-D conformation adopted by the interaction of discontinuous amino acid residues. A linear or a sequential epitope is an epitope that is recognized by antibodies by its linear sequence of amino acids, or primary structure and is formed by the 3-D conformation adopted by the interaction of contiguous amino acid residues.

[0285] In specific embodiments of the invention the antibody or fragment thereof is a monoclonal antibody or a fragment thereof.

[0286] In one embodiment of the invention the anti-DPP3 antibody or the anti-DPP3 antibody fragment is a human or humanized antibody or derived therefrom. In one specific embodiment one or more (murine) CDR’s are grafted into a human antibody or antibody fragment.

[0287] In certain embodiments of the invention, the DPP3 binder is a monoclonal human or humanized CDR-grafted antibody or antibody fragment thereof comprising a heavy chain (H chain) comprising:

[0288] GFSLSTSGMS (SEQ ID NO: 6),

[0289] IWWNDNK (SEQ ID NO: 7),

[0290] ARNYSYDY (SEQ ID NO: 8) and / or further comprising a light chain (L chain) comprising:

[0291] RSLVHSIGSTY (SEQ ID NO: 9),

[0292] KVS (no assigned SEQ ID NO),

[0293] SQSTHVPWT (SEQ ID NO: 10).

[0294] In certain embodiments of the invention, the DPP3 binder is a monoclonal human or humanized CDR-grafted antibody or antibody fragment thereof comprising a heavy chain comprising at least one CDR selected from the group comprising:

[0295] GFSLSTSGMS (SEQ ID NO: 6),

[0296] IWWNDNK (SEQ ID NO: 7),

[0297] ARNYSYDY (SEQ ID NO: 8) and a light chain comprising at least one CDR selected from the group comprising:

[0298] RSLVHSIGSTY (SEQ ID NO: 9),

[0299] KVS (no assigned SEQ ID NO), T75369WO BOEHMERT & BOEHMERT

[0300] SQSTHVPWT (SEQ ID NO: 10).

[0301] The anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold according to the present invention in particular embodiments exhibits an affinity towards human DPP3 in such that affinity constant is greater than 10'7M, preferred 10'8M, preferred affinity is greater than 10'9M, most preferred higher than IO'10M. Aperson skilled in the art knows that it may be considered to compensate lower affinity by applying a higher dose of compounds and this measure would not lead out-of-the-scope of the invention. The affinity constants may be determined according to the method as described in Example 5.

[0302] In certain embodiments of the invention, the DPP3 binder is a monoclonal antibody or antibody fragment thereof comprising the following sequence as a variable heavy chain: SEQ ID NO: 4

[0303] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDN KSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS and comprising the following sequence as a variable light chain: SEQ ID NO: 5

[0304] DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK.

[0305] In certain embodiments of the invention, the DPP3 binder is a monoclonal human or humanized CDR-grafted antibody or antibody fragment thereof comprising the following sequence as a heavy chain: SEQ ID NO: 11

[0306] MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSV GWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDT GTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI< VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG and comprising the following sequence as a light chain: SEQ ID NO: 12

[0307] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLY WYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQST HVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK T75369WO BOEHMERT & BOEHMERT

[0308] VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC, or fragments thereof comprising the CDRs as defined herein and comprising at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, of SEQ ID NO: 11 and SEQ ID NO: 12, respectively; more particularly such fragments are Fab fragments; more particularly, such fragments comprise the CDRs of SEQ ID NOs 6 to 10 and KVS as defined above (i.e. the CDR sequences are conserved).

[0309] In specific embodiments of the invention the antibody comprises the following sequence as a heavy chain: SEQ ID NO: 11 or a sequence that is > 95% identical to it, preferably > 98%, preferably > 99% and comprises the following sequence as a light chain: SEQ ID NO: 12 or a sequence that is > 95% identical to it, preferably > 98%, preferably > 99%, or fragments thereof comprising the CDRs as defined herein and comprising at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, of SEQ ID NO: 11 and SEQ ID NO: 12, respectively; more particularly such fragments are Fab fragments; more particularly such antibodies comprise the CDRs of SEQ ID NOs 6 to 10 and KVS as defined above (i.e. the CDR sequences are conserved).

[0310] In certain embodiments of the invention, the DPP3 binder is a monoclonal human or humanized CDR-grafted antibody or antibody fragment thereof comprising the following sequence as a heavy chain: SEQ ID NO: 68

[0311] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDN KSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTGTYYCARNYSYDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG and comprising the following sequence as a light chain: SEQ ID NO: 69

[0312] DIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRF

[0313] SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAP T75369WO BOEHMERT & BOEHMERT

[0314] SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, or fragments thereof comprising the CDRs as defined herein and comprising at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, of SEQ ID NO: 68 and SEQ ID NO: 69, respectively; more particularly such fragments are Fab fragments; more particularly, such fragments comprise the CDRs of SEQ ID NOs 6 to 10 and KVS as defined above (i.e. the CDR sequences are conserved).

[0315] In specific embodiments of the invention the antibody comprises the following sequence as a heavy chain: SEQ ID NO: 68 or a sequence that is > 95% identical to it, preferably > 98%, preferably > 99% and comprises the following sequence as a light chain: SEQ ID NO: 69 or a sequence that is > 95% identical to it, preferably > 98%, preferably > 99%, or fragments thereof comprising the CDRs as defined herein and comprising at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, of SEQ ID NO: 68 and SEQ ID NO: 69, respectively; more particularly such fragments are Fab fragments; more particularly such antibodies comprise the CDRs of SEQ ID NOs 6 to 10 and KVS as defined above (i.e. the CDR sequences are conserved).

[0316] To assess the identity between two amino acid sequences, a pairwise alignment is performed. Identity defines the percentage of amino acids with a direct match in the alignment.

[0317] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 67.

[0318] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a light chain Fab fragment comprising SEQ ID NO: 12.

[0319] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 67 and comprises a light chain Fab fragment comprising SEQ ID NO: 12. T75369WO BOEHMERT & BOEHMERT

[0320] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 67.

[0321] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a light chain variable region portion from SEQ ID NO: 12.

[0322] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 67 and comprises a light chain variable region portion from SEQ ID NO: 12.

[0323] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof is a fragment, wherein the fragment is aScFv and wherein the ScFv comprises a heavy chain variable region portion from SEQ ID NO: 67 and comprises a light chain variable region portion from SEQ ID NO: 12, and wherein the heavy chain variable region portion from SEQ ID NO: 67 and a light chain variable region portion from SEQ ID NO: 12 are linked together via linker.

[0324] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof is a fragment, wherein the fragment is a Fab fragment and wherein the Fab fragment comprises a heavy chain Fab fragment comprising SEQ ID NO: 67 and comprises a light chain Fab fragment comprising SEQ ID NO: 12.

[0325] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 72.

[0326] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, T75369WO BOEHMERT & BOEHMERT wherein said antibody or fragment thereof comprises a light chain Fab fragment comprising SEQ ID NO: 69.

[0327] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 72 and comprises a light chain Fab fragment comprising SEQ ID NO: 69.

[0328] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 72.

[0329] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a light chain variable region portion from SEQ ID NO: 69.

[0330] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 72 and comprises a light chain variable region portion from SEQ ID NO: 69.

[0331] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof is a fragment, wherein the fragment is a ScFv and wherein the ScFv comprises a heavy chain variable region portion from SEQ ID NO: 72 and comprises a light chain variable region portion from SEQ ID NO: 69, and wherein the heavy chain variable region portion from SEQ ID NO: 72 and a light chain variable region portion from SEQ ID NO: 69 are linked together via linker.

[0332] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof is a fragment, wherein the fragment is a Fab fragment and wherein the Fab fragment comprises a heavy chain Fab fragment comprising SEQ ID NO: 72 and comprises a light chain Fab fragment comprising SEQ ID NO: 69. T75369WO BOEHMERT & BOEHMERT

[0333] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 4.

[0334] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a light chain variable region comprising SEQ ID NO: 5.

[0335] In one embodiment of the present invention, the DPP3 binder for use in therapy or prevention of endothelial dysfunction is an anti-DPP3 Antibody or fragment thereof binding to DPP3, wherein said antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 4 and comprises a light chain variable region comprising SEQ ID NO: 5.

[0336] In particular embodiments, the level of Pro-Adrenomedullin or a fragment thereof is above a threshold in a sample of bodily fluid of said patient before administration of the DPP3 binder, and said level decreases in a sample of bodily fluid of said patient after administration of said DPP3 binder; more particularly within a time frame of 48 hours, more particularly within a time frame of 24 hours upon administration of said DPP3 binder.

[0337] In particular embodiments, the level of Pro-Adrenomedullin or a fragment thereof is above a threshold in a sample of bodily fluid of said patient before administration of the DPP3 binder, and said level decreases below said threshold in a sample of bodily fluid of said patient after administration of said DPP3 binder; more particularly within a time frame of 48 hours, more particularly within a time frame of 24 hours upon administration of said DPP3 binder.

[0338] In certain embodiments of the invention, where a level of Pro-Adrenomedullin or a fragment thereof in a sample of a bodily fluid of a patient is above a threshold, the patient is identified as having a level of Pro- Adrenomedullin or a fragment thereof above said threshold, in particular in said sample of bodily fluid.

[0339] Therefore, as an example, embodiments of the invention concern the DPP3 binder for use in therapy or prevention of endothelial dysfunction in a critically ill patient according to the invention, wherein said patient is identified having a level of Pro-Adrenomedullin or a fragment thereof above a threshold, in particular in a sample of bodily fluid obtained from said patient. T75369WO BOEHMERT & BOEHMERT

[0340] Methods for obtaining monoclonal antibodies

[0341] In all of the following embodiments, the term monoclonal antibody is meant to include monoclonal antibodies, as well as fragments of monoclonal antibodies, such as the ones detailed herein, more particularly monoclonal antibodies.

[0342] Hybridoma

[0343] In a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain a multitude of hybridomas, ii) isolating from said multitude of hybridomas a hybridoma producing a desired monoclonal antibody.

[0344] In certain embodiments, the antibody according to the present invention is a monoclonal antibody obtainable by isolating from a multitude of hybridomas a hybridoma producing a desired monoclonal antibody, wherein said multitude of hybridomas were produced by fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain multitude of hybridomas.

[0345] A desired monoclonal antibody is in particular a monoclonal antibody binding the antigen, in particular with a binding affinity of at least 107M’1, preferred 108M’1, more preferred affinity is greater than 109M’1, most preferred greater than IO10M’1.

[0346] In certain embodiments of the method for obtaining an antibody, in step i) the animal is a mammal, particularly a rabbit, a mouse or a rat, more particularly a mouse, more particularly a Balb / c mouse.

[0347] In certain embodiments of the method for obtaining an antibody, in step i) the antibodysecreting cell is a splenocyte, more particularly an activated B-cell.

[0348] In certain embodiments of the method for obtaining an antibody, in step i) fusing involves the use of polyethylene glycol.

[0349] In certain embodiments of the method for obtaining an antibody, in step i) the myeloma is derived from a mammal, in certain embodiments from the same species of mammal from which the multitude of antibody-secreting cells is obtained. In certain specific embodiments of the method for obtaining an antibody, in step i) the myeloma cells are of the cell line SP2 / 0. T75369WO BOEHMERT & BOEHMERT

[0350] In certain embodiments of the method for obtaining an antibody, said fusing in step i) comprises PEG-assisted fusion, Sendai virus-assisted fusion or electric current-assisted fusion.

[0351] In certain embodiments of the method for obtaining an antibody, said isolating in step ii) comprises performing an antibody capture assay, an antigen capture assay, and / or a functional screen.

[0352] In certain embodiments of the method for obtaining an antibody, in step ii) isolating the hybridoma producing a desired monoclonal antibody may involve cloning and re-cloning the hybridomas using the limiting-dilution technique.

[0353] In one embodiment, said antigen capture assay comprises: a) binding the produced antibodies to a substrate, particularly a solid substrate, b) allowing antigen to bind to said antibodies, c) removing unbound antigen by washing, d) detecting bound antigen; or said antigen capture assay comprises: a) allowing an antigen to bind the produced antibodies to form an antibody-antigen complex, b) binding said antibody-antigen complex to a substrate, particularly a solid substrate, c) removing unbound antigen by washing, d) detecting bound antigen.

[0354] It is apparent to the skilled person that in the context of the aforementioned antibody capture assay, the terms “substrate” and in particular “solid substrate” relate to a support, and in particular a solid support, i.e. a solid phase.

[0355] In one embodiment, said isolating of step ii) comprises performing an enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, cell staining, immunoprecipitation, and / or a western blot.

[0356] In one embodiment, said detecting of the antibody or the antigen is accomplished with an immunoassay.

[0357] In one embodiment, the animal is a transgenic animal, in particular a transgenic mouse (wherein in particular the mouse immunoglobulin (Ig) gene loci have been replaced with human loci within the transgenic animal genome), such as HuMabMouse or XenoMouse. T75369WO BOEHMERT & BOEHMERT

[0358] In one embodiment, the antigen comprises a peptide as described herein in Table 4, which in certain embodiments (in particular for immunization) may be conjugated to a protein, particularly a serum protein, more particularly a serum albumin, more particularly BSA.

[0359] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) fusing splenocytes cells from a Balb / c mouse previously immunized with a peptide as described herein in Table 4 with SP2 / 0 myeloma cells using polyethylene glycol, to obtain a multitude of hybridomas, ii) isolating from said multitude of hybridomas a hybridoma producing a desired monoclonal antibody; more preferably, the method comprises:

[0360] 1) growing hybridomas for a first period (in particular 2 weeks) in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT- Supplement]

[0361] 2) followed replacing HAT medium with HT Medium for a multitude of passages (in particular 3)

[0362] 3) followed by returning to the normal cell culture medium for a second time period, in particular until the end of three weeks after fusion

[0363] 4) primary screening of cell culture supernatants for antigen-specific IgG antibodies

[0364] 5) propagating microcultures of cells that tested positive in 4)

[0365] 6) retesting cell culture supernatants of microcultures for antigen-specific IgG antibodies

[0366] 7) cloning and re-cloning cultures that tested positive in 6), using the limiting-dilution technique

[0367] 8) optionally determining the isotypes of clones obtained from 7)

[0368] 9) optionally purifying antibodies via Protein A

[0369] Phage Display

[0370] In a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) isolating at least one antibody having affinity to an antigen from an antibody gene library; ii) generating at least one cell strain expressing said at least one antibody; T75369WO BOEHMERT & BOEHMERT iii) isolating the at least one antibody from a culture of the at least one cell strain obtained in step ii).

[0371] In the embodiments of the present invention, an antibody having affinity to an antigen, in particular to the DPP3 antigens as detailed herein, is in particular an antibody with a binding affinity of at least 107M’1, preferred 108M’1, more preferred affinity is greater than 109M’1, most preferred greater than IO10M'1. A person skilled in the art knows that it may be considered to compensate lower affinity by applying a higher dose of compounds and this measure would not lead out-of-the-scope of the invention.

[0372] In a certain embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by isolating at least one antibody from a culture derived from at least one cell strain which expressed at least one antibody having affinity to an antigen from an antibody gene library.

[0373] In one embodiment, the antigen comprises a peptide as described herein in Table 4, which in certain embodiments may be bound to a solid phase.

[0374] In certain embodiments of the method for obtaining an antibody, in step i) the antibody gene library is a naive antibody gene library, particularly a human naive antibody gene library, more particularly in said library the antibodies are presented via phage display, i.e. on phages comprising a nucleotide sequence encoding for such respective antibody; more particularly the library HAL 7, HAL 8, or HAL 9, more particularly a library comprising the human naive antibody gene libraries HAL7 / 8 or, in further embodiments an equivalent library thereof.

[0375] In certain embodiments of the method for obtaining an antibody, in step i) screening comprises the use of an antigen, particularly an antigen containing a tag, more particularly a biotin tag, linked thereto via two different spacers. In particular embodiments, such panning strategy includes a mix of panning rounds with non-specifically bound antigen and antigen bound specifically via the tag, in the case of a biotin tag, bound to streptavidin. In this way, the background of non-specific binders may be minimized.

[0376] In certain embodiments of the method for obtaining an antibody, in step i), in embodiments wherein the library is a phage display library, the antibody is isolated by isolating a phage presenting said antibody (and comprising a nucleotide sequence encoding for the antibody). T75369WO BOEHMERT & BOEHMERT

[0377] In certain embodiments of the method for obtaining an antibody, in step ii) said cell strain is generated via introduction of a nucleotide sequence encoding for the antibody), in embodiments wherein the library in step i) is a phage display library, the isolated phage from step i) may be used to produce a bacterial strain, e.g. an Escherichia coli (E. coli) strain, expressing the antibody.

[0378] In certain embodiments of the method for obtaining an antibody, in step iv); in embodiments wherein the library in step i) is a phage display library and wherein a bacterial strain is produced in step ii), antibody may be isolated from the supernatant of the culture.

[0379] It is understood that, as used in describing the methods for obtaining an antibody, the term “one antibody” in the expression “at least one antibody” in particular may include more than one antibody molecule of antibodies having the same amino acid sequence. This understanding applies, mutatis mutandis, to the term “one cell strain”.

[0380] In certain embodiments of the method for obtaining an antibody, more than one antibody (referring to a multitude of antibodies having distinct amino acid sequences, respectively) is isolated in step i) and accordingly more than one cell strain is generated in step ii). Such method may involve the selection of clones that are positive for binding to the antigen, e.g. via a binding assay, e.g. an ELISA assay involving the antigen, and cells positive for binding to the antigen may be isolated to produce monoclonal cell strains.

[0381] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) isolating at least one antibody having affinity to an antigen from an antibody gene library comprising the human naive antibody gene libraries HAL7 / 8, by eluting phages carrying said antibody from the library; ii) generating at least one bacterial strain, e.g., E. coli cell strain, expressing said at least one antibody; iii) isolating the at least one antibody from the supernatant a culture of the at least one E. coli cell strain obtained in step ii).

[0382] In a further aspect, an antibody fragment according to the present invention is produced by a method involving enzymatic digestion of an antibody. T75369WO BOEHMERT & BOEHMERT

[0383] In certain embodiments, this method produces e.g. Fab or F(ab)2 antibody fragments. In certain embodiments, this method involves digestion with pepsin or papain, which are optionally immobilized on a surface.

[0384] In certain embodiments, antibodies may be humanized by CDR-grafting, in particular by a process involving the steps: extracting RNA from hybridomas expressing an antibody of interest (e.g. obtained by a method as described herein); amplifying said extracted RNA via RT-PCR, in particular with primer sets specific for the heavy and light chains of the antibody of interest, to obtain a DNA product; further amplifying said DNA product via PCR, in particular using semi-nested primer sets specific for antibody variable regions; determining the sequence of the DNA product; aligning said sequence with homologous human framework sequences to determine a humanized sequence for the variable heavy chain and the variable light chain sequences (of the desired antibody).

[0385] In certain embodiments, antibodies may be humanized by aligning the sequence of a DNA product that was obtained by amplifying RNA extracted from hybridomas expressing an antibody of interest via RT-PCR, in particular with primer sets specific for the heavy and light chains of the antibody of interest and further amplifying the DNA obtained therefrom via PCR, in particular using semi-nested primer sets specific for antibody variable regions, with homologous human framework sequences to determine a humanized sequence for the variable heavy chain and the variable light chain sequences (of the desired antibody).

[0386] In certain embodiments, antibodies may be humanized by determining the complementary determining regions (CDR), which may be accomplished by analysing the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen;

[0387] - transplanting said CDR sequences into a human framework region.

[0388] In certain embodiments, antibodies may be humanized by transplanting CDR sequences, which may preferably have been determined by analysing the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen, into a human framework region. T75369WO BOEHMERT & BOEHMERT

[0389] In certain embodiments variations in the amino acid sequence of the CDRs or FRs may be introduced to maintain structural interactions with the antigen (which may otherwise be abolished by introducing the human FR sequences), for instance by a random approach using phage display libraries or via directed approach guided by molecular modelling.

[0390] The DNA sequences encoding for antibodies determined as detailed herein can be transferred by known genetic engineering techniques into cells and used for production of the antibody.

[0391] More particularly, the DNA sequences encoding for antibodies determined as detailed herein can be transferred by known recombinant genetic engineering techniques into expression vectors and introduced into cells (e.g., via transfection or transformation) and used for production of the antibody, whereby the cells are the production cells.

[0392] Producing antibodies

[0393] In a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by a method comprising: culturing a cell strain comprising a nucleotide sequence encoding for the antibody; isolating the antibody from said culture.

[0394] In a further certain aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by isolating the antibody from a culture of a cell strain comprising a nucleotide sequence encoding for said antibody.

[0395] In certain embodiments of said method, the cell strain is produced as described herein above and may comprise bacterial cells, such as gram-negative bacteria, e.g. E. coh, Proteus mirabilis, o Pseudomonas putidas, gram-positive bacteria, e.g. Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacilli such as Lactobacillus zeae / casei or Lactobacillus paracasei, or Streptomyces, such as Streptomyces lividans eucariotic cells such as yeast, e.g. Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica, fugi, such as filamentous fungi, e.g. of the genus Trichoderma of Aspergillus, such as A. niger (e.g. subgenus A. awamori) and Aspergillus oryzae, Trichoderma reesei, Chrysosporium, such as C. luckno ense protozoae, such as Leishmania, e.g. L. larenlolae: insect cells, such as insect cells transfected a Baculovirus, e.g. AcNPV, such as insect cell lines from Spodoptera frugiperda, e.g. Sf-9 or Sf-21, Drosophila melanogaster, e.g. DS2, or Trichopulsia ni, e.g. High Five cells (BTI-TN-5B1-4); mammalian cells such as hamster, e.g. Chinese hamster ovary such as K1-, T75369WO BOEHMERT & BOEHMERT

[0396] DukX Bl 1-, DG44, Led 3, or BHK, mouse, e.g. mouse myeloma such as NSO, Homo sapiens, e.g. Per.C6, AGE1.HN, HEK293.

[0397] In certain embodiments of said method, the cells may be hybridoma cells, e.g. as described herein.

[0398] In a further aspect, procizumab (“PCZ”) is a monoclonal antibody obtainable by methods known in the art, or methods described herein. PCZ comprises two heavy chain sequences comprising SEQ ID NO: 68 and two light chain sequences comprising SEQ ID NO: 69. The respective SEQ ID NO: 11 for the heavy chain and SEQ ID NO: 12 for the light chain contain their respective N-terminal signal peptide sequences that can be used to manufacture the PCZ. The signal peptide sequence for the heavy chain is SEQ ID NO: 70 (MDPKGSLSWRILLFLSLAFELSYG) and for the light chain is SEQ ID NO: 71 (METDTLLLWVLLLWVPGSTG). As the PCZ is produced by the cell, the signal peptide sequence is cleaved off by cell before the protein is secreted. Culturing cells that express PCZ may be used to manufacture the protein at various scales. PCZ may be generated, for example, by any of the methods disclosed above used in the development and identification of the antibody. The DNA encoding the generated PCZ may be isolated and identified, for example, by DNA sequencing of the gene encoding the antibody against DPP3. Recombinant DNA technology well-known in the art may be used to manipulate the DNA. For example, the DNA encoding PCZ may be modified to make PCZ fragments and / or full-length PCZ to be combined with, for example, expression plasmid DNA. Further, the DNA can be codon optimized. The expression plasmid DNA encoding the PCZ or a fragment thereof may then be introduced, for example, by transfecting or in alternative embodiments transforming, into cells used for culturing and expressing the PCZ or a fragment thereof. The culturing cells expressing the PCZ or a fragment thereof may be used to manufacture the PCZ or a fragment thereof at various scales. The culturing cells may be, for example, Chinese Hamster Ovary (CHO) cells (including strains K1-, DukX B 11-, DG44, Lecl3), Human Embryonic Kidney 293 (HEK293) cells, baby hamster kidney (BHK21) cells, murine myeloma cells (NSO and Sp2 / 0), Vero cells, MRC-5 cells, PerC.6 cells, AGE1.HN, or any other cells known in the art that are used to culture therapeutic proteins for manufacturing at various scales. Other non-mammalian cell lines or other cells may be used for culturing PCZ or a fragment thereof, such as, S. cerevisiae and E. coli and the others described above. The post translational modification of the protein should be taken into account for the choice of the cells for culturing. T75369WO BOEHMERT & BOEHMERT

[0399] In certain embodiments of said method, culturing may take place in a static suspension culture, an agitated suspension culture, a membrane-based culture, a matrix-based culture or a high cell density bioreactor; a vessel for such culturing may be selected from the group comprising a T- flask, a roller culture, a spinner culture, a stirred tank bioreactor, an airlift bioreactor, a static membrane-based or matrix-based culture system, a suspension bioreactor, a fluidized bed bioreactor, a ceramic bioreactor, a perfusion system, a hollow fiber bioreactor.

[0400] In certain embodiments of said method, the cells may be immobilized on a matrix.

[0401] A high cell density bioreactor is in particular a culture system capable of generating cell densities greater than 108cells / ml.

[0402] In a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by a method comprising: generating a transgenic plant or animal comprising a nucleotide sequence encoding for the antibody; isolating the antibody from said plant or animal or a secretion or product of said plant or animal.

[0403] In a certain further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by isolating the antibody from a transgenic plant or transgenic animal or a secretion or product of a transgenic plant or transgenic animal having a nucleotide sequence encoding for the antibody.

[0404] Said animal may e.g. be selected from a chicken, a mouse, a rat, a rabbit, a cow, a goat, a sheep, a pig; said secretion or product may e.g. be milk or an egg. Said plant may e.g. be selected from tobacco (N. tabacum or N. benthamiana), duckweed (Lemna minor), Chlamydomonas reinhardtii, rice, Arabidopsis thaliana, alfalfa (Medicago sativa), lettuce, maize.

[0405] The antibodies can in certain embodiments be isolated by physicochemical fractionation, e.g. size exclusion chromatography, precipitation, e.g. using ammonium sulphate, ion exchange chromatography, immobilized metal chelate chromatography gel filtration, zone electrophoresis; based on their classification e.g. binding to bacterial proteins A, G, or L, jacalin; antigen-specific affinity purification via immobilized ligands / antigens; if necessary, low molecular weight components can be removed by methods like dialysis, desalting, and diafiltration. T75369WO BOEHMERT & BOEHMERT

[0406] In some embodiments the antibody is encoded by a nucleotide sequence where the nucleotide sequence is a reverse transcription of an amino acid sequence from an antibody produced by one of the processes described herein.

[0407] A person of ordinary skill in the art would appreciate that the biosimilarity of an antibody to an anti-DPP3 antibody or fragment thereof claimed herein can be determined by, for example, following the U.S. FDA’s Guidance “Questions and Answers on Biosimilar Development and the BPCI Act” dated September 2021, incorporated in full herein.

[0408] In a specific embodiment the level of Pro- Adrenomedullin or a fragment thereof are measured with an immunoassay using antibodies or fragments of antibodies binding to Pro- Adrenomedullin or fragments thereof.

[0409] The above-mentioned threshold values might show a certain amount of variation, depending on the assay used to determine the level of the respective pro- ADM fragment, if these have been calibrated differently from the assay systems used in the present invention. Therefore, the above-mentioned thresholds shall apply in particular for the assay systems used in the present invention, and shall apply for such differently calibrated assays accordingly, taking into account the differences in calibration. One possibility of quantifying the difference in calibration is a method comparison analysis (correlation) of the assay in question (e.g. assay for measurement of proADM or fragments thereof) with the respective biomarker assay used in the present invention by measuring the respective biomarker (pro-ADM or a fragment thereof) in samples using both assays. Assuming a linear correlation between the assays, another possibility is to determine with the assay in question, given this test has sufficient analytical sensitivity, the median biomarker level of a representative normal population, compare results with the median biomarker levels as described in the literature (e.g. bio-ADM: Weber et al. 2017. J Appl Lab Med 2(2): 222-233; MR-proADM: Smith et al. 2009. Clin Chem 55:1593-1595; PAMP-Gly: Hashida et al. 2004. Clinical Biochemistry 37 14 21; mature PAMP: Hashida et al. 2004. Clinical Biochemistry 37 14 21) and recalculate the calibration based on the difference obtained by this comparison, e.g. by applying a factor. Hence, the x-fold of e.g. the mean or median (or a specific percentile) of the level of Pro- Adrenomedullin or a fragment thereof in a healthy population can be used as a threshold level using a differently calibrated assay, taking into account the above precautions.

[0410] Methods to quantify fragments derived from proADM have been described, e.g. the measurement of mature ADM (Weber et al. 2017. J Appl Lab Med 2(2): 222-233), MR- T75369WO BOEHMERT & BOEHMERT proADM (Morgenthaler et al. 2005. Clin Chem 51710): 1823-9}, mature PAMP (Hashida et al. 2004. Clinical Biochemistry 37 14- 21;), PAMP-Gly (Hashida et al. 2004. Clinical Biochemistry 37 14 21: } and CT-proADM (EP 2 111 552}. For example, a commercial homogeneous time-resolved fluoroimmunoassay for the measurement of MR-proADM in plasma on a fully automated system is available (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) (Caruhel etal. 2009. Clin Biochem 42(7-8) :725-8).

[0411] In certain embodiments the level of MR-proADM is determined by an assay comprising incubating a sample with a) a solution comprising a first anti -MR-proADM antibody binding specifically to a first region of MR-proADM and, wherein said first antibody is labelled, e.g. with a fluorophore, e.g. Cy5, and b) a solution comprising a second anti -MR-proADM antibody, binding specifically to a second region of MR-proADM, wherein said second antibody is labelled, e.g. with a fluorescent donor, e.g. a rare earth cryptate moiety; and subsequently measuring a signal, e.g. fluorescence emitted by the label on the first antibody, to determine the amount of bound first antibody and thus the amount of MR-proADM in said sample.

[0412] In other certain embodiments the level of MR-proADM is determined by a sandwich immunoassay, wherein an anti-MR-proADM antibody is immobilized on a surface, a liquid comprising MR-proADM and a second, labeled anti-MR-proADM antibody is added, and after a washing step, the binding of the second antibody is measured based on detection of the label, e.g. by determining chemiluminescence.

[0413] In certain embodiments the level of_CT-proADM is determined by a sandwich immunoassay, wherein an anti-CT-proADM antibody is immobilized on a surface, a liquid comprising CT- proADM and a second, labeled anti-CT-proADM antibody is added, and after a washing step, the binding of the second antibody is measured based on detection of the label, e.g. by determining chemiluminescence.

[0414] The plasma median concentration of mature ADM (bio- ADM) in a normal (healthy) population was 13.7 pg / ml, the lowest value 11 pg / ml and the 99thpercentile 43 pg / ml (Weber et al. 2017. J Appl Lab Med 2(2): 222-233}.

[0415] The plasma median MR-proADM concentration in normal (healthy) subjects was 0.41 (interquartile range 0.23 - 0.64) nmol / L (Smith etal. 2009. Clin Chem 55: 1593-1595} using the automated sandwich fluorescence assay for the detection of MR-proADM as described in Caruhel et al. (Caruhel et al. 2009. Clin Biochem 42: 725-8}. T75369WO BOEHMERT & BOEHMERT

[0416] The plasma median concentration of CT-proADM in normal healthy subjects (n=200) was 77.6 pmol / L (min 46.6 pmol / L, max 136.2 pmol / L) and the 95% percentile was 113.8 pmol / L (EP 2 111 552 Bl}.

[0417] The plasma mean concentration of PAMP-Gly in normal healthy subjects (n=51) was 1.15 pmol / L + / - 0.38 pmol / L (Hashida et al. 2004. Clinical Biochemistry 37 : 14- 21}.

[0418] The plasma mean concentration of mature PAMP in normal healthy subjects (n=51) was 0.51 pmol / L + / - 0.19 pmol / L (Hashida et al. 2004. Clinical Biochemistry 37 : 14- 21}.

[0419] The threshold level is a level, which allows for allocating the patient into a group of patients who have been diagnosed and / or are having an increased risk of an adverse event (e.g., mortality), or into a group of patients who have not been diagnosed and / or have a decreased risk of an adverse event, or into a severity group. Thus, the threshold level shall allow for differentiating between a patient who is diagnosed and / or is having an increased risk of an adverse event and a patient who is not diagnosed and / or is having a decreased risk of an adverse event.

[0420] It is known in the art how threshold levels can be determined. Threshold levels are predetermined values and are set to meet routine requirements in terms of, e.g., specificity and / or sensitivity. These requirements can vary. It may for example be that sensitivity or specificity, respectively, has to be set to certain limits, e.g., 80%, 90%, 95% or 98%, respectively.

[0421] The sensitivity and specificity of a diagnostic and / or prognostic test depends on more than just the analytical "quality" of the test, they also depend on the definition of what constitutes an abnormal result. In practice, Receiver Operating Characteristic curves (ROC curves), are typically calculated by plotting the value of a variable versus its relative frequency in "reference group" (i.e. patients who do not have the disease) and "disease" populations (i.e. patients who have the disease). For any particular marker, a distribution of marker levels for patients with and without disease will likely overlap. Under such conditions, a test does not absolutely distinguish patients with and without disease (e.g., endothelial dysfunction) with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the T75369WO BOEHMERT & BOEHMERT perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results do not necessarily give an accurate number. As long as one can rank results, one can create a ROC curve. For example, results of a test on "disease" samples might be ranked according to degree (e.g. l=low, 2=normal, and 3=high). This ranking can be correlated to results in the "reference" group, and a ROC curve created. These methods are well known in the art (See, e.g., Hanley et al.1982. Radiology 143: 29-36). Preferably, ROC curves result in an Area under the ROC curve (AUC) of greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. The term "about" in this context refers to + / - 5% of a given measurement.

[0422] The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off threshold selected, the value of (1 - specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

[0423] In addition to the normal range, other methods may be used to determine thresholds for a specific indication, depending on the intended use and application / clinical setting. Such methods include e.g. the Youden optimum, thresholds that maximize overall accuracy, the odds ratio, or the positive or negative predictive value. In some situations, thresholds achieving a pre-specified level of sensitivity or specificity (e.g. 80%, 90%, 95% or 99%) can be appropriate for the clinical application. The choice of methods depends on the clinical application, which weights the costs of false positive and false negative results based on the test result consequences for the patient and the health care system, as well as the clinical need. Finally, multiple approaches may be combined to define a consensus threshold.

[0424] A“reference group” may be a healthy population, e.g., with no signs and symptoms of a disease. A reference group may consist of more than one reference subjects.

[0425] Particular threshold values are for instance the 90th, 95th, or 99th percentile of a reference group (e.g. healthy population). By using a higher percentile than the 75th percentile, one reduces the number of false positive subjects identified, but one might miss to identify subjects, who are at moderate, albeit still increased risk. Thus, one might adopt the threshold value T75369WO BOEHMERT & BOEHMERT depending on whether it is considered more appropriate to identify most of the subjects at risk at the expense of also identifying "false positives", or whether it is considered more appropriate to identify mainly the subjects at high risk at the expense of missing several subjects at moderate risk.

[0426] For example, the 75thpercentile, more particular the 90thpercentile, even more particular a 95thpercentile, most particular the 99thpercentile values can be used for the upper limits of the normal range.

[0427] Biomarkers can also be used for the prediction of a future event or risk (e.g. prediction of mortality risk). For time-to-event data threshold levels can be obtained for instance from a Kaplan-Meier analysis, where the occurrence of a disease is correlated with e.g. the tertiles, quartiles, quintiles of the markers (e.g. endothelial function markers) in the population. There are also equivalent methods available to the ROC methods described before, based on i.e. timedependent ROC analysis or generalizations of the area under the ROC curve (C index).

[0428] The threshold level may vary depending on various physiological parameters such as age, gender, or sub-population, as well as on the means used for the determination of Pro- Adrenomedullin and fragments thereof referred to herein.

[0429] In another specific embodiment of the present invention said level of Pro- Adrenomedullin or a fragment thereof is determined more than once in different samples taken from said patient at different time-points. In another specific embodiment of the present invention the difference between said level of Pro- Adrenomedullin or a fragment thereof in different samples taken from said patient at different time-points is determined. The difference may be determined as absolute or relative difference.

[0430] In certain embodiment of the present invention said level of Pro- Adrenomedullin or a fragment thereof is determined at least twice.

[0431] In another specific embodiment of the present invention said at least second determination of the level of Pro- Adrenomedullin or a fragment thereof is conducted within 2, 4, 6, or 12 hours of the first determination, or alternatively within 24 or 48 hours.

[0432] Bio- ADM levels may be determined using an immunoassay as described in Weber et al. 2017 (Weber et al. 2017. J ALM 2(2): 222-233). T75369WO BOEHMERT & BOEHMERT

[0433] In certain embodiments of the present invention the level of pro-Adrenomedullin or fragment thereof is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to pro-Adrenomedullin or fragment thereof.

[0434] In certain embodiments of the present invention the determination of the level of pro- Adrenomedullin or fragment thereof comprises the use of a capture-binder that binds specifically to pro-Adrenomedullin or fragment thereof wherein said capture-binder may be selected from the group comprising an antibody, an antibody fragment, and a non-IgG scaffold.

[0435] In certain embodiments of the present invention the level of pro-Adrenomedullin or fragment thereof is determined in a bodily fluid sample of said subject and wherein said determination comprises the use of a capture-binder that binds specifically to pro-Adrenomedullin or fragment thereof and wherein said capture-binder is an antibody.

[0436] In certain embodiments of the present invention the level of pro-Adrenomedullin or a fragment thereof is determined in a bodily fluid sample of said subject and wherein said determination comprises the use of a capture-binder that binds specifically to pro-Adrenomedullin or a fragment thereof, wherein said capture-binder is immobilized on a surface.

[0437] The level of pro-Adrenomedullin or fragments thereof may be measured an immunoassay, wherein said immunoassay maybe a sandwich immunoassay, preferably a fully automated assay.

[0438] In one embodiment the analytical assay sensitivity of said assay for ADM-Gly is able to quantify ADM-Gly of healthy subjects and is 20 pg / ml, preferably 15 pg / ml and more preferably 10 pg / ml.

[0439] In one embodiment the analytical assay sensitivity of said assay for ADM-Gly is able to quantify ADM-Gly of healthy subjects and is a 0.7-fold, preferably a 0.6-fold and more preferably 0.4-fold of the median of a healthy population.

[0440] In one embodiment the analytical assay sensitivity of said assay for PAMP is able to quantify PAMP of healthy subjects and is < 0.5 pmol / L, preferably < 0.25 pmol / L and more preferably < 0.1 pmol / L.

[0441] In one embodiment the analytical assay sensitivity of said assay for PAMP is able to quantify PAMP of healthy subjects and is a 1.0-fold, preferably a 0.5-fold and more preferably a 0.2-fold of the mean of a healthy population. T75369WO BOEHMERT & BOEHMERT

[0442] In one embodiment the analytical assay sensitivity of said assay for the detection of CT- proADM is able to quantify CT-proADM of healthy subjects and is < 100 pmol / L, preferably

[0443] < 75 pmol / L and more preferably < 50 pmol / L.

[0444] In one embodiment the analytical assay sensitivity of said assay for the detection of CT- proADM is able to quantify CT-proADM of healthy subjects and is a 1.3-fold, preferably a 1.0- fold and more preferably a 0.6-fold of the median of a healthy population.

[0445] In one embodiment the analytical assay sensitivity of said assay for the detection of mature ADM is able to quantify mature of healthy subjects and is < 40 pg / ml, preferably < 25 pg / ml and more preferably < 10 pg / ml.

[0446] In one embodiment the analytical assay sensitivity of said assay for the detection of mature ADMis able to quantify mature of healthy subjects and is a 1.6-fold, preferably a 1.0-fold and more preferably a 0.4-fold of the median of a healthy population.

[0447] In one embodiment the analytical assay sensitivity of said assay is able to quantify MR- proADM of healthy subjects and is < 0.5 nmol / L, preferably < 0.4 nmol / L and more preferably

[0448] < 0.2 nmol / L.

[0449] In one embodiment the analytical assay sensitivity of said assay is able to quantify MR- proADM of healthy subjects and is a 1.2-fold, preferably a 1.0-fold and more preferably a 0.5- fold of the median of a healthy population.

[0450] Pharmaceutical compositions

[0451] As used herein, “pharmaceutical formulation” and “pharmaceutical composition” are used interchangeably.

[0452] Further subject matter of the present invention is a pharmaceutical composition for use in the treatment of a patient in need thereof as detailed in the embodiments of the invention, said composition comprising a DPP3 binder as detailed in the embodiments of the invention.

[0453] In certain embodiments of the invention relating to a pharmaceutical composition for use in the treatment of a patient in need thereof, the level of Pro-Adrenomedullin or fragments thereof in a sample of bodily fluid obtained from said patient is above a threshold.

[0454] In certain embodiments, said pharmaceutical formulation is a solution, preferably a ready-to- use solution. T75369WO BOEHMERT & BOEHMERT

[0455] In certain embodiments, said pharmaceutical formulation is in a freeze-dried state.

[0456] In certain embodiments, said pharmaceutical formulation is to be administered intra-muscular.

[0457] In certain embodiments, said pharmaceutical formulation is to be administered intra-vascular.

[0458] In certain embodiments, said pharmaceutical formulation is to be administered via infusion.

[0459] In certain embodiments, said pharmaceutical formulation is to be administered systemically.

[0460] In one embodiment, the anti-DPP3 antibody is contained in a pharmaceutical formulation. In one embodiment, the anti-DPP3 antibody pharmaceutical formulation is a concentrated solution (i.e., to be diluted prior to administration) for infusion and contains the anti-DPP3 antibody as an active substance together with one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are those that are compatible with the anti-DPP3 antibody. The pharmaceutically acceptable excipients for use with the anti-DPP3 antibody include stabilizers, antioxidants, tonicity modifiers, and buffers. Suitable stabilizers include sugars, sugar alcohols, amino acids, surfactants, polyols, and stabilizing proteins. Examples of sugar stabilizers are sucrose, glucose, lactose, trehalose, and maltose; examples of sugar alcohol stabilizers include mannitol, xylitol, and sorbitol; examples of amino acid stabilizers include arginine, histidine, glycine, proline, alanine, lysine, leucine, and methionine; and examples of surfactant stabilizers include Polysorbate 20 and Polysorbate 80 (i.e., Tween 20 and Tween 80, respectively), Poloxamers (i.e., poloxamer 188 and Pluronic F68, 127), and PEGylated surfactants; examples of stabilizing polyols include glycerol, polyethylene glycol (PEG, low MW) and examples of stabilizing proteins include transferrin, human serum albumin, and bovine serum albumin. Antioxidants useful in an anti-DPP3 concentrated solution include methionine, ascorbic acid, reduced glutathione, cysteine, Thio glycerol, sodium thiosulfate, platinum, N-acetyl-DL-tryptophan, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Examples of suitable tonicity modifiers include sodium chloride, potassium chloride, calcium chloride, mannitol, sorbitol, and glycerol. Buffers suitable in a concentrated solution include histidine, citrate, phosphate (e.g., phosphate- buffered saline), acetate, succinate, MES, HEPES, and Tris (e.g. tris-buffered saline). Examples of suitable pHs are in the range of 4.8 to 8.0, preferred in the range of 5.0 to 6.5, or more preferred in the range of 5.5 to 6.8; or at a pH of about 5.0, about 5.5, about 6.0, or about 6.5; preferred at a pH of 6.5. In one embodiment the anti-DPP3 antibody is lyophilized and is formulated with bulking agents (i.e., lyoprotectants) and suitable bulking agents include T75369WO BOEHMERT & BOEHMERT sucrose, trehalose, mannitol, glycine, and dextran. The excipients can serve multiple purposes within a single formulation and are not limited to a single function. Multiple excipients can be included in the formulation to fulfill similar purposes. In one embodiment, the anti-DPP3 antibody is formulated with 10 mM Histidine-HCl, 250 mM Trehalose, and 20 mM L- Methionine pH 6.5 buffer.

[0461] In one embodiment, the anti-DPP3 antibody pharmaceutical formulation may be stored at 2-8 °C. Before use, the vials of the concentrated solution are equilibrated to room temperature and then diluted with 0.9% saline , water for injection (WFI), phosphate buffered with saline (PBS), or WFI formulated with a tonicity modifier(s) (e.g., sodium chloride, potassium chloride, calcium chloride, mannitol, sorbitol, and glycerol) prior to infusion. In one embodiment the diluent has the same or equivalent excipient composition as is used for the concentrated pharmaceutical form, albeit, without the anti-DPP3 antibody. In another embodiment the diluent has the same or equivalent excipient composition and concentrations of the excipients as used for the concentrated pharmaceutical form, albeit, without the anti-DPP3 antibody for infusion.

[0462] In particular embodiments of the present invention, said pharmaceutical composition is formulated to be administered or is administered orally, epicutaneously, subcutaneously, intradermally, sublingually, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. orally)” likewise reads on “formulated for (e.g. oral) administration”.

[0463] In the most preferred embodiments of the present invention, said pharmaceutical composition is formulated to be administered or is administered, subcutaneously, intradermally, intramuscularly, intraarterially or intravenously, or via intraperitoneal administration.

[0464] According to the present invention the administered dosage might be a single bolus injection delivering the described amount of the compounds to be administered or a continuous infusion of the compounds delivering the desired amount of compounds over a distinct period of time taking into account the velocity of infusion. Thereby the compounds might be administered as a combined injection and / or infusion or as several parallel injections and / or infusion, wherein T75369WO BOEHMERT & BOEHMERT one or all compounds are delivered as an injection and / or infusions or a distinct compound is injected and another distinct compound is infused.

[0465] Critically ill / diseases

[0466] In certain embodiments of the invention, the patient is a critically ill patient. “Critically ill” means in particular that said patient is suffering from an acute disease or acute condition which is life-threatening and in which death is possible or imminent. In in specific embodiments said critically ill patient is an ICU patient.

[0467] In one embodiment said patient is (i) a patient suffering from severe infection, sepsis, heart failure, chronic heart failure, acute heart failure, myocardial infarction, stroke, (ii) a patient with organ dysfunction or organ failure, (iii) a patient undergoing major surgery, (iv) a patient with trauma, (v) a patient with shock and / or a patient running into shock, or (vi) a patient with cancer optionally receiving an anti-cancer therapy.

[0468] Heart failure (HF) is a cardiac condition that occurs, when a problem with the structure or function of the heart impairs its ability to supply sufficient blood flow to meet the body's needs. It can cause a large variety of symptoms, particularly shortness of breath (SOB) at rest or during exercise, signs of fluid retention such as pulmonary congestion or ankle swelling and objective evidence of an abnormality of the structure or function of the heart at rest.

[0469] Heart failure is a clinical syndrome characterized by a constellation of symptoms and signs caused by cardiac dysfunction. It is one of the major causes of morbidity and mortality in the developed countries, with a prevalence of 1-2%. Heart failure can be grouped into chronic HF and acute HF. Patients with chronic HF can be grouped into stable chronic HF, worsening signs and symptoms of chronic HF and acute decompensation of chronic HF. Acute heart failure (AHF) is defined as a rapid onset of signs and symptoms of heart failure resulting in the need for urgent therapy or hospitalization. AHF can present as acute de novo HF (new onset of AHF in a patient without previous cardiac dysfunction) or acute decompensation of chronic HF.

[0470] Patients with chronic heart failure (HF) may include patients with worsening signs and symptoms of chronic heart failure and acute decompensation of chronic heart failure.

[0471] Chronic HF with worsening signs and symptoms is in particular characterized by:

[0472] (i) the presence of structural or functional failure of the heart that impairs its ability to supply sufficient blood flow to meet body's needs, T75369WO BOEHMERT & BOEHMERT

[0473] (ii) volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of cardiac output (manifested by hypotension, renal insufficiency and / or a shock syndrome) and whereas the patient is not in need of urgent therapy and does not require hospitalization, but is in need of therapy adjustment.

[0474] Chronic heart failure may also decompensate (termed acute decompensated heart failure or acute decompensated chronic heart failure), which is most commonly the result from an intercurrent illness (such as pneumonia), myocardial infarction, arrhythmias, uncontrolled hypertension or a patient's failure to maintain fluid restriction, diet or medication.

[0475] New onset acute HF and acute decompensated chronic HF are characterized by:

[0476] (i) the presence of structural or functional failure of the heart that impairs its ability to supply sufficient blood flow to meet body's needs,

[0477] (ii) volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of cardiac output (manifested by hypotension, renal insufficiency and / or a shock syndrome) and whereas the patient is in need of urgent therapy or therapy adjustment and does require hospitalization.

[0478] The above definitions of acute heart failure that either new-onset AHF or acute decompensated HF or acute decompensated chronic HF or worsening signs / symptoms of chronic heart failure are in line with Voors et al., European Journal of Heart Failure (2016), 18, 716 - 726.

[0479] In one embodiment of the invention said organ dysfunction is defined as cardiovascular dysfunction, respiratory dysfunction (lung), kidney dysfunction, liver dysfunction, central nervous system dysfunction or dysfunction of the coagulation system.

[0480] “Organ dysfunction” is a condition where an organ does not perform its expected function. Organ dysfunction in the context of the present invention includes a dysfunction of the respective organ to such a degree that normal homeostasis cannot be maintained without external clinical intervention, which may be termed “organ failure”. Said organ failure may pertain an organ selected from the group comprising kidney, liver, heart, lung, nervous system. “Multi-organ dysfunction” denotes an organ dysfunction of at least two organs.

[0481] By contrast, organ function represents the expected function of the respective organ within physiologic ranges. The person skilled in the art is aware of the respective function of an organ during medical examination. T75369WO BOEHMERT & BOEHMERT

[0482] In one embodiment of the invention said organ support is selected from kidney support, respiratory (lung) support, cardiovascular support and / or liver support.

[0483] In another embodiment respiratory (lung) support is defined as non-invasive, invasive mechanical ventilation or ECMO.

[0484] Mechanical ventilation or assisted ventilation is the medical term for artificial ventilation where mechanical means are used to assist or replace spontaneous breathing. In one embodiment mechanical ventilation involves a machine called a ventilator or the breathing may be assisted manually by a suitably qualified professional, such as an anesthesiologist, respiratory therapist, Registered Nurse, or paramedic, by compressing a bag valve mask device. Mechanical ventilation is termed "invasive" if it involves any instrument inside the trachea through the mouth. In one embodiment mechanical ventilation is invasive mechanical ventilation comprising an endotracheal tube or the skin, such as a tracheostomy tube. In a further embodiment mechanical ventilation is non-invasive ventilation comprising face or nasal masks. In a further embodiment non-invasive ventilation is used in appropriately selected conscious patients.

[0485] Extracorporeal membrane oxygenation (ECMO), also known as extracorporeal life support (ECLS), is an extracorporeal technique for providing prolonged cardiac and respiratory support to persons whose heart and lungs are unable to provide an adequate amount of gas exchange or perfusion to sustain life. The technology for ECMO is largely derived from cardiopulmonary bypass, which provides shorter-term support with arrested native circulation. ECMO works by removing blood from the person's body and artificially removing carbon dioxide from, and adding oxygen to, the patient's red blood cells.

[0486] Pulmonary function is assessed, particularly in patients under invasive ventilation in critical care, with the Horowitz index (synonyms: oxygenation after Horowitz, Horowitz coefficient, P / F ratio). It is useful for evaluating the extent of damage to the lungs. The Horowitz index (PaO2 / FiO2 ratio) is a well-known marker of acute pulmonary injury and predicts mortality in patients with acute respiratory failure. In one embodiment, the Horowitz index is defined as the ratio of partial pressure of oxygen in blood (PaO2), in millimeters of mercury, and the fraction of oxygen in the inhaled air (FiO2) - the PaO2 / FiO2 ratio. In healthy lungs the Horowitz index depends on age and usually falls between 350 and 500. In an embodiment of the present invention, the Horowitz index has a value below 300, wherein a Horowitz index of 300 is the threshold for mild lung injury, wherein a Horowitz index of 200 is of the threshold for a T75369WO BOEHMERT & BOEHMERT moderately severe lung injury and wherein a Horowitz index of below 100 is the threshold for a severe injury.

[0487] In one embodiment said requirement of organ support is respiratory (lung) support, wherein requirement for respiratory (lung) support by non-invasive and invasive mechanical ventilation is defined by a Horovitz Index (P / F ratio) of 200 or below, preferably 150 or below, preferred 100 or below.

[0488] Kidney support is defined as renal replacement therapy (RRT).

[0489] Renal replacement therapy (RRT) replaces the normal blood-filtering function of the kidneys andrelates to a therapy that is employed to replace the normal blood-filtering function of the kidneys. Renal replacement therapy may refer to dialysis (e.g. hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration. Such techniques are various ways of diverting the blood into a machine, cleaning it, and then returning it to the body. The hemodialysis, hemofiltration, and hemodiafiltration may be continuous or intermittent and can use an arteriovenous route (in which blood leaves from an artery and returns via a vein) or a venovenous route (in which blood leaves from a vein and returns via a vein). This results in various types of RRT. For example, the renal replacement therapy may be selected from the group of, but not limited to continuous renal replacement therapy (CRRT), continuous hemodialysis (CHD), continuous arteriovenous hemodialysis (CAVHD), continuous venovenous hemodialysis (CVVHD), continuous hemofiltration (CHF), continuous arteriovenous hemofiltration (CAVH or CAVHF), continuous venovenous hemofiltration (CVVH or CVVHF), continuous hemodiafiltration (CHDF), continuous arteriovenous hemodiafiltration (CAVHDF), continuous venovenous hemodiafiltration (CVVHDF), intermittent renal replacement therapy (IRRT), intermittent hemodialysis (H D), intermittent venovenous hemodialysis (IVVHD), intermittent hemofiltration (H F), intermittent venovenous hemofiltration (IVVH or IVVHF), intermittent hemodiafiltration (IHDF) and intermittent venovenous hemodiafiltration (IVVHDF).

[0490] In a specific embodiment of the invention renal replacement therapy is selected from the group comprising dialysis (hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration.

[0491] From the definition of the cardiovascular component of the SOFA Score it can be delineated that pharmacological support of the cardiovascular system is required, if the mean arterial pressure decreases below 70 mm Hg. Support is given by administration of vasoactive drugs T75369WO BOEHMERT & BOEHMERT such as dopamine, epinephrine or norepinephrine. In severe cardiovascular dysfunction, doses given are dopamine > 15 pg / kg / min OR epinephrine > 0.1 pg / kg / min OR norepinephrine > 0.1 pg / kg / min.

[0492] In one embodiment said requirement of organ support is support of the cardiovascular system, wherein said support of the cardiovascular system is selected from vasoactive drugs such as dopamine, epinephrine, norepinephrine, a norepinephrine equivalent, phenylephrine and vasopressin.

[0493] Declining liver function can be described by increasing blood concentrations of bilirubin. In severe liver dysfunction, bilirubin can exceed 12 mg / dL. Extracorporeal Liver Support (ECLS) is a system that performs the essential functions of the liver via filtration and detoxification of blood within an external device with the end goal of reducing mortality or to bridge a patient to liver transplant. In some cases, ECLS can temporarily mitigate sequelae of liver failure such as hepatic encephalopathy and multi-organ failure long enough for the native liver to recover function on its own. The two main modalities of ECLS are divided into artificial (MARS, Prometheus, SPAD and HepaWash) and bio-artificial categories (HepatAssist and ELAD) (Nguyen et al. 2022).

[0494] In another embodiment said patient is an intensive care unit (ICU) patient. The term “ICU- patient” relates, without limitation, to a patient who has been admitted to an intensive care unit. An intensive care unit, which can also be termed an intensive therapy unit or intensive treatment unit (ITU) or critical care unit (CCU), is a special department of a hospital or health care facility that provides intensive treatment medicine. ICU-patients usually suffer from severe and lifethreatening illnesses and injuries, which require constant, close monitoring and support from specialist equipment and / or medications in order to ensure normal bodily functions. Common conditions that are treated within ICUs include, without limitation, acute respiratory distress syndrome (ARDS), trauma, organ dysfunction or organ failure, sepsis and shock.

[0495] The severity of a disease may be defined as the extent of organ system derangement or physiologic decompensation for a patient. The severity may be classified into different stages using for example scoring systems.

[0496] Organ dysfunction may be defined by the sequential organ failure assessment score (SOFA- Score) or the components thereof. The SOFA score, previously known as the sepsis-related organ failure assessment score (Singer et al. 2016) is used to track a person's status during the T75369WO BOEHMERT & BOEHMERT stay in an intensive care unit (ICU) to determine the extent of a person's organ function or rate of failure. The score is based on six different scores, one each for the respiratory, cardiovascular, hepatic, coagulation, renal and neurological systems each scored from 0 to 4 with an increasing score reflecting worsening organ dysfunction. The criteria for assessment of the SOFA score are described for example in Lamden et al. (for_review see (Lambden et al. 2019). SOFA score may traditionally be calculated on admission to ICU and at each 24-h period that follows.

[0497] The quick SOFA Score (quickSOFA or qSOFA) was introduced by the Sepsis-3 group in February 2016 as a simplified version of the SOFA Score as an initial way to identify patients at high risk for poor outcome with an infection (Angus et al. 2016. Critical Care Medicine. 44 (3): ell3-el21). The qSOFA simplifies the SOFA score drastically by only including its 3 clinical criteria and by including "any altered mentation" instead of requiring a Glasgow Coma Scale (GCS) <15. qSOFA can easily and quickly be repeated serially on patients. The score ranges from 0 to 3 points. One point is given for: low blood pressure (SBP <100 mmHg), high respiratory rate ((> 22 breaths / min) and altered mentation (GCS < 15). The presence of 2 or more qSOFA points near the onset of infection was associated with a greater risk of death or prolonged intensive care unit stay. These are outcomes that are more common in infected patients who may be septic than those with uncomplicated infection. Based upon these findings, the Third International Consensus Definitions for Sepsis recommends qSOFA as a simple prompt to identify infected patients outside the ICU who are likely to be septic (Seymour et al. 2016. JAMA 315(8):762-774).

[0498] In one embodiment said trauma is trauma burn trauma, polytrauma.

[0499] In one embodiment said patient has a shock or is running into shock, wherein said shock is selected from the group comprising shock due to hypovolemia, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.

[0500] In one embodiment said patient has a shock and / or is running into shock, wherein

[0501] • in case of cardiogenic shock said patient may have suffered an acute coronary syndrome (e.g., acute myocardial infarction) or wherein said patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture or massive pulmonary embolism, or

[0502] • in case of hypovolemic shock said patient may have suffered a hemorrhagic disease including gastrointestinal bleed, trauma, vascular etiologies (e.g., ruptured abdominal T75369WO BOEHMERT & BOEHMERT aortic aneurysm, tumor eroding into a major blood vessel) and spontaneous bleeding in the setting of anticoagulant use or a non-hemorrhagic disease including vomiting, diarrhea, renal loss, skin losses / insensible losses (e.g., burns, heat stroke) or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma, or

[0503] • in case of obstructive shock said patient may have suffered a cardiac tamponade, tension pneumothorax, pulmonary embolism or aortic stenosis, or

[0504] • in case of distributive shock said patient may have septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.

[0505] In a preferred embodiment said patient is a patient suffering from heart failure, chronic heart failure, acute heart failure, myocardial infarction, stroke.

[0506] In another preferred embodiment said patient is a patient suffering from severe infection, sepsis, organ dysfunction or organ failure or shock.

[0507] In another preferred embodiment said patient is a patient undergoing major surgery or a patient with trauma.

[0508] In yet another preferred embodiment said patient is a patient with shock or a patient running into shock.

[0509] In yet another preferred embodiment said patient is a patient with cancer optionally receiving an anti-cancer therapy.

[0510] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection (see Singer, M., et al. (2016). The third international consensus definitions for sepsis and septic shock (Sepsis-3). Jama, 315(8), 801-810.). Organ dysfunction can be identified as an acute change in total SOFA score >2 points consequent to the infection. The baseline SOFA score can be assumed to be zero in patients not known to have preexisting organ dysfunction. A SOFA score >2 reflects an overall mortality risk of approximately 10% in a general hospital population with suspected infection. Even patients presenting with modest dysfunction can deteriorate further, emphasizing the seriousness of this condition and the need for prompt and appropriate intervention, if not already being instituted.

[0511] Sepsis is a life-threatening condition that arises when the body’s response to an infection injures its own tissues and organs. Patients with suspected infection who are likely to have a prolonged T75369WO BOEHMERT & BOEHMERT

[0512] ICU stay or to die in the hospital can be promptly identified at the bedside with qSOFA, i.e., alteration in mental status, systolic blood pressure <100 mm Hg, or respiratory rate >22 / min.

[0513] Shock is characterized by decreased oxygen delivery and / or increased oxygen consumption or inadequate oxygen utilization leading to cellular and tissue hypoxia. It is a life-threatening condition of circulatory failure and most commonly manifested as hypotension (systolic blood pressure less than 90 mm Hg or MAP less than 65 mmHg). Shock is divided into four main types based on the underlying cause: hypovolemic, cardiogenic, obstructive, and distributive shock (Vincent, J. L., & De Backer, D. (2013). Circulatory shock. New England Journal of Medicine, 369(18), 1726-1734).

[0514] Septic shock is a potentially fatal medical condition that occurs when sepsis, which is organ injury or damage in response to infection, leads to dangerously low blood pressure and abnormalities in cellular metabolism. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) defines septic shock as a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone. Patients with septic shock can be clinically identified by a vasopressor requirement to maintain a mean arterial pressure of 65 mm Hg or greater and serum lactate level greater than 2 mmol / L (>18 mg / dL) in the absence of hypovolemia. This combination is associated with hospital mortality rates greater than 40% (Singer et al. (2016). The third international consensus definitions for sepsis and septic shock (Sepsis-3). Jama, 315(8), 801-810). The third international consensus definitions for sepsis and septic shock (Sepsis-3). Jama, 315(8), 801-810). The primary infection is most commonly caused by bacteria, but also may be by fungi, viruses or parasites. It may be located in any part of the body, but most commonly in the lungs, brain, urinary tract, skin or abdominal organs. It can cause multiple organ dysfunction syndrome (formerly known as multiple organ failure) and death. Frequently, people with septic shock are cared for in intensive care units. It most commonly affects children, immunocompromised individuals, and the elderly, as their immune systems cannot deal with infection as effectively as those of healthy adults. The mortality rate from septic shock is approximately 25-50%.

[0515] Cardiogenic shock (CS) is defined as a state of critical end-organ hypoperfusion due to reduced cardiac output. Notably, CS forms a spectrum that ranges from mild hypoperfusion to profound shock. Established criteria for the diagnosis of CS are: (i) systolic blood pressure, <90 mmHg for >30 min or vasopressors required to achieve a blood pressure >90 mmHg; (ii) pulmonary congestion or elevated left-ventricular filling pressures; (iii) signs of impaired organ perfusion T75369WO BOEHMERT & BOEHMERT with at least one of the following criteria: (a) altered mental status; (b) cold, clammy skin; (c) oliguria (< 0.5 mL / kg / h or <30 mL / h); (d) increased serum-lactate (Reynolds, H. R., et al. (2008) Circulation, 117(5), 686-697). Acute myocardial infarction (AMI) with subsequent ventricular dysfunction is the most frequent cause of CS accounting for approximately 80% of cases. Mechanical complications such as ventricular septal (4%) or free wall rupture (2%), and acute severe mitral regurgitation (7%) are less frequent causes of CS after AMI (Hochman, J. S. et al. (2000) Journal of the American College of Cardiology, 36(3 S 1 ), 1063-1070).

[0516] Non-AMI-related CS may be caused by decompensated valvular heart disease, acute myocarditis, arrhythmias, etc. with heterogeneous treatment options. This translates in 40,000 to 50,000 patients per year in the USA and 60,000 to 70,000 in Europe. Despite advances in treatment mainly by early revascularization with subsequent mortality reduction, CS remains the leading cause of death in AMI with mortality rates still approaching 40-50% according to recent registries and randomized trials (Goldberg, R. J. et al. (2009) Circulation, 119(9), 1211— 1219.).

[0517] Heart failure (HF) is a cardiac condition that occurs, when a problem with the structure or function of the heart impairs its ability to supply sufficient blood flow to meet the body's needs. It can cause a large variety of symptoms, particularly shortness of breath (SOB) at rest or during exercise, signs of fluid retention such as pulmonary congestion or ankle swelling and objective evidence of an abnormality of the structure or function of the heart at rest.

[0518] Heart failure is a clinical syndrome characterized by a constellation of symptoms and signs caused by cardiac dysfunction. It is one of the major causes of morbidity and mortality in the developed countries, with a prevalence of 1-2%. Heart failure can be grouped into chronic HF and acute HF. Patients with chronic HF can be grouped into stable chronic HF, worsening signs and symptoms of chronic HF and acute decompensation of chronic HF. Acute heart failure (AHF) is defined as a rapid onset of signs and symptoms of heart failure resulting in the need for urgent therapy or hospitalization. AHF can present as acute de novo HF (new onset of AHF in a patient without previous cardiac dysfunction) or acute decompensation of chronic HF.

[0519] Patients with chronic heart failure (HF) may include patients with worsening signs and symptoms of chronic heart failure and acute decompensation of chronic heart failure.

[0520] Chronic HF with worsening signs and symptoms is in particular characterized by: T75369WO BOEHMERT & BOEHMERT

[0521] (i) the presence of structural or functional failure of the heart that impairs its ability to supply sufficient blood flow to meet body's needs,

[0522] (ii) volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of cardiac output (manifested by hypotension, renal insufficiency and / or a shock syndrome) and whereas the patient is not in need of urgent therapy and does not require hospitalization, but is in need of therapy adjustment.

[0523] Chronic heart failure may also decompensate (termed acute decompensated heart failure or acute decompensated chronic heart failure), which is most commonly the result from an intercurrent illness (such as pneumonia), myocardial infarction, arrhythmias, uncontrolled hypertension or a patient's failure to maintain fluid restriction, diet or medication.

[0524] New onset acute HF and acute decompensated chronic HF are characterized by:

[0525] (i) the presence of structural or functional failure of the heart that impairs its ability to supply sufficient blood flow to meet body's needs,

[0526] (ii) volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of cardiac output (manifested by hypotension, renal insufficiency and / or a shock syndrome) and whereas the patient is in need of urgent therapy or therapy adjustment and does require hospitalization.

[0527] The above definitions of acute heart failure that either new-onset AHF or acute decompensated HF or acute decompensated chronic HF or worsening signs / symptoms of chronic heart failure are in line with Voors et al., European Journal of Heart Failure (2016), 18, 716 - 726.

[0528] Numerous ways to treat cancer have been developed, and there are more to come in the future (Wolfgang Link, pages 7-76 in “Principles of Cancer Treatment and Anticancer Drug Development”, ISBN 978-3-030-18721-7, published September 20, 2019; Alecsandru Ioan Baba and Cornel Catoi in “Comparative Oncology”; Bucharest (RO): The Publishing House of the Romanian Academy; 2007. ISBN-10: 973-27-1457-3; https: / / www.cancer.gov / about- cancer / treatment / types). In the present application, all of these shall be comprised under “cancer therapies”. Ways of treating cancer depend on the type of cancer and on how advanced it is. Treatments might be given only once, or repeatedly, as a single type of treatment, or as a combination of different types of treatments. Cancer therapies include (https: / / www.cancer.gov / about-cancer / treatment / types): Chemotherapy, hormone therapy, hyperthermia, immunotherapy (including T-cell transfer therapy of the type tumor-infiltrating T75369WO BOEHMERT & BOEHMERT lymphocytes (or TIL) therapy and Chimeric Antigen Receptor (CAR) T-cell therapy), photodynamic therapy, radiation therapy, stem cell transplant, surgery, targeted therapy.

[0529] A bodily fluid according to the present invention is in particular embodiments a blood sample. A blood sample more particularly is selected from the group comprising whole blood, serum, and plasma. More particularly said sample is selected from the group comprising human citrate plasma, heparin plasma, and EDTA plasma.

[0530] In certain embodiments of the invention, the compound(s) as defined herein for the use in a treatment as defined herein relates to a) a method of treatment comprising administering said compound to the patient in need thereof in said treatment, or b) to the use of said compound(s) for the production of a medicament or pharmaceutical composition for use in the said treatment, or c) for a pharmaceutical composition comprising said compound and confectioned for the use in said treatment, or d) to the use of said compound in said treatment.

[0531] The following embodiments form also part of the present invention:

[0532] 1. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient wherein the level of Pro-Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient is above a threshold.

[0533] 2. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to item 1, wherein said Pro-Adrenomedullin or a fragment thereof is selected from the group comprising Pro-Adrenomedullin (SEQ ID No. 33), PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT-proADM (SEQ ID NO: 39).

[0534] 3. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to item 1 or 2, wherein said fragment of Pro-Adrenomedullin is MR-proADM and the threshold of the level of MR-proADM is between 0.5 and 2 nmol / L, more preferred between 0.6 and 1.5 nmol / L, even more preferred between 0.7 and 1 nmol / L, most preferred said threshold is 0.8 nmol / L, or wherein said threshold of the level of MR-proADM is an x-fold of the median level of MR-proADM in a healthy population, particularly the threshold level of MR- proADM is in the range between 1.2-fold and 4.9-fold, more preferred between 1.5-fold T75369WO BOEHMERT & BOEHMERT and 3.7-fold, even more preferred between 1.7-fold and 2.4-fold, most preferred said threshold is 2.0-fold of the median of the level of MR-proADM in a healthy population.

[0535] 4. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to item 1 or 2, wherein said fragment of Pro-Adrenomedullin is mature PAMP and the threshold of the level of mature PAMP is between 0.7 and 1.2 pmol / L, more preferred between 0.8 and 1.0 pmol / L, most preferred said threshold is 0.9 pmol / L, or wherein said threshold level of mature PAMP is an x-fold of the mean level of mature PAMP in a healthy population, particularly the threshold level of mature PAMP is in the range between the 1.4-fold and 2.4-fold, more particular in the range between 1.6-fold and 2.0-fold, most particular said threshold is the 1.8-fold of the mean of the level of mature PAMP in a healthy population.

[0536] 5. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to item 1 or 2, wherein said fragment of Pro-Adrenomedullin is PAMP-Gly and the threshold of the level of PAMP-Gly is between 1.5 and 2.4 pmol / L, even more preferred between 1.8 and 2.2 pmol / L, most preferred said threshold is 2 pmol / L, or said threshold level of PAMP-Gly is an x-fold of the mean level of PAMP-Gly in a healthy population, particularly in the range between the 1.3-fold and 2.1-fold, more particular in the range between 1.6-fold and 1.9-fold, most particular said threshold is the 1.7-fold of the mean of the level of PAMP-Gly in a healthy population.

[0537] 6. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to item 1 or 2, wherein said fragment of Pro-Adrenomedullin is ADM-Gly and the threshold of the level of ADM-Gly is between 25 and 125 pg / ml, more preferred between 30 and 100 pg / ml, even more preferred between 35 and 75 pg / ml, most preferred said threshold is 40 pg / ml, or said threshold level of ADM-Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly the threshold level of ADM-Gly is in the range between the 0.9-fold and 4.6-fold, more particular in the range between the 1.1-fold and 3.7-fold, more particular in the range between 1.3-fold and 2.8-fold, most particular said threshold is the 1.5-fold of the median of the level of ADM-Gly in a healthy population, or wherein said patient is a cancer patient receiving or about to receive IETC, in particular CAR T-cell therapy, said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly, and the threshold of the level of ADM-Gly is between 40 and 175 pg / ml, more preferred between 40 and 125 pg / ml, most preferred 40 pg / ml, T75369WO BOEHMERT & BOEHMERT or wherein said patient is a cancer patient receiving or about to receive IETC, in particular CAR T-cell therapy, said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly the threshold level of ADM-Gly is in the range between 1.5-fold and 6.5-fold, more preferred between 1.5-fold and 4.6-fold, most preferred said threshold is 1.5-fold of the median of the level of ADM-Gly in a healthy population.

[0538] 7. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to item 1 or 2, wherein said fragment of Pro-Adrenomedullin is mature ADM and the threshold of the level of mature ADM is between 35 and 125 pg / ml, more preferred between 40 and 100 pg / ml, even more preferred between 50 and 90 pg / ml, most preferred said threshold is 70 pg / ml. or said threshold level of mature ADM, and / or the threshold of the level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, particularly the threshold level of mature ADM is in the range between the 2.6-fold and 9.1-fold, more particular in the range between the 2.9- fold and 7.3-fold, more particular in the range between 3.6-fold and 6.6-fold, most particular said threshold is the 5.1 -fold of the median of the level of mature ADM in a healthy population, or wherein said patient is a cancer patient receiving or about to receive IETC, in particular CAR T-cell therapy, said fragment of Pro-Adrenomedullin is mature ADM and said threshold level of mature ADM, and the threshold of the level of ADM-Gly is between 25 and 150 pg / ml, more preferred between 30 and 125 pg / ml, most preferred 30 pg / ml, or wherein said patient is a cancer patient receiving or about to receive IETC, in particular CAR T-cell therapy, said fragment of Pro- Adrenomedullin is mature ADM and said threshold level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, particularly the threshold level of mature ADM is in the range between 1.8-fold and 10.9-fold, more preferred between 2.2-fold and 9.1-fold, most preferred said threshold is 2.2-fold of the median of the level of mature ADM in a healthy population.

[0539] 8. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to item 1 or 2, wherein said fragment of Pro- Adrenomedullin is CT-proADM and the threshold of the level of CT-proADM is between 75 and 350 pmol / L, more preferred between 100 and 250 pmol / L, even more preferred between 125 and 200 pmol / L, most preferred said threshold is 150 pmol / L, or said threshold level of CT- T75369WO BOEHMERT & BOEHMERT proADM, and / or the threshold of the level of CT-proADM is an x-fold of the median level of CT-proADM in a healthy population, particularly the threshold level of CT- proADM is in the range between the l.O-fold and 4.5-fold, more particular in the range between the 1.3-fold and 3.2-fold, more particular in the range between 1.6-fold and 2.6-fold, most particular said threshold is the 1.9-fold of the median of the level of CT- proADM in a healthy population.

[0540] 9. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 1 to 8, wherein said sample is a bodily fluid sample selected from the group comprising whole blood, plasma and serum.

[0541] 10. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 1 to 9, wherein said patient is a patient suffering from a severe infectious disease, inflammation, sepsis, shock, pulmonary diseases including ARDS, cardiovascular diseases including acute myocardial infarction, acute or chronic heart failure, organ dysfunction, a hepatic disease, a renal disease, an infectious disease, an autoimmune disease including ulcerative colitis, arthritis other chronic inflammatory diseases, other chronic inflammatory diseases, age-related macular degeneration, diabetic macular edema, a psychiatric disease, a neurodegenerative disease, or cancer, in particular wherein the patient is a cancer patient receiving immune effector cell therapy, in particular chimeric antigen receptor T-cell therapy (CAR T-cell therapy).

[0542] 11. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 1 to 10, wherein said patient suffers from organ organ dysfunction, in particular kidney dysfunction, lung dysfunction, heart dysfunction, or liver dysfunction.

[0543] 12. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 1 to 11, wherein said patient suffers from shock, in particular selected from the group comprising cardiogenic shock, septic shock, and anaphylactic shock.

[0544] 13. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 1 to 12, wherein the DPP3 binder is selected from the group comprising small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments, and anti-DPP3 non-Ig scaffolds. T75369WO BOEHMERT & BOEHMERT

[0545] 14. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 1 to 13, wherein said DPP3 binder is an anti-DPP3 antibody or anti- DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID No. 1 :

[0546] MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSP EAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNY KSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLR HLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPY YEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKA KAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYR DPFGSRGEFEGFVAVVNKAMSAKFERLASAEQLLKELPWPPTFEKDKFLTPDF TSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLE EDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPET GEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADA EDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGL VTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRAL YEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAA GLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA.

[0547] 15. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 1 to 14, wherein said DPP3 binder is an anti-DPP3 antibody or anti- DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in

[0548] SEQ ID No. 2: CETVINPETGEQIQSWYRSGE, particularly SEQ ID NO: 3: INPET G.

[0549] 16. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 14 to 15, wherein said antibody is a monoclonal antibody or monoclonal antibody fragment.

[0550] 17. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 14 to 16, wherein said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity determining regions (CDRs) in the heavy chain comprise the sequences:

[0551] CDR1 : SEQ ID NO: 6: GFSLSTSGMS T75369WO BOEHMERT & BOEHMERT

[0552] CDR2: SEQ ID NO: 7: IWWNDNK

[0553] CDR3: SEQ ID NO: 8: ARNYSYDY and wherein the complementarity determining regions (CDRs) in the light chain comprise the sequences:

[0554] CDR1: SEQ ID NO: 9: RSLVHSIGSTY

[0555] CDR2: KVS

[0556] CDR3: SEQ ID NO: 10: SQSTHVPWT.

[0557] 18. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 14 to 17, wherein said antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.

[0558] 19. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 14 to 18, wherein said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence: SEQ ID NO: 11 :

[0559] MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTS GMSVGWIRQPPGI<ALEWLAHIWWNDNI<SYNPALI<SRLTITRDTSI<NQVVLT MTNMDPVDTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, or a sequence that is > 80% identical to it, > 90% identical to it, > 95% identical to it, preferably > 98%, preferably > 99%, and wherein the light chain comprises the sequence: SEQ ID NO: 12:

[0560] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGS TYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVG VYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN T75369WO BOEHMERT & BOEHMERT

[0561] FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

[0562] VYACEVTHQGLS SP VTKSFNRGEC or a sequence that is > 80% identical to it, > 90% identical to it, > 95% identical to it, preferably > 98%, preferably > 99%, wherein more particularly the CDRs of SEQ ID NOs 6 to 10 and KVS as defined above (i.e. the CDR sequences are conserved).

[0563] 20. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 19, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 67.

[0564] 21. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 19, wherein said antibody or fragment thereof comprises a light chain Fab fragment comprising SEQ ID NO: 12.

[0565] 22. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 19, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 67 and comprises light chain Fab fragment comprising SEQ ID NO: 12.

[0566] 23. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according according to items 13 to 19, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 67.

[0567] 24. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 19, wherein said antibody or fragment thereof comprises a light chain variable region portion from SEQ ID NO: 12.

[0568] 25. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 19, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 67 and comprises a light chain variable region portion from SEQ ID NO: 12.

[0569] 26. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 19, wherein said antibody or fragment thereof is an ScFv comprising a heavy chain variable region portion from SEQ ID NO: 67 and comprises a light chain variable region portion from SEQ ID NO: 12, and wherein the heavy chain T75369WO BOEHMERT & BOEHMERT variable region portion from SEQ ID NO: 67 and the light chain variable region portion from SEQ ID NO: 12 are linked together via linker.

[0570] 27. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 19, wherein said antibody or fragment thereof is a fragment, wherein the fragment is a Fab fragment and wherein the Fab fragment comprises a heavy chain Fab fragment comprising SEQ ID NO: 67 and comprises a light chain variable fragment comprising SEQ ID NO: 12.

[0571] 28. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18, wherein said antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 4.

[0572] 29. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to any of items 13 to 18 or 28, wherein said antibody or fragment thereof comprises a light chain variable region comprising SEQ ID NO: 5.

[0573] 30. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18, 28 or 29, wherein said antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 4 and comprises a light chain variable region comprising SEQ ID NO: 5.

[0574] 31. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 1 to 13, wherein said DPP3 binder is a small molecule and is selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, or a derivative or analog of any of the aforementioned compounds, and benzimidazol derivatives (i.e. compounds having a benzimidazole scaffold).

[0575] 32. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18, wherein said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence: SEQ ID NO: 68:

[0576] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIW WNDNI<SYNPALI<SRLTITRDTSI<NQVVLTMTNMDPVDTGTYYCARNYSYDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS T75369WO BOEHMERT & BOEHMERT

[0577] HEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<E YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG, or a sequence that is > 80% identical to it, > 90% identical to it, > 95% identical to it, preferably > 98%, preferably > 99%, and wherein the light chain comprises the sequence: SEQ ID NO: 69:

[0578] DIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC or a sequence that is > 80% identical to it, > 90% identical to it, > 95% identical to it, preferably > 98%, preferably > 99%, wherein more particularly the CDRs of SEQ ID NOs 6 to 10 and KVS as defined above (i.e. the CDR sequences are conserved).

[0579] 33. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18 and 32, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 72.

[0580] 34. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18 and 33, wherein said antibody or fragment thereof comprises a light chain Fab fragment comprising SEQ ID NO: 69.

[0581] 35. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18 and 32, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 72 and comprises light chain Fab fragment comprising SEQ ID NO: 69.

[0582] 36. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according according to items 13 to 18 and 32, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 72. T75369WO BOEHMERT & BOEHMERT

[0583] 37. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18 and 36, wherein said antibody or fragment thereof comprises a light chain variable region portion from SEQ ID NO: 69.

[0584] 38. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18 and 32, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 72 and comprises a light chain variable region portion from SEQ ID NO: 69.

[0585] 39. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18 and 32, wherein said antibody or fragment thereof is an ScFv comprising a heavy chain variable region portion from SEQ ID NO: 72 and comprises a light chain variable region portion from SEQ ID NO: 69, and wherein the heavy chain variable region portion from SEQ ID NO: 72 and the light chain variable region portion from SEQ ID NO: 69 are linked together via linker.

[0586] 40. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to items 13 to 18 and 32, wherein said antibody or fragment thereof is a fragment, wherein the fragment is a Fab fragment and wherein the Fab fragment comprises a heavy chain Fab fragment comprising SEQ ID NO: 72 and comprises a light chain variable fragment comprising SEQ ID NO: 69.

[0587] The following embodiments pertaining to an aspect B of the invention also form part of the present invention:

[0588] 1. A method of therapy guidance or therapy stratification or therapy monitoring of a treatment of a patient with a DPP3 binder, the method comprising:

[0589] • providing a sample of bodily fluid of said patient,

[0590] • determining the level of Pro-Adrenomedullin or a fragment thereof in said sample, and

[0591] • comparing said level of Pro-Adrenomedullin or a fragment thereof to a threshold, T75369WO BOEHMERT & BOEHMERT wherein the level of Pro-Adrenomedullin or a fragment thereof in said sample being above said threshold is indicative that said patient requires treatment with a DPP3 binder or wherein if said level of Pro-Adrenomedullin or a fragment thereof in said sample is above said threshold, said patient is treated with a DPP3 binder.

[0592] 2. The method according to item 1 of aspect B, wherein said Pro-Adrenomedullin or a fragment thereof is selected from the group comprising Pro-Adrenomedullin (SEQ ID NO: 33), mature PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT-proADM (SEQ ID NO: 39).

[0593] 3. The method according to items 1 or 2 of aspect B, wherein said fragment of Pro- Adrenomedullin is MR-proADM and the threshold of the level of MR-proADM is between 0.5 and 2 nmol / L, more preferred between 0.6 and 1.5 nmol / L, even more preferred between 0.7 and 1 nmol / L, most preferred said threshold is 0.8 nmol / L or wherein said threshold of the level of MR-proADM is an x-fold of the median level of MR-proADM in a healthy population, particularly the threshold level of MR- proADM is in the range between 1.2-fold and 4.9-fold, more preferred between 1.5-fold and 3.7-fold, even more preferred between 1.7-fold and 2.4-fold, most preferred said threshold is 2.0-fold of the median of the level of MR-proADM in a healthy population.

[0594] 4. The method according to items 1 or 2 of aspect B, wherein said fragment of Pro- Adrenomedullin is mature PAMP and the threshold of the level of mature PAMP is between 0.7 and 1.2 pmol / L, more preferred between 0.5 and 1.0 pmol / L, most preferred said threshold is 0.9 pmol / L, or wherein said threshold level of mature PAMP is an x-fold of the mean level of mature PAMP in a healthy population, particularly the threshold level of mature PAMP is in the range between the 1.4-fold and 2.4-fold, more particular in the range between 1.6-fold and 2.0-fold, most particular said threshold is the 1.8-fold of the mean of the level of mature PAMP in a healthy population.

[0595] 5. The method according to items 1 or 2 of aspect B, wherein said fragment of Pro- Adrenomedullin is PAMP-Gly and the threshold of the level of PAMP-Gly is between 1.5 and 2.4 pmol / L, even more preferred between 1.8 and 2.2 pmol / L, most preferred said threshold is 2 pmol / L, or said threshold level of PAMP-Gly is an x-fold of the mean level of PAMP-Gly in a healthy population, particularly the threshold level of PAMP- T75369WO BOEHMERT & BOEHMERT

[0596] Gly is in the range between the 1.3-fold and 2.1-fold, more particular in the range between 1.6-fold and 1.9-fold, most particular said threshold is the 1.7-fold of the mean of the level of PAMP-Gly in a healthy population.

[0597] 6. The method according to items 1 or 2 of aspect B, wherein said fragment of Pro- Adrenomedullin is ADM-Gly and the threshold of the level of ADM-Gly is between 25 and 125 pg / ml, more preferred between 30 and 100 pg / ml, even more preferred between 35 and 75 pg / ml, most preferred said threshold is 40 pg / ml, or said threshold level of ADM-Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly the threshold level of ADM-Gly is in the range between the 0.9-fold and 4.6-fold, more particular in the range between the 1.1-fold and 3.7-fold, more particular in the range between 1.3-fold and 2.8-fold, most particular said threshold is the 1.5-fold of the median of the level of ADM-Gly in a healthy population, or wherein said patient is a cancer patient receiving or about to receive ITEC, in particular CAR T-cell therapy, said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM- Gly, and the threshold of the level of ADM-Gly is between 40 and 175 pg / ml, more preferred between 40 and 125 pg / ml, most preferred 40 pg / ml, or wherein said patient is a cancer patient receiving or about to receive ITEC, in particular CAR T-cell therapy, said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM- Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly the threshold level of ADM-Gly is in the range between 1.5-fold and 6.5-fold, more preferred between 1.5-fold and 4.6-fold, most preferred said threshold is 1.5-fold of the median of the level of ADM-Gly in a healthy population.

[0598] 7. The method according to items 1 or 2 of aspect B, wherein said fragment of Pro- Adrenomedullin is mature ADM and the threshold of the level of mature ADM is between 35 and 125 pg / ml, more preferred between 40 and 100 pg / ml, even more preferred between 50 and 90 pg / ml, most preferred said threshold is 70 pg / ml, or said threshold level of mature ADM, and / or the threshold of the level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, in the range between the 2.6-fold and 9.1-fold, more particular in the range between the 2.9-fold and 7.3-fold, more particular in the range between 3.6-fold and 6.6-fold, most particular said threshold is the 5.1 -fold of the median of the level of mature ADM in a healthy population, or said patient is a cancer patient receiving or about to receive ITEC, in particular CAR T- T75369WO BOEHMERT & BOEHMERT cell therapy, said fragment of Pro-Adrenomedullin is mature ADM and said threshold level of mature ADM, and the threshold of the level of ADM-Gly is between 25 and 150 pg / ml, more preferred between 30 and 125 pg / ml, most preferred 30 pg / ml, or wherein said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro-Adrenomedullin is mature ADM and said threshold level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, particularly the threshold level of mature ADM in the range between 1.8- fold and 10.9-fold, more preferred between 2.2-fold and 9.1-fold, most preferred said threshold is 2.2-fold of the median of the level of mature ADM in a healthy population.

[0599] 8. The method according to items 1 or 2 of aspect B, wherein said fragment of Pro- Adrenomedullin is CT-proADM and the threshold of the level of CT-proADM is between 75 and 350 pmol / L, more preferred between 100 and 250 pmol / L, even more preferred between 125 and 200 pmol / L, most preferred said threshold is 150 pmol / L, or said threshold level of CT-proADM, and / or the threshold of the level of CT-proADM is an x-fold of the median level of CT-proADM in a healthy population, particularly the threshold level of CT-proADM is in the range between the 1.0-fold and 4.5-fold, more particular in the range between the 1.3-fold and 3.2-fold, more particular in the range between 1.6-fold and 2.6-fold, most particular said threshold is the 1.9-fold of the median of the level of CT-proADM in a healthy population.

[0600] 9. The method according to items 1 to 8 of aspect B, wherein said sample is a bodily fluid sample selected from the group comprising whole blood, plasma, and serum.

[0601] 10. The method according to items 1 to 9 of aspect B, wherein said patient is a patient suffering from a severe infectious disease, inflammation, sepsis, shock, pulmonary diseases including ARDS, cardiovascular diseases including acute myocardial infarction, acute or chronic heart failure, organ dysfunction, a hepatic disease, a renal disease, an infectious disease, an autoimmune disease including ulcerative colitis, arthritis other chronic inflammatory diseases, other chronic inflammatory diseases, age- related macular degeneration, diabetic macular edema, a psychiatric disease, a neurodegenerative disease, or cancer, in particular wherein the patient is a cancer patient receiving immune effector cell therapy, particularly chimeric antigen receptor T-cell therapy (CAR T-cell therapy). T75369WO BOEHMERT & BOEHMERT

[0602] 11. The method according to items 1 to 10 of aspect B, wherein said patient suffers from organ organ dysfunction, in particular kidney dysfunction, lung dysfunction, heart dysfunction, or liver dysfunction.

[0603] 12. The method according to items 1 to 11 of aspect B, wherein said patient suffers from shock, in particular selected from the group comprising cardiogenic shock, septic shock, and anaphylactic shock.

[0604] 13. The method according to items 1 to 12 of aspect B, wherein the DPP3 binder is selected from the group comprising small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments, and anti-DPP3 non-Ig scaffolds.

[0605] 14. The method according to items 1 to 13 of aspect B, wherein said DPP3 binder is an anti- DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID No. 1 : MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSP EAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNY KSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLR HLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPY YEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKA KAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYR DPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDF TSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLE EDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPET GEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADA EDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGL VTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRAL YEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAA GLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA.

[0606] 15. The method according to items 1 to 14 of aspect B, wherein said DPP3 binder is an anti- DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID No. 2: CETVINPETGEQIQSWYRSGE, particularly SEQ ID NO: 3: INPET G. T75369WO BOEHMERT & BOEHMERT

[0607] 16. The method according to items 1 to 15 of aspect B, wherein said antibody is a monoclonal antibody or monoclonal antibody fragment.

[0608] 17. The method according to items 1 to 16 of aspect B, wherein said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity determining regions (CDRs) in the heavy chain comprise the sequences:

[0609] CDR1: SEQ ID NO: 6: GFSLSTSGMS

[0610] CDR2: SEQ ID NO: 7: IWWNDNK

[0611] CDR3: SEQ ID NO: 8: ARNYSYDY and wherein the complementarity determining regions (CDRs) in the light chain comprise the sequences:

[0612] CDR1: SEQ ID NO: 9: RSLVHSIGSTY

[0613] CDR2: KVS

[0614] CDR3: SEQ ID NO: 10: SQSTHVPWT.

[0615] 18. The method according to items 1 to 17 of aspect B, wherein said antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.

[0616] 19. The method according to items 1 to 18 of aspect B, wherein said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence: SEQ ID NO: 11:

[0617] MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTS GMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLT MTNMDPVDTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, T75369WO BOEHMERT & BOEHMERT or a sequence that is > 80% identical to it, > 90% identical to it, > 95% identical to it, preferably > 98%, preferably > 99%. and wherein the light chain comprises the sequence: SEQ ID NO: 12:

[0618] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGS

[0619] TYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVG VYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

[0620] VYACEVTHQGLS SP VTKSFNRGEC or a sequence that is > 80% identical to it, > 90% identical to it, > 95% identical to it, preferably > 98%, preferably > 99%, wherein more particularly the CDRs of SEQ ID NOs 6 to 10 and KVS as defined above (i.e. the CDR sequences are conserved).

[0621] 20. The method according to items 13 to 19 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 67.

[0622] 21. The method according to items 13 to 20 of aspect B, wherein said antibody or fragment thereof comprises a light chain Fab fragment comprising SEQ ID NO: 12.

[0623] 22. The method according to items 13 to 21 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 67 and comprises light chain Fab fragment comprising SEQ ID NO: 12.

[0624] 23. The method according to items 13 to 22 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 67.

[0625] 24. The method according to items 13 to 23 of aspect B, wherein said antibody or fragment thereof comprises a light chain variable region portion from SEQ ID NO: 12.

[0626] 25. The method according to items 13 to 24 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 67 and comprises a light chain variable region portion from SEQ ID NO: 12.

[0627] 26. The method according to items 13 to 25 of aspect B, wherein said antibody or fragment thereof is an ScFv comprising a heavy chain variable region portion from SEQ ID NO: 67 and comprises a light chain variable region portion from SEQ ID NO: 12, and T75369WO BOEHMERT & BOEHMERT wherein the heavy chain variable region portion from SEQ ID NO: 67 and the light chain variable region portion from SEQ ID NO: 12 are linked together via linker.

[0628] 27. The method according to items 13 to 26 of aspect B, wherein said antibody or fragment thereof is a fragment of aspect B, wherein the fragment is a Fab fragment and wherein the Fab fragment comprises a heavy chain Fab fragment comprising SEQ ID NO: 67 and comprises a light chain variable fragment comprising SEQ ID NO: 12.

[0629] 28. The method according to items 13 to 18 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 4.

[0630] 29. The method according to any of items 13 to 18 or 28 of aspect B, wherein said antibody or fragment thereof comprises a light chain variable region comprising SEQ ID NO: 5.

[0631] 30. The method according to items 13 to 18, 28 or 29 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 4 and comprises a light chain variable region comprising SEQ ID NO: 5.

[0632] 31. The method according to items 1 to 13 of aspect B, wherein said DPP3 binder is a small molecule and is selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, or a derivative or analog of any of the aforementioned compounds, and benzimidazol derivatives (i.e. compounds having a benzimidazole scaffold).

[0633] 32. The method according to items 1 to 18 of aspect B, wherein said antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence: SEQ ID NO: 68:

[0634] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIW WNDNI<SYNPALI<SRLTITRDTSI<NQVVLTMTNMDPVDTGTYYCARNYSYDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<E YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG, T75369WO BOEHMERT & BOEHMERT or a sequence that is > 80% identical to it, > 90% identical to it, > 95% identical to it, preferably > 98%, preferably > 99%, and wherein the light chain comprises the sequence: SEQ ID NO: 69:

[0635] DIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYK

[0636] VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTK

[0637] VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

[0638] GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC or a sequence that is > 80% identical to it, > 90% identical to it, > 95% identical to it, preferably > 98%, preferably > 99%, wherein more particularly the CDRs of SEQ ID NOs 6 to 10 and KVS as defined above (i.e. the CDR sequences are conserved).

[0639] 33. The method according to items 13 to 18 and 32 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 72.

[0640] 34. The method according to items 13 to 18 and 33 of aspect B, wherein said antibody or fragment thereof comprises a light chain Fab fragment comprising SEQ ID NO: 69.

[0641] 35. The method according to items 13 to 18 and 32 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain Fab fragment comprising SEQ ID NO: 72 and comprises light chain Fab fragment comprising SEQ ID NO: 69.

[0642] 36. The method according to items 13 to 18 and 32 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 72.

[0643] 37. The method according to items 13 to 18 and 36 of aspect B, wherein said antibody or fragment thereof comprises a light chain variable region portion from SEQ ID NO: 69.

[0644] 38. The method according to items 13 to 18 and 32 of aspect B, wherein said antibody or fragment thereof comprises a heavy chain variable region portion from SEQ ID NO: 72 and comprises a light chain variable region portion from SEQ ID NO: 69.

[0645] 39. The method according to items 13 to 18 and 32 of aspect B, wherein said antibody or fragment thereof is an ScFv comprising a heavy chain variable region portion from SEQ ID NO: 72 and comprises a light chain variable region portion from SEQ ID NO: 69, T75369WO BOEHMERT & BOEHMERT and wherein the heavy chain variable region portion from SEQ ID NO: 72 and the light chain variable region portion from SEQ ID NO: 69 are linked together via linker.

[0646] 40. The method according to items 13 to 18 and 32 of aspect B, wherein said antibody or fragment thereof is a fragment of aspect B, wherein the fragment is a Fab fragment and wherein the Fab fragment comprises a heavy chain Fab fragment comprising SEQ ID NO: 72 and comprises a light chain variable fragment comprising SEQ ID NO: 69.

[0647] Examples

[0648] The murine anti-hDPP3 antibody and humanized anti-hDPP3 antibody, in particular those used in the in vitro and in vivo tests of the utility of the antibodies are as described in more detail in the examples detailing antibody generation. The murine and humanized antibody comprise the CDRs according to SEQ ID Nos 6-10; more particularly, the mouse antibody comprises heavy and light chain variable regions of SEQ ID NOs 4 and 5; and more particularly, the humanized antibody comprises heavy and light chain sequences of SEQ ID NOs 11 and 12 and even more particularly, _the humanized antibody comprises heavy and light chain sequences of SEQ ID NOs 68 and 69.

[0649] Example 1 - DPP3 antibody administration in patients with endothelial dysfunction

[0650] Three critically ill patients with refractory shock and multi-organ failure, having elevated plasma levels of bio- ADM, which indicates endothelial dysfunction, were included in the study. The patients were treated with humanized anti-hDPP3 antibody, and bio-ADM and ADM-Gly were measured in serial plasma samples gained from these patients.

[0651] Patient description:

[0652] Two women (patients 01 and 03) and one man (patient 02) were treated. The patients’ age ranged between 64 and 84 years. All three patients had pre-existing conditions and were in utmost critical condition with signs of multi-organ failure, with patient 01 and 02 suffering from combined septic and cardiogenic shock (AMI in patient 01 was untreated due to hemodynamic instability) and patient 03 from right-sided cardiogenic shock. All three were suffering from renal failure, in need of renal replacement therapy, two out of three (patients 01 and 02) were intubated and under invasive mechanical ventilation. All three patients were assessed and T75369WO BOEHMERT & BOEHMERT approved for treatment with humanized anti-hDPP3 antibody by an independent international patient selection board, which considered patient 01 as having a 24-hour probability of mortality of >90%, as attested by a critical care physician, and patients 02 and 03 as already having entered process of dying.

[0653] Intervention:

[0654] Patients received a single dose of humanized anti-hDPP3 antibody infusion at a dose of 10 mg / kg body weight over a 2-hour period, humanized anti-hDPP3 antibody was administered between 1 and 3 days after transfer to intensive care unit (ICU), separately from any concomitant drugs (meaning no other drugs were administered during humanized anti-hDPP3 antibody administration) using a dedicated lumen of a central venous catheter.

[0655] Therapy was well tolerated in all three patients, no immediate adverse reactions were noted, and no adverse events were found to be associated with humanized anti-hDPP3 antibody.

[0656] Bio- ADM and ADM-Gly were measured in plasma samples (as described in Example 8) gained from these patients immediately prior to dosing with humanized anti-hDPP3 antibody, as well as 24 and 48 hours thereafter. All patients presented at baseline before administration of humanized anti-hDPP3 antibody with highly supranormal concentrations of bio-ADM (>400 pg / mL), ADM-Gly (>2,000 pg / mL) and MR-proADM (>10 nmol / L). Concentrations of all three, bio-ADM, ADM-Gly and MR-proADM, declined already 24 hours after treatment with humanized anti-hDPP3 antibody, and decline continued steadily throughout the observation period (Figure 1). The degree of decline was most pronounced for ADM-Gly.

[0657] The finding of rapidly and steadily declining bio-ADM, ADM-Gly and MR-proADM concentrations following treatment with humanized anti-hDPP3 antibody is highly surprising. A previous observational study performed in patients with cardiogenic shock (Takagi et al. 2021) had demonstrated that under standard of care therapy, concentrations of bio-ADM do not decline in the same time frame assessed in Example 1, neither in 30-day survivors nor nonsurvivors.

[0658] The development of required doses of vasopressor to achieve target mean arterial pressure recorded in the patients was in line with the observation of declining bio-ADM, ADM-Gly and MR-proADM concentrations after treatment with humanized anti-hDPP3 antibody. If disturbed endothelial function in the shock patients was improved by the treatment with humanized anti- hDPP3 antibody, it would be expected that the requirement for administration of exogenous norepinephrine, a strong inotrope, to achieve a target mean arterial pressure would be reduced upon treatment with humanized anti-hDPP3 antibody. Prior to the treatment with humanized T75369WO BOEHMERT & BOEHMERT anti-hDPP3 antibody, the three patients received norepinephrine doses of 0.8, 1.4, and 0.6 pg / kg / min (mean: 0.9 pg / kg / min), respectively. 24 hours after the treatment, the required doses of norepinephrine dropped to 67%, and after 48 hours down to 31%, compared to the starting dose (Figure. 2).

[0659] Overall, it can be concluded that treatment of patients having endothelial dysfunction with humanized anti-hDPP3 antibody leads to reduction of endothelial dysfunction.

[0660] Example 2 - DPP3 binder administration in septic shock

[0661] A porcine septic shock model was used to investigate the influence of treatment with humanized anti-hDPP3 antibody on the development of endothelial dysfunction, as assessed by serial measurement of plasma ADM. Previously, an ADM cut-off of 70 pg / mL has been established and validated clinically for distinguishing subjects with increased endothelial dysfunction and mortality risk from those with lower or no endothelial dysfunction and mortality risk (Mebazaa et al. 2018) (Caironi et al. 2017).

[0662] For the experiment, 16 pigs were used, half for the treatment with humanized anti-hDPP3 antibody, the other half as controls. Septic shock was induced by an intraperitoneal instillation of 3 g / kg of autologous feces. Four to six hours later, eight animals each were treated with humanized anti-hDPP3 antibody, another eight animals received standard treatment, and plasma ADM was measured in serial blood draws gained at five timepoints of the observation period.

[0663] As graphically presented in Figure 3, in the humanized anti-hDPP3 antibody group only 2 out of 8 animals (=25%) developed ADM concentrations above 70 pg / mL, whereas 5 out of 8 (=63%) did in the control group.

[0664] Experimental details:

[0665] An established porcine model of septic shock (Carrara, Herpain, et al. 2020) (Carrara, Niccolo, et al. 2020) with 16 pigs (Sus scrofa domesticus, RA-SE Genetics, Belgium), weighing 55 + / - 5 kg, was used. The animals were randomized the day before the experiment to either humanized anti-hDPP3 antibody administration in addition to standard treatment or standard treatment (Control). The experimental protocol and study time-points are illustrated in Figure

[0666] 4

[0667] Briefly, after anesthesia and surgery, animals were left to stabilize for two hours; this timepoint was then considered as baseline. Sepsis was then induced by an intraperitoneal instillation of 3 g / kg of autologous feces, previously collected from the animal's cage, via two peritoneal drains. T75369WO BOEHMERT & BOEHMERT

[0668] Animals were kept in a hypotensive state for one hour to enforce the organ dysfunction. Fluid resuscitation was then started with 10 mL / kg*h of balanced crystalloid (Plasmalyte, Baxter, USA) and 10 mL / kg*h of colloid (Geloplasma, Fresenius Kabi, France) over a period of 20 minutes, to restore an optimal preload based on an arterial pulse pressure variation (PPV) < 13%. Then, norepinephrine was started, targeting a MAP between 65 and 75 mmHg. At this point, broad spectrum antibiotic with 2 g amoxicillin-clavulanic acid was initiated and repeated 8 hours later. In parallel, peritoneal drains were opened to remove peritoneal fluid and peritoneal lavage was performed with the infusion of 1 L of crystalloids, previously warmed to 40°C, through the peritoneal drains. The abdominal wall was opened surgically (without opening the peritoneum) to limit the increase in intra-abdominal pressure and prevent abdominal compartment syndrome (Kirkpatrick et al. 2013). Fluids were titrated to maintain a PPV < 13% in the case of a decrease in MAP. Full resuscitation with source control, fluids, and norepinephrine was continued for 12 hours, with hemodynamic time-points recorded every hour (labeled as Hl to H12) and biological time-points at 4, 8, and 12 hours after the start of norepinephrine (labeled as H4, H8, and H12, respectively). At the biological time points and at the time points Baseline and Shock, EDTA-plasma samples were gained by drawing blood in EDTA tubes and centrifugation at a fixed temperature of 4°C and 2500 rotations per minute for a duration of 15 minutes (SL 8R, ThermoFisher Scientific, Germany). EDTA plasma samples were stored frozen at -80°C until measurement of ADM.

[0669] In the interventional group, once the shock time point was reached, a continuous infusion of humanized anti-hDPP3 antibody was started at 9.3 mg / kg humanized anti-hDPP3 antibody, an G1 immunoglobulin inhibiting cDPP3 activity, was generated as previously described (Deniau et al. 2020) in Chinese hamster ovary cells and purified via Protein A chromatography followed by a polishing step consisting of an anion-exchange membrane with a consecutive multi-modal chromatography step. The antibody underwent ultra- and diafiltration during which it was concentrated to 20 mg / mL and formulated into its final formulation buffer. The antibody solution was placed under aseptic conditions into 20R DIN glass vials, labelled, packed, and provided by 4TEEN4 Pharmaceuticals GmbH (Hennigsdorf, Germany), humanized anti- hDPP3 antibody was stored at 2-8 °C under temperature control.

[0670] Example 3 - Endothelial dysfunction and CAR T-cell therapy

[0671] A cohort of seventeen patients with advanced relapsed and / or refractory lymphoma or leukemia was treated with CAR T-cells following an established procedure as described (Ayala Ceja et T75369WO BOEHMERT & BOEHMERT al. 2024). Blood samples were collected immediately prior to the administration of T-cells as well as after the treatment, triggered by clinical events including diagnosis and treatment of complications related to a cytokine release syndrome (CRS). CRS was defined and graded according to American Society for Transplantation and Cellular Therapy (ASTCT) guidelines (D. W. Lee et al. 2019). The grading system has defined a range of 5 grades, with grade 5 being the most severe conditions defined as death due to CRS in which another cause is not the principal factor leading to this outcome. Blood samples for measurement of ADM-Gly were gained just before the administration of T-cells in the frame of CAR-T therapy (baseline), and then at several time points in the days following the administration of T-cells in the frame of the CAR-T therapy.

[0672] Baseline concentrations of bio- ADM and ADM-Gly are shown in Table 22 and Figure 5 . The median bio-ADM concentration was 17.5 pg / mL, the mean bio-ADM concentration was 40.8 pg / mL. The median ADM-Gly concentration was 41.7 pg / mL, the mean ADM-Gly concentration was 77.4 pg / mL. From the seventeen patients studied, 58.8% had ADM-Gly concentrations above 40 pg / mL.

[0673] Table 2: Baseline ADM-Gly levels of patients treated with CAR T-cell therapy, and their CRS grading resulting from the CAR T-cell therapy. T75369WO BOEHMERT & BOEHMERT

[0674] Baseline concentrations of bio- ADM and ADM-Gly were correlated to a certain extent with the grading of the cytokine release syndrome (Spearman r=0.43 and r=0.31, respectively) and were higher in patients with a higher CRS grading (Figure 6).

[0675] Both markers, bio-ADM and ADM-Gly, were also measured in the follow-up of the patients. As blood draws were not taken at the exact same time points after administration of T-cells in the frame of CAR T-cell therapy for all patients, data from bio-ADM and ADM-Gly measurements were grouped in time intervals of four days, e.g. > 0 and <4 days, > 4 and <8 days, > 8 and <12 days, > 12 and <16 days, > 16 days, after administration of T-cells in the frame of CAR T-cell therapy. For both markers, bio-ADM and ADM-Gly, it was observed that concentrations increased after administration of T-cells in the frame of CAR T-cell therapy until the time interval > 4 and <8 days, followed by a decline, only for patients who developed severe side-effects (defined as CRS score >2) (Figure 7). In contrast, patients who did not or only very moderately develop such effects (defined as CRS <2), exhibited constant low levels of bio- ADM and ADM-Gly over the entire observation period.

[0676] The maximal concentrations of bio-ADM and ADM-Gly achieved in the observation periods are listed in Table 3.

[0677] Table 3: Maximal concentrations of ADM-Gly achieved in the observation period. T75369WO BOEHMERT & BOEHMERT

[0678] The proportion of these maximal concentrations of ADM-Gly above the healthy normal range (95thpercentile; threshold 40 pg / mL) was 94%.

[0679] The same analysis was done for the six patients with CRS grade 3 or 4: there the proportion was 100%.

[0680] The results of the current example demonstrate that CAR-T therapy is frequently associated with endothelial barrier dysfunction, as reflected by increased bio-ADM and ADM-Gly concentrations, and that this increase is more pronounced both at baseline and in the time course following the CAR-T therapy in patients suffering from severe cytokine release syndrome.

[0681] Consequently, such patients would benefit from a therapy improving the endothelial function.

[0682] Additionally, as endothelial dysfunction has been shown in the previous examples to constitute an indication for the treatment with a DPP3 binder, such as the humanized anti-hDPP3 antibody, patients suffering from side effects due to CAR-T therapy represent a target group for the treatment with a DPP3 binder, such as the humanized anti-hDPP3 antibody.

[0683] Example 4 - Generation of antibodies and antibody characterization

[0684] Generation of antibodies and determination DPP3 binding ability: Several murine antibodies were produced and screened by their ability of binding human DPP3 in a specific binding assay (see Table 4).

[0685] Peptides / conjugates for immunization:

[0686] DPP3 peptides for immunization were synthesized, see Table 4, (JPT Technologies, Berlin, Germany) with an additional N-terminal cysteine (if no cysteine is present within the selected DPP3 -sequence) residue for conjugation of the peptides to Bovine Serum Albumin (BSA). The peptides were covalently linked to BSA by using Sulfolink-coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, MA, USA).

[0687] Immunization of mice, immune cell fusion and screening:

[0688] Balb / c mice were intraperitoneally (i.p.) injected with 84 pg GST-hDPP3 or 100 pg DPP3- peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold Adjuvant), 84 pg or 100 pg at day 14 (emulsified in complete Freund’s adjuvant) and 42 pg or 50 pg at day 21 and 28 (in incomplete Freund’s adjuvant). At day 49 the animal received an intravenous (i.v.) injection of T75369WO BOEHMERT & BOEHMERT

[0689] 42 pg GST-hDPP3 or 50 pg DPP3-peptide-BSA-conjugates dissolved in saline. Three days later the mice were sacrificed and the immune cell fusion was performed.

[0690] Splenocytes from the immunized mice and cells of the myeloma cell line SP2 / 0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37°C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT- Supplement], After one week, the HAT medium was replaced with HT Medium for three passages followed by returning to the normal cell culture medium. The cell culture supernatants were primarily screened for recombinant DPP3 binding IgG antibodies two weeks after fusion. Therefore, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 pl cell culture supernatant per well for 2 hours at room temperature. After washing of the plate, 50 pl / well peroxidase (POD)-rabbit anti mouse IgG was added and incubated for 1 h at RT. After a next washing step, 50 pl of a chromogen solution (3,7 mM o- phenylen-diamine in citrate / hydrogen phosphate buffer, 0.012% H2O2) were added to each well, incubated for 15 minutes at RT and the chromogenic reaction stopped by the addition of 50 pl 4N sulfuric acid. Absorption was detected at 490 mm. The positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and re-cloned using the limiting-dilution technique and the isotypes were determined.

[0691] Mouse monoclonal antibody production:

[0692] Antibodies raised against GST-tagged human DPP3 or DPP3-peptides were produced via standard antibody production methods (Marx et al. 1997 and purified via Protein A. The antibody purities were > 90% based on SDS gel electrophoresis analysis.

[0693] Characterization of antibodies - binding to hDPP3 and / or immunization peptide:

[0694] To analyse the capability of DPP3 / immunization peptide binding by the different antibodies and antibody clones a binding assay was performed:

[0695] Solid phase: Recombinant GST-tagged hDPP3 (SEQ ID NO: 1) or a DPP3 peptide (immunization peptide, SEQ ID NO: 2) was immobilized onto a high binding microtiter plate surface (96-Well polystyrene microplates, Greiner Bio-One international AG, Austria, 1 pg / well in coupling buffer [50 mM Tris, 100 mM NaCl, pH7,8], Ih at RT). After blocking with 5% bovine serum albumin, the microplates were vacuum dried.

[0696] Labelling procedure (tracer) T75369WO BOEHMERT & BOEHMERT

[0697] 100 pg (100 pl) of the different antiDPP3 antibodies (detection antibody, 1 mg / ml in PBS, pH 7.4) were mixed with 10 pl acridinium NHS-ester (1 mg / ml in acetonitrile, In Vent GmbH, Germany; EP 0 353 971) and incubated for 30 min at room temperature. Labelled antiDPP3 antibody was purified by gel-filtration HPLC on Shodex Protein 5 pm KW-803 (Showa Denko, Japan). The purified labelled antibody was diluted in assay buffer (50 mmol / 1 potassium phosphate, 100 mmol / 1 NaCl, 10 mmol / 1 Na2-EDTA, 5 g / 1 bovine serum albumin, 1 g / 1 murine IgG, 1 g / 1 bovine IgG, 50 pmol / 1 amastatin, 100 pmol / 1 leupeptin, pH 7.4). The final concentration was approx. 5-7* 106relative light units (RLU) of labelled compound (approx. 20 ng labelled antibody) per 200 pl. acridinium ester chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). hDPP3 binding assay: the plates were filled with 200 pl of labelled and diluted detection antibody (tracer) and incubated for 2-4 h at 2-8 °C. Unbound tracer was removed by washing 4 times with 350 pl washing solution (20 mM PBS, pH 7.4, 0.1 % Triton X-100). Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0698] Characterization of antibodies - hDPP3 -inhibition analysis:

[0699] To analyse the capability of PPP3 inhibition by the different antibodies and antibody clones a PPP3 activity assay with known procedure (Jones et al., 1982) was performed. Recombinant GST-tagged hPPP3 was diluted in assay buffer (25 ng / ml GST-PPP3 in 50 mM Tris-HCl, pH7,5 and 100 pM ZnCh) and 200 pl of this solution incubated with 10 pg of the respective antibody at room temperature. After 1 hour of pre-incubation, fluorogenic substrate Arg-Arg- PNA (20 pl, 2mM) was added to the solution and the generation of free PNA over time was monitored using the Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37 °C.

[0700] Fluorescence of PNA is detected by exciting at 340 nm and measuring emission at 410 nm. Slopes (in RFU / min) of increasing fluorescence of the different samples are calculated. The slope of GST-hPPP3 with buffer control is appointed as 100 % activity. The inhibitory ability of a possible capture-binder is defined as the decrease of GST-hPPP3 activity by incubation with said capture-binder in percent.

[0701] Table 4 represents a selection of obtained antibodies and their binding rate in Relative Light Units (RLU) as well as their relative inhibitory ability (%; Table 4). The monoclonal antibodies T75369WO BOEHMERT & BOEHMERT raised against the below depicted DPP3 regions, were selected by their ability to bind recombinant DPP3 and / or immunization peptide, as well as by their inhibitory potential.

[0702] All antibodies raised against the GST-tagged, full-length form of recombinant hDPP3 show a strong binding to immobilized GST-tagged hDPP3. Antibodies raised against the SEQ ID NO: 2 peptide bind as well to GST-hDPP3. The SEQ ID NO: 1 antibodies also strongly bind to the immunization peptide.

[0703] Table 4: List of antibodies raised against full-length or sequences of hDPP3 and their ability to bind hDPP3 (SEQ ID NO: 1) or immunization peptide (SEQ ID NO: 2) in RLU, as well as the maximum inhibition of recombinant GST-hDPP3.

[0704] Example 5 - Development of the humanized anti-hDPP3 antibody

[0705] Murine antibodies raised against SEQ ID NO: 2 were characterized in more detail (epitope mapping, binding affinities, specificity, inhibitory potential). Here the results for murine clone 1967 of SEQ ID NO: 2 (murine anti-hDPP3 antibody) are shown as an example.

[0706] Determination of murine anti-hDPP3 antibody epitope on DPP3 :

[0707] For epitope mapping of murine anti-hDPP3 antibody a number of N- or C-terminally biotinylated peptides were synthesized (peptides & elephants GmbH, Hennigsdorf, Germany). These peptides include the sequence of the full immunization peptide (SEQ ID NO: 2) or T75369WO BOEHMERT & BOEHMERT fragments thereof, with stepwise removal of one amino acid from either C- or N-terminus (see Table 5 for a complete list of peptides).

[0708] High binding 96 well plates were coated with 2 pg Avidin per well (Greiner Bio-One international AG, Austria) in coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). Plates were then washed and filled with specific solutions of biotinylated peptides (10 ng / well; buffer - IxPBS with 0.5% BSA).

[0709] Murine anti-hDPP3 antibody was labelled with a chemiluminescence label according to Example 4.

[0710] The plates were filled with 200 pl of labelled and diluted detection antibody (tracer) and incubated for 4 h at room temperature. Unbound tracer was removed by washing 4 times with 350 pl washing solution (20 mM PBS, pH 7.4, 0.1 % Triton X-100).

[0711] Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). Binding of murine anti-hDPP3 antibody to the respective peptides is determined by evaluation of the relative light units (RLU). Any peptide that shows a significantly higher RLU signal than the unspecific binding of murine anti-hDPP3 antibody is defined as murine anti-hDPP3 antibody binder. The combinatorial analysis of binding and non-binding peptides reveals the specific DPP3 epitope of murine anti-hDPP3 antibody.

[0712] Determination of binding affinities using Octet:

[0713] The experiment was performed using Octet Red96 (ForteBio). Murine anti-hDPP3 antibody was captured on kinetic grade anti-humanFc (AHC) biosensors. The loaded biosensors were then dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM). Association was observed for 120 seconds followed by 180 seconds of dissociation (Figure 9). The buffers used for the experiment were assay buffer (PBS with 0.1% BSA, 0.02% Tween-21), regeneration buffer (10 mM Glycine buffer (pH 1.7)) and neutralization buffer (PBS with 0.1% BSA, 0.02% Tween-21), respectively. Kinetic analysis was performed using a 1 : 1 binding model and global fitting.

[0714] Western Blot analysis of Binding specificity of murine anti-hDPP3 antibody:

[0715] Blood cells from human EDTA-blood were washed (3x in PBS), diluted in PBS and lysed by repeated freeze-thaw-cycles. The blood cell lysate had a total protein concentration of 250 pg / ml, and a DPP3 concentration of 10 pg / ml. Dilutions of blood cell lysate (1 :40, 1 :80, 1 : 160 T75369WO BOEHMERT & BOEHMERT and 1 :320) and of purified recombinant human His-DPP3 (31.25-500 ng / ml) were subjected to SDS-PAGE and Western Blot. The blots were incubated in 1.) blocking buffer (IxPBS-T with 5% skim milk powder), 2.) primary antibody solution (murine anti-hDPP3 antibody 1 :2,000 in blocking buffer) and 3.) HRP labelled secondary antibody (goat anti mouse IgG, 1 : 1,000 in blocking buffer). Bound secondary antibody was detected using the Amersham ECL Western Blotting Detection Reagent and the Amersham Imager 600 UV (both from GE Healthcare) (Figure 10).

[0716] DPP3 inhibition assay: To analyse the capability of DPP3 inhibition by murine anti-hDPP3 antibody a DPP3 activity assay with known procedure (Jones and Kapralou 1982) was performed as described herein above. The inhibitory ability murine anti-hDPP3 antibody is defined as the decrease of GST-hDPP3 activity by incubation with said antibody in percent. The resulting lowered DPP3 activities are shown in an inhibition curve in Figure 8.

[0717] Epitope mapping: The analysis of peptides that murine anti-hDPP3 antibody binds to and does not bind to revealed the DPP3 sequence INPETG (SEQ ID NO: 3) as necessary epitope for Procizumab binding (see Table 5). Similar results were obtained for the humanized anti-hDPP3 antibody.

[0718] Binding affinity: murine anti-hDPP3 antibody binds with an affinity of 2.2*1 O'9M to recombinant GST-hDPP3 (kinetic curves see Figure 9).

[0719] Table 5: Peptides used for Epitope mapping of murine anti-hDPP3 antibody. T75369WO BOEHMERT & BOEHMERT

[0720] The immunization peptides #1 through #17 have SEQ ID NO: 13 through SEQ ID NO: 29 (see herein below).

[0721] Specificity and inhibitory potential:

[0722] The only protein detected with murine anti-hDPP3 antibody as primary antibody in lysate of blood cells was DPP3 at 80 kDa (Figure 10). The total protein concentration of the lysate was 250 pg / ml whereas the estimated DPP3 concentration is about 10 pg / ml. Even though there is 25 times more unspecific protein in the lysate, murine anti-hDPP3 antibody binds and detects specifically DPP3 and no other unspecific binding takes place.

[0723] Murine anti-hDPP3 antibody inhibits 15 ng / ml DPP3 in a specific DPP3 activity assay with an IC50 of about 15 ng / ml (Figure 8).

[0724] Chimerization / Humanization:

[0725] The murine monoclonal anti-hDPP3 antibody, with the ability of inhibiting DPP3 activity by 70 %, was chosen as possible therapeutic antibody and was also used as template for chimerization and humanization. The sequence of the variable region of murine anti-hDPP3 antibody in the heavy chain is shown in SEQ ID No. 4. The sequence of the variable region of murine anti-hDPP3 antibody in the light chain is shown in SEQ ID No. 5.

[0726] Humanization of murine antibodies may be conducted according to the following procedure:

[0727] For humanization of an antibody of murine origin the antibody sequence is analysed for the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modelling (Almagro and Fransson, 2008. Humanization of antibodies. Front Biosci. 13:1619-33}.

[0728] With the above context, the variable region can be connected to any subclass of constant regions (IgG, IgM, IgE. IgA), or only scaffolds, Fab fragments, Fv, Fab and F(ab)2. The murine T75369WO BOEHMERT & BOEHMERT antibody variant contains an IgG2a backbone. For chimerization and humanization a human IgGlk backbone was used.

[0729] For epitope binding the Complementarity Determining Regions (CDRs) are of importance.

[0730] The CDRs for the heavy chain and the light chain of the murine anti-hDPP3 antibody as well as for the humanized anti-hDPP3 antibody are shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 for the heavy chain and SEQ ID NO: 9, sequence KVS and SEQ ID NO: 10 for the light chain, respectively.

[0731] Sequencing of the humanized anti-hDPP3 antibody revealed an antibody heavy chain (H chain) according to SEQ ID NO: 11 and an antibody light chain (L chain) according to SEQ ID NO: 12 containing their respective N-terminal signal peptide sequences.

[0732] Example 6 - Generation of anti-ADM antibodies and determination of their affinity constants

[0733] We developed mouse monoclonal antibodies binding to the N-terminal (NT-ADM), mid- regional (MR- ADM) and C-terminal (CT- ADM) part of bio- ADM and their affinity constants were determined (Table 6). Peptides were supplied by JPT Peptide Technologies GmbH (Berlin, Germany). Peptides were coupled to BSAusing the Sulfo-SMCC crosslinking method. The crosslinking procedure was performed according the manufacturer's instructions (Thermo Fisher / Pierce).

[0734] Generation of murine antibodies

[0735] A Balb / c mouse was immunized with 100 pg Peptide-BSA-Conjugate at day 0 and 14 (emulsified in 100 pl complete Freund’s adjuvant) and 50 pg at day 21 and 28 (in 100 pl incomplete Freund’s adjuvant). Three days before the fusion experiment was performed, the animal received 50 pg of the conjugate dissolved in 100 pl saline, given as one intraperitoneal and one intra venous injection.

[0736] Splenocytes from the immunized mouse and cells of the myeloma cell line SP2 / 0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37°C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement). After two weeks the HAT medium is replaced with HT medium for three passages followed by returning to the normal cell culture medium. T75369WO BOEHMERT & BOEHMERT

[0737] The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion. The positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined (Lane, 1985. J. Immunol. Meth. 81: 223-228; Ziegler etal. 1996. Horm. Metab. Res. 28: 11-15}.

[0738] Table 6: Affinity constants of monoclonal antibodies directed to different regions of ADM

[0739] Monoclonal antibody production

[0740] Antibodies were produced via standard antibody production methods (Marx et al, 1997. Monoclonal Antibody Production, ATLA 25, 121} and purified via Protein A. The antibody purities were > 95% based on SDS gel electrophoresis analysis.

[0741] Affinity Constants

[0742] To determine the affinity of the antibodies to Adrenomedullin, the kinetics of binding of Adrenomedullin to immobilized antibody was determined by means of label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using an anti-mouse Fc antibody covalently coupled in high density to a CM5 sensor surface according to the manufacturer's instructions (mouse antibody capture kit; GE Healthcare).

[0743] Labelling procedure (tracer)

[0744] 100 pg (100 pl) of antibody (1 mg / ml in PBS, pH 7.4,) was mixed with 10 pl Akridinium NHS-ester (1 mg / ml in acetonitrile, In Vent GmbH, Germany) (EP 0353971} and incubated for 20 min at room temperature. Labelled CT-H was purified by Gel-filtration HPLC on Bio-Sil® T75369WO BOEHMERT & BOEHMERT

[0745] SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified labeled antibody was diluted in (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 5 g / L Bovine Serum Albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 pL. Akridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

[0746] Solid phase

[0747] Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with antibody (1.5 pg antibody / 0.3 mL 100 mmol / L NaCl, 50 mmol / L TRIS / HC1, pH 7.8). After blocking with 5% bovine serum albumin, the tubes were washed with PBS, pH 7.4 and vacuum dried.

[0748] Calibrators

[0749] Synthetic human ADM (hADM) (Bachem, Switzerland) was linearly diluted using 50 mM Tris / HC1, 250 mM NaCl, 0.2% Triton X-100, 0.5% BSA, 20 tabs / L Protease Complete Protease Inhibitor Cocktail Tablets (Roche AG); pH 7.8. Calibrators were stored at -20 °C before use.

[0750] Example 7 - Determination of the antibody combination that yields high signal / noise ratios

[0751] ADM Immunoassay

[0752] 50 pl of sample (or calibrator) was pipetted into coated tubes, after adding labelled second antibody (200 pl), the tubes were incubated for 2 h at room temperature. Unbound tracer was removed by washing 5 times (each 1 ml) with washing solution (20 mM PBS, pH 7.4, 0.1 % Triton X-100). Tube-bound chemiluminescence was measured by using the LB 953 (Berthold Technologies GmbH & Co. KG).

[0753] All antibodies were used in a sandwich immunoassay as coated tube and labelled antibody and combined in the following variations (see Table 7). Incubation was performed as described under hADM-Immunoassay. Results are given in ratio of specific signal (at 10 ng / ml ADM) / background (sample without ADM) signal. T75369WO BOEHMERT & BOEHMERT

[0754] Table 7: Signal to noise-ratio for anti- ADM antibody combinations

[0755] Surprisingly, we found the combination of MR- ADM and CT-ADM tracer as combination for highest signal / noise ratio. This combination is measuring specifically bio-ADM (C-terminal amidated ADM), as the tracer antibody is directed against the C-terminal amidated end of bio- ADM.

[0756] Subsequently, we used this antibody-combination for further investigations to measure bio- ADM. We used anti-MR-ADM as solid phase antibody and anti-CT-ADM as labelled antibody. A typical dose / signal curve is shown in Figure 11. The analytical sensitivity (average of 10 runs, ADM-free sample + 2SD) of the assay was 2 pg ADM / ml.

[0757] Example 8 - Measurement of biomarkers

[0758] 8, 1, Measurement of bio-ADM

[0759] Quantification of bio-ADM was conducted using the sphingotest bio-ADM assay as described elsewhere (Weber, J. et al. Sandwich Immunoassay for Bioactive Plasma Adrenomedullin. J. Appl. Lab. Med. An AACC Publ. 2, 222-233 (2017)) using the antibody combination as described in Example 7. Briefly, 96-well high binding polystyrene microtiter plates (Greiner Bio-One International AG) were coated (18 h at 20 °C) with monoclonal anti-ADM antibody, directed towards amino-acids 21-32 (SEQ ID NO: 41) of bio-ADM (anti-MR-ADM AK; 1 pg / 0.2 mL per well in 50 mM Tris-HCl, 100 mM NaCl, pH 7.8). After blocking with 30 g / L Karion, 5 g / L BS A (protease free), 6.5 mmol / L monopotassium phosphate, 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum-dried.

[0760] 100 pL of samples / calibrators were pipetted into coated microtiter plates. Afterwards 150 pL of MACN labelled tracer antibody (anti-CT-ADM AK; directed towards the amidated C-terminus of bio-ADM; SEQ ID NO: 42) the microtiter plates were incubated for Ih at 22 °C under agitation at 600 rpm. Unbound tracer was removed by washing 5 times (each 350 pL per well) with washing solution (20 mM PBS, 1 g / L Triton X-100, pH 7.4). Well-bound chemiluminescence was measured for 1 s per well by using the Centro LB 960 microtiter plate luminescence reader (Berthold Technologies). The assay was calibrated using dilutions of T75369WO BOEHMERT & BOEHMERT synthetic human bio- ADM (American Peptide Company). The lowest calibrator did not contain bio- ADM, but a concentration of 2 pg / mL was assigned to facilitate logarithmic evaluation. The calibrators were lyophilized in 20 mmol K2PO4, 6 mmol / L Na-EDTA, 5 g / L BSA, 100 pmol / L leupeptin, 50 pmol / L amastatin, 10 pg / mL of anti-N-terminal ADM antibody (SEQ ID NO: 40), pH 8.0, and reconstituted in H2O before use.

[0761] Healthy subjects (n=100, average age 56 years) were measured using the bio- ADM assay. The median value was 24.7 pg / ml, the lowest value 11 pg / ml and the 99thpercentile 43 pg / ml. Since the assay sensitivity was 2 pg / ml, 100% of all healthy subjects were detectable using the described bio- ADM assay.

[0762] 8,2, Measurement of ADM-Gly

[0763] An antibody directed towards the glycine extended C-terminus of ADM-Gly (AK835 / G4) using an immunization peptide according to SEQ ID NO: 43 was developed using the methods as described in Example 6.

[0764] Quantification of ADM-Gly was conducted as follows: 96-well high binding polystyrene microtiter plates (Greiner Bio-One International AG) were coated (18 h at 20 °C) with monoclonal anti-ADM antibody, directed towards amino-acids 21-32 (SEQ ID NO: 41) of ADM-Gly (anti -MR- ADM- AK, 1 pg / 0.2 mL per well in 50 mM Tris-HCl, 100 mM NaCl, pH 7.8). After blocking with 30 g / LKarion, 5 g / L BSA (protease free), 6.5 mmol / L monopotassium phosphate, 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum-dried. 50 pL of samples / calibrators were pipetted into coated microtiter plates. Afterwards 200 pL of MACN labelled tracer antibody (AK835 / G4, directed towards the glycine extended C-terminus of ADM-Gly, SEQ ID NO: 43). AK835 / G4 had no cross-reactivity with bio-ADM) the microtiter plates were incubated for 18h at 4°C under agitation at 600 rpm. Unbound tracer was removed by washing 5 times (each 350 pL per well) with washing solution (20 mM PBS, 1 g / L Triton X-100, pH 7.4). Well-bound chemiluminescence was measured for 1 s per well by using the Centro LB 960 microtiter plate luminescence reader (Berthold Technologies). The assay was calibrated using dilutions of synthetic human ADM-Gly (Peptides and Elephants, Hennigsdorf, Germany). The lowest calibrator did not contain ADM-Gly, but a concentration of 1 pg / mL was assigned to facilitate logarithmic evaluation. The calibrators were lyophilized in 20 mmol K2PO4, 6 mmol / L Na-EDTA, 5 g / L BSA, 100 pmol / L leupeptin, 50 pmol / L amastatin, 10 pg / mL of anti-N-terminal ADM-antibody (SEQ ID NO: 40), pH 8.0, and reconstituted in H2O before use. A typical standard curve is shown in Figure 12. A normal distribution of ADM-Gly from n=128 self-reported healthy individuals is shown in Figure 13. T75369WO BOEHMERT & BOEHMERT

[0765] The median value was 27.1 pg / ml, the lowest value 10.1 pg / ml and the 97.5thpercentile 58.9 pg / ml.

[0766] 8,3, Measurement of MR-proADM

[0767] Quantification of MR-proADM was conducted using the MR-proADM Kryptor assay (B.R.A.H.M.S Thermofisher Scientific, Hennigsdorf, Germany) as described elsewhere (Morgenthaler et al. 2005).

[0768] Purified anti-MR-proADM sheep polyclonal antibody (Morgenthaler et al. 2005) specific for amino-acids 68-86 was coupled to Cy5 fhiorophore (General Electric Healthcare). Purified anti-MR-proADM sheep polyclonal antibody specific for amino-acids 83-94 (Morgenthaler et al. 2005) was coupled to europium cryptate TBP-mono-MP (CISBIO, Bagnols / Ceze, France). The coupling reactions were performed according to the manufacturer prescribed coupling protocols.

[0769] The stock Cy5-conjugated antibody solution was diluted at 4 pg / mL with assay buffer (100 mmol / L sodium phosphate, 1 g / L protease free bovine serum albumin, 483 mmol / L potassium fluoride, 0.645 g / L non-specific bovine IgG, 1.12% sheep sera, pH 7.1). The EuC-conjugated antibody solution was diluted at 1.05 pg / mL with assay buffer (300 mmol / L sodium phosphate, 3 g / L protease free bovine serum albumin, 1 g / L trehalose, 5 g / L mannitol, pH 7.1) and freeze- dried. The EuC conjugate was reconstituted automatically on the KRYPTOR with 483 mmol / L potassium fluoride. The diluent contained in the kit was composed of newborn calf serum (Trina Bioreactives AG, Nanikon, Switzerland) with 7.7 nmol / L sodium azide and 5.4 nmol / L EDTA. After reconstitution, the reagents can be stored up to 15 days in the refrigerated zone of the machine. The calibrator was composed of 2 nmol / L of the synthetic peptide representing positions 45-92 of preproADM (JERINI AG, Berlin, Germany) diluted in human normal plasma (Scantibodies Laboratory Inc., Villebon / Yvette, France) with 150 mmol / L EDTA. The immunoassay was performed by incubating 26 pL of sample / calibrator, 62 pL of Cy5- conjugated antibody solution and 62 pL of EuC-conjugated antibody solution at 37 °C on KRYPTOR compact automate (Cezanne SAS, Nimes, France), according to the manufacturer's instructions. The reaction time of the assay is 29 min. The specific fluorescence was measured by simultaneous dual wavelength measurement at 665 and 620 nm using KRYPTOR compact automate. A homogeneous sandwich fluoroimmunoassay can be used equally on the two automates KRYPTOR and KRYPTOR compact (B.R.A.H.M.S Thermofisher Scientific, Hennigsdorf / Berlin, Germany). T75369WO BOEHMERT & BOEHMERT

[0770] The plasma median MR-proADM concentration in normal (healthy) subjects was 0.41 (interquartile range 0.23 - 0.64) nmol / L (Smith etal. 2009. Clin Chem 55: 1593-1595} using the automated sandwich fluorescence assay for the detection of MR-proADM as described in Caruhel et al. (Caruhel et al. 2009. Clin Biochem 42: 725-8}.

[0771] Example 9 - Development of anti-DPP3 antibodies with a high success rate using SELMA

[0772] One method for the development of monoclonal antibodies against a specific antigen is the primary screening of hybridoma cells, wherein those cells that produce the antibodies of interest must be identified, selected, and separated from the other cells. While effective, the result of this method is that only a portion of the hybridoma cells that are tested are successful. This method involves separating the pool of hybridomas to ensure that the antibodies they produce are monoclonal, testing to determine whether they bind to the target antigen, and selecting binding candidates for the desired application. One aspect of this method involves the separation of the hybridoma cell and the antibody produced by this cell, e.g. the antibody gets secreted into the culture medium and thereby is detached from its parent cell. This separation requires the initial pool of hybridoma cells to be separated by sufficient dilution or by sorting with a cell sorter to generate single cell cultures, subsequent culturing, and separate testing of the culture supernatants containing the antibodies. This method allows the link to be made between the positively identified antibody and the corresponding producing hybridoma cell line.

[0773] In this Example, an alternative method is described that will capture the released antibody on the cell surface of the corresponding hybridoma cell. It is expected that doing will enable the rapid identification and selection of the desired hybridomas while discarding the others, thereby resulting in a high success rate. SELMA™ technology (Single-cell ELISA-linked MACS / Affinity) has been developed and shown to achieve a high success rate (see WO2015161835, which is incorporated by reference; Listek, M., etal, 2020, Scientific Reports, 10(1), 1664,' Listek, M., et al., 2023, ACS Applied Materials & Interfaces, 15(37), 43219 43222}. In this Example, the SELMA method is utilized to identify, select, and isolate hybridoma cell lines producing monoclonal anti-DPP3 antibodies with a high success rate.

[0774] Overview: T75369WO BOEHMERT & BOEHMERT

[0775] A schematic overview is provided in Figure 14. The SELMA method uses genetically engineered myeloma cells for the generation of hybridoma cells that express the specific engineered protein on the surface of the cell (the features of this protein are described in more detail below). These modified myeloma cells are fused with the B-cells of immunized mice to create a new line of transgenic hybridoma cells possessing the new artificial surface protein. The overall effect of these changes is to allow the new hybridomas to “capture” the antibodies they produce. The captured antibody and the corresponding cell may be labelled with a fluorescent dye and thus be sorted from the pool of hybridoma cells. This allows the identification of the secreted antibodies and the corresponding mother cell in one simple screening step. By an automated flow cytometry -based cell sort, the desired antibody producing cells may be selected with a high success rate that reduces experimentation and timelines, minimizing trial and error. Using the SELMA method, the antigen attaches to the marker on the cell surface. In turn, the antibody binds to the immobilized antigen — thus physically connecting the antibody to its producing cell.

[0776] Method:

[0777] To induce antigen-specific B lymphocytes in mice, the mice are immunized with purified native human DPP3 (e.g., SEQ ID NO: 1) or with a DPP3 peptide (e.g. SEQ ID NO: 2) (4TEEN4 Pharmaceuticals, Hennigsdorf, Germany). The primary immunization contains 100 pg of DPP3 and is administered intraperitoneally together with an adjuvant. After 4-8 weeks, a second immunization without adjuvant is performed, and 7 days later the spleen cells of the mice are isolated and counted. At the same time, cells of the modified myeloma cell line intended for the fusion are also prepared and used in the appropriate amount for the fusion, as described below.

[0778] The genetic modification of the myeloma cells comprises the transfection with a vector encoding a specific protein expressed at the cell surface: The genetic construct contains a truncated variant of the human immature epidermal growth factor (EGF)-receptor (EGF-R), a hemagglutinin epitope (HA) and a biotin acceptor peptide (AP; “AviTag”). To express the HA- AP-EGF -receptor fusion protein on the surface of myeloma cells, the signal peptide of the immature human EGF-receptor is inserted at the N-terminus of the cloned hemagglutinin epitope (HA) containing a biotin acceptor peptide (AP) sequence, and a truncated variant of the mature humane EGF-receptor (aa 1-651) at its C-terminus. The construct is controlled by the human elongation factor 1 -alpha (EFla)-promoter. T75369WO BOEHMERT & BOEHMERT

[0779] Spleen cells and myeloma cells are used in a ratio of 3: 1 for a fusion. For this, the cells are washed and mixed together in fusion buffer (125 mM NaCl, 5 mM KC1, 4 mM CaCh, 2.5 mM MgCh, and 5 mM Tris-HCl, pH 7.4, adjusted). The cell pellet is resuspended in 200 pl fusion buffer and mixed with 200 pl PEG8000 solution. The cell solution is then transferred to an electroporation cuvette with an electrode spacing of 0.2 cm and the electroporation is carried out. The cells are fused by electroporation at a DC voltage of 500-700 V and a pulse duration of 20 ps. The cells then incubate for another 3 min in the cuvette and are then added to selection medium: DMEM, with 20% fetal calf serum, 2 mM glutamine, 50 pM beta-mercaptoethanol, 200 pM hypoxanthine, 12 pM azaserine, 32 pM 2 ' deoxythymidine, and 5 pg / mL puromycin, and are transferred and cultured until sorted.

[0780] In preparation of the in vitro biotinylation of the cells via the transgenically mediated acceptor peptide, the cell supernatant is discarded and the cells are washed twice with PBS supplemented with 5 mM MgCh. Following that, the cells are incubated with a mixture consisting of 5 mM MgCh, 1 mM ATP, 10 pM biotin, and 0.789 pg per 106cells BirAbiotin ligase (GeneCopoeia™ Source BioScience LifeSciences, Nottingham UK) for 30 min. To get rid of the reaction mixture, the cells are washed four times with PBS containing 5 mM MgCh. Thereafter, the cells are ready for being loaded with the DPP3 -streptavidin conjugate.

[0781] The DPP3 -streptavidin conjugate is prepared by coupling purified native human DPP3 (e.g., SEQ ID NO: 1) to streptavidin via the homobifunctional crosslinker glutaraldehyde. 3 mg of DPP3 are mixed with 2 mg of streptavidin and then mixed with 0.25% glutaraldehyde in 1 mL lx PBS for 2 hours at 4°C. The reaction mixture is then dialyzed against 5 L of IxPBS for 1 hour.

[0782] For the coupling of the DPP3 -streptavidin conjugate to the cells, 33 pg of the DPP3 -streptavidin conjugate are added to the previously biotinylated 106cells and are incubated for 20 minutes at 22°C. The cells are then centrifuged and taken up in fresh cell culture medium. The cells are incubated for four hours at 37°C and 8% CO2 to start production of antibodies. The cells are transferred to a reaction vessel and pelleted at 200g for 8 min with a 1% bovine serum albumin solution to which 2 mM ethylenediaminetetraacetic acid (EDTA) has been added. The cell pellet is again resuspended in blocking solution (IxPBS, 5% neonatal calf serum) and labeled with fluorescein isothiocyanate goat anti-mouse antibody (Jackson ImmunoResearch Europe Ltd., Ely, UK) in a concentration of 1 pg per 106cells; incubation time is for 1 hour on ice in the dark. T75369WO BOEHMERT & BOEHMERT

[0783] This is followed by another washing step and the cell pellets are taken up in 300 pl buffer for a fluorescence-activated cell sorter, namely 0.5% bovine serum albumin, 0.01% NaNs in PBS. For the fluorescence-activated cell sorting, a BD FACS Aria III and Flow Jo software are used. The positively labeled cells that produce the desired antibody that has bound its antigen DPP3 on the cell are then sorted out by the sorting function of the flow cytometer. The fraction obtained will be pelleted by centrifugation.

[0784] The cell pellet is then taken up in complete medium, RPMI 1640, 10% fetal calf serum, 2 mM glutamine, 50 pM beta-mercaptoethanol, and seeded onto 96-well cell culture plates. The cell culture plates already contain a feeder cell population that is isolated from the mouse peritoneum, which supports the growth of the freshly fused and sorted hybridoma cells.

[0785] The antibodies harvested from the cell culture supernatants are later re-tested for binding to DPP3. Antibodies from the vast majority of cell cultures will test positive for binding to the DPP3 antigen.

[0786] Example 10: Development of anti-DPP3 antibodies and selection based on their inhibitory activity

[0787] One method for the development and selection of monoclonal antibodies against inter alia a conjugated peptide derived from the DPP3 amino acid sequence SEQ ID NO: 2 is described herein see above) and additionally described in WO2017182561.

[0788] In this Example, a new method is described that will allow for the selection of inhibitory antibodies directly based on functional features. By utilizing this method, it is expected that almost any monoclonal antibody generated using SEQ ID NO: 2 as the immunogen, which is nested within the catalytic site, will have the ability to strongly inhibit DPP3 enzymatic activity in vitro.

[0789] Peptide / conjugate for immunization:

[0790] The SEQ ID NO: 2 DPP3 peptide for immunization (peptides & elephants, Hennigsdorf, Germany) was synthesized with an additional N-terminal cysteine residue — as compared to the corresponding amino acid sequence in human DPP3 — for conjugation of the peptide to bovine thyroglobulin (BTG). The peptide was covalently linked to BTG by using Sulfolink-coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the T75369WO BOEHMERT & BOEHMERT instruction manual of Perbio-science. (Thermo Scientific, Cat. No. 20401 (MAN0011276_SulfoLnk_Coupling_Resin_UG.pdf))

[0791] Procedure for Immobilizing a Peptide or Protein Having Free Sulfhydryls

[0792] A. Additional Materials Required

[0793] • Column: Choose a glass or plastic column size appropriate for the volume of SulfoLink Resin to be used. The Disposable Column Trial Pack (Product No. 29925) contains accessories plus two each of three different column sizes, appropriate for 0.5-10mL resin bed volumes. Alternatively, several centrifuge-ready Thermo Scientific Pierce Columns are available for resin bed volumes from 25 pl to lOmL.

[0794] • Coupling Buffer: 50mM Tris, 5mM EDTA-Na; pH 8.5. Prepare a volume equal to 20 times the volume of SulfoLink Resin to be used.

[0795] • Quenching Reagent: L-cysteine»HCl (Product No. 44889)

[0796] • Wash Solution: IM sodium chloride (NaCl)

[0797] • Storage Buffer: Phosphate-buffered saline (PBS) or other suitable buffer containing 0.05% sodium azide (NaN 3)

[0798] B. Prepare SulfoLink Resin Column

[0799] 1. Equilibrate SulfoLink Coupling Resin and all other reagents to room temperature.

[0800] 2. Stir or swirl bottle to evenly suspend the resin, and then use a wide-bore pipette to transfer an appropriate volume of the 50% resin slurry to an empty column. For example, transfer 2mL of resin slurry to obtain a ImL resin bed.

[0801] 3. Equilibrate column with four resin-bed volumes of Coupling Buffer. Replace the bottom column cap.

[0802] Note: When using gravity-flow columns, do not allow the resin bed to become dry at any time throughout the procedure. Add more solution or replace the bottom cap on the column whenever the buffer drains down to the top of the resin bed.

[0803] C. Couple Peptide / Protein to Resin

[0804] 1. Dissolve prepared (i.e., reduced) peptide / protein in Coupling Buffer and add it to the column. Use l-2mL of peptide or protein solution per milliliter of SulfoLink Coupling Resin. If desired, retain a small amount of the peptide or protein solution for later comparison to the coupling reaction flow-through fraction to estimate coupling efficiency. T75369WO BOEHMERT & BOEHMERT

[0805] 2. Replace the top cap and mix column (by rocking or end-over-end mixing) at room temperature for 15 minutes.

[0806] 3. Stand the column upright and incubate the column at room temperature for an additional 30 minutes without mixing.

[0807] 4. Sequentially remove top and bottom column caps and allow the solution to drain from the column into a clean tube.

[0808] 5. Place the column over a new collection tube and wash column with three resin-bed volumes of Coupling Buffer.

[0809] 6. Determine the coupling efficiency by comparing the protein / peptide concentrations (e.g., by absorbance at 280nm) of the noncoupled fraction (Step 4) to the starting sample (Step 1).

[0810] D. Block Nonspecific Binding Sites on Resin

[0811] 1. Replace the bottom cap on column. Prepare a solution of 50mM L-Cysteine»HCl in Coupling Buffer.

[0812] 2. Add one resin-bed volume of 50mM cysteine solution to the column. Mix for 15 minutes at room temperature, and incubate the reaction without mixing for an additional 30 minutes.

[0813] E. Washing the Column

[0814] 1. Sequentially remove the top and bottom caps and allow the column to drain.

[0815] 2. Wash the column with at least six resin-bed volumes of Wash Solution (IM NaCl).

[0816] 3. Wash the column with two resin-bed volumes of degassed Storage Buffer.

[0817] Immunization of mice, immune cell fusion and screening:

[0818] Five Balb / c mice were intraperitoneally (i.p.) injected with 100 pg DPP3-peptide-BTG- conjugate at day 0 (emulsified in TiterMax® Gold adjuvant (Sigma-Aldrich, Taufkirchen, Germany), 100 pg at day 14 (emulsified in complete Freund’s adjuvant), and 50 pg at days 21 and 28 (emulsified in incomplete Freund’s adjuvant). On day 49 the animals received an intravenous (i.v.) injection of 50 pg DPP3-peptide-BTG-conjugate dissolved in saline. Three days later, the mice were sacrificed and the immune cell fusion was performed.

[0819] For the immune cell fusion, splenocytes from the immunized mice and cells of the myeloma cell line SP2 / 0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37°C. After washing, T75369WO BOEHMERT & BOEHMERT the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement). After one week, the HAT medium was replaced with HT medium for three passages followed by returning to the normal cell culture medium.

[0820] For the primary screening of cell culture supernatants a dual test procedure was applied as follows, which comprised measurement of binding to DPP3, and, in parallel, measurement of inhibitory enzymatic activity: The available total amount of 200 pL cell culture supernatant per hybridoma cell line candidate was split into half and subjected to a binding test and an inhibition test, respectively, as shown in Figure 15. Two previously described anti-DPP3 antibodies were used as controls (murine antibodies clone #2555 and clone #1967 mentioned in Table 3 of WO2017182561; 4TEEN4 Pharmaceuticals, Henningsdorf, Germany).

[0821] Binding test:

[0822] 96-well microtiter plates (white; high binding; Greiner Bio-One International GmbH, Austria) were coated with goat anti-mouse IgG (Arista, Fortis Life Science, Boston, Massachusetts, USA), as follows: 100 pl of goat anti-mouse IgG diluted at 5 pg / mL in coating buffer (71.5 mM NaHCOs, 28.5 mMNa2CO3) were added per well, and plates were incubated at 4°C overnight. Plates were rinsed once with 300 pL / well blocking buffer (6.5 mMKH2PO4, 3.5 mMNa2HPO4, 3% Karion, 0.5% BSA), and another 300 pL of blocking buffer were added, and plates were incubated for one hour at 22°C. Blocking solution was discarded, and plates were washed once with wash buffer (20 mM PBS, pH 7.4, 0.1 % Tween 20).

[0823] 100 pL of each undiluted cell culture supernatant were added and incubated for 90 minutes at 22°C under agitation at 600 rpm to capture the IgG from the supernatant.

[0824] Due to the limited and defined binding capacity of the goat anti-mouse IgG solid phase, this step ensured that the same amounts of antibodies per supernatant were immobilized for further testing, as excess amounts beyond the binding capacity were discarded, making further analysis independent from the varying original antibody concentrations in the supernatants.

[0825] After washing of the plates with wash buffer (4 x 350 pL per well), human native DPP3, labeled with acridinium NHS-ester was added. Purified human native DPP3 (SEQ ID NO: 1; 4TEEN4 Pharmaceuticals, Hennigsdorf, Germany) was labeled with acridinium NHS-ester as follows: 100 pg (100 pl) of a purified human native DPP3 (1 mg / ml in PBS, pH 7.4) were mixed with 10 pl acridinium NHS-ester (1 mg / ml in acetonitrile, In Vent GmbH, Germany; EP 0 353 971) T75369WO BOEHMERT & BOEHMERT and incubated for 30 min at room temperature. Labelled DPP3 was purified by gel-filtration HPLC on Shodex Protein 5 pm KW-803 (Showa Denko, Japan). The purified labeled DPP3 was diluted in assay buffer (PBS, 5 g / L bovine serum albumin, 0.075 g / L murine IgG, 1 g / L bovine IgG, 0.1% Tween 20, pH 7.4). The final concentration was approximately 7*106relative light units (RLU) of labeled compound (approximately 20 ng labeled DPP3) per 100 pl. The labeled acridinium ester chemiluminescence was measured as described here. Labeled DPP3 was added to the wells. After incubation for 90 minutes at 22°C under agitation at 600 rpm, the plates were washed with wash buffer (4 x 350 pL per well), and solid phase-bound acridinium ester chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0826] Inhibition test:

[0827] 96-well microtiter plates (black; high binding; Greiner Bio-One International GmbH, Austria) were coated with goat anti-mouse IgG (Arista, Fortis Life Science, Boston, Massachusetts, USA), as follows: 100 pl of goat anti-mouse IgG diluted at 5 pg / mL in coating buffer (71.5 mM NaHCOs, 28.5 mMNa2COs) were added per well, and plates were incubated at 4°C overnight. Plates were rinsed once with 300 pL / well blocking buffer (6.5 mMKH2PO4, 3.5 mMNa2HPO4, 3% Karion, 0.5% BSA), and another 300 pL of blocking buffer were added, and plates were incubated for one hour at 22°C. The blocking buffer solution was discarded, and plates were washed once with wash buffer (20 mM PBS, pH 7.4, 0.1 % Tween 20).

[0828] 100 pL of each undiluted cell culture supernatant were added and incubated for 90 minutes at 22°C under agitation at 600 rpm to capture the IgG from the supernatant.

[0829] Due to the limited and defined binding capacity of the goat anti-mouse IgG solid phase, this step ensured that the same amounts of antibodies per supernatant were immobilized for further testing, as excess amounts beyond the binding capacity were discarded, making further analysis independent from the varying original antibody concentrations in the supernatants.

[0830] After washing of the plates with wash buffer (4 x 350 pL per well), 100 pl of purified human native DPP3 was added per well (1 pg / mL in PBS, 1 g / L bovine serum albumin, 0.075 g / L murine IgG, 1 g / L bovine IgG, pH 7.4), and plates were incubated for 90 minutes at 22°C under agitation at 600 rpm. Plates were washed with wash buffer (4 x 350 pL per well), and 100 pL per well of substrate solution was added (100 pM Arg-Arg-B-naphthylamide in 50 mM Tris, 50 pM C0CI2, 0.125% Triton-X, pH 7.8). After adding substrate, the increase of fluorescence was T75369WO BOEHMERT & BOEHMERT monitored for 1 h at 37°C in Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG, Germany) using excitation wavelength of 340 nm and emission wavelength of 410 nm. Slopes of increasing fluorescence, determined as relative fluorescence units (RFU) per minute, represent the enzymatic activity of immobilized DPP3, and the RFU are directly proportional with molar amount of the B-naphthylamine generated (76,994, 123 RFU / pmol).

[0831] Relating inhibition test results with binding test results:

[0832] Cell culture supernatants with no binding test signal above background were discarded for further analysis, as these do not contain detectable anti-DPP3 antibodies. For all others, the enzymatic DPP3 activity determined in the inhibition test (RFU / min) was divided by the binding test signal (RLU). This ratio represents the relative inhibitory activity of an anti-DPP3 antibody: A non-inhibitory or weakly inhibiting antibody would give a binding signal and a high DPP3 enzymatic activity signal. In contrast, a strongly inhibiting antibody would give a binding signal and a low DPP3 enzymatic activity signal. As discussed below, known weakly inhibiting as well as strong inhibiting anti-DPP3 antibodies have been run in parallel to the cell culture supernatants to facilitate interpretation of the test results obtained for cell culture supernatants.

[0833] Results of testing:

[0834] For the validation of the binding test and the inhibition test described above, available strongly inhibiting (AK1967) and a weakly inhibiting (AK2555; CDRs are SEQ ID NO: 73 to SEQ ID NO: 78 as shown herein below; variable regions of the heavy and light chains are SEQ ID NO: 79 to SEQ ID NO: 80, respectively, as shown herein below) mouse monoclonal anti-DPP3 antibodies were assessed in both tests at several dilutions in cell culture medium (see Table 8). For both antibodies it was evident that results in both tests were essentially independent from the antibody concentration used: Signals obtained for 100 ng / mL and 10,000 ng / mL differed by maximally a factor of approximately 2. Antibody AK2555 was generated using full-length DPP3 (SEQ ID NO: 1) as the immunogen in mice and is a murine antibody. (See, e.g., the incorporated by reference US Patent No. 11, 726, 094, Example 3. )

[0835] For the strongly inhibiting antibody (AK1967) the ratio of activity divided by binding was approximately 0.0006, whereas for the weakly inhibiting antibody (AK2555), it was T75369WO BOEHMERT & BOEHMERT approximately 1,000-fold higher (Table 8). These results demonstrated that the design of both tests and the evaluation fulfill the needs of the analysis in this Example.

[0836] Table 8: Results of binding test and inhibition test obtained for a strongly inhibiting (AK1967) and a weakly inhibiting (AK2555) monoclonal anti-DPP3 antibody.

[0837] Results of the binding test and the inhibition test obtained for the cell culture supernatants are shown in Table 9. Only cell culture supernatants exhibiting more than 3-fold the binding observed in the absence of the cell culture supernatant, e.g. the buffer control providing the unspecific binding (UB), were considered, and ranked by their binding test signal. From the 768 cell culture supernatants obtained from the cell fusion, 30 exhibited a binding signal that was at least 3-fold higher than the UB. Of note, all of these, without exception, showed a very low activity signal, and the ratio of activity divided by binding was similarly low as observed for the strongly inhibiting reference antibody PCZ, which had been developed earlier by the same immunization technique as described in the example.

[0838] Table 9: Results of binding test and inhibition test obtained for the cell culture supernatants in the primary screening of the example. T75369WO BOEHMERT & BOEHMERT

[0839] The results demonstrated that by application of the test designs described in this Example, monoclonal anti-DPP3 antibodies may be selected directly based on the functional feature that is their ability to inhibit DPP3 enzymatic activity, and secondly, that any monoclonal antibody raised against SEQ ID NO: 2 has the ability to strongly inhibit DPP3 enzymatic activity in vitro.

[0840] References

[0841] Abramic, Marija, and Dejan Agic. 2022. “Survey of Dipeptidyl Peptidase III Inhibitors: From Small Molecules of Microbial or Synthetic Origin to Aprotinin.” Molecules 27 (9). https: / / doi.org / 10.3390 / molecules27093006.

[0842] Arrigo, Mattia, Mari ell Jessup, Wilfried Mullens, Nosheen Reza, Ajay M Shah, Karen Sliwa, T75369WO BOEHMERT & BOEHMERT and Alexandre Mebazaa. 2020. “Acute Heart Failure.” Nature Reviews Disease Primers 6 (1): 16.

[0843] Ayala Ceja, Melanie, Mobina Khericha, Caitlin M Harris, Cristina Puig-Saus, and Yvonne Y Chen. 2024. “CAR-T Cell Manufacturing: Major Process Parameters and next- Generation Strategies.” Journal of Experimental Medicine 221 (2): e20230903.

[0844] Balint, Laszlo, Nathan P Nelson-Maney, Yanna Tian, Stephen D Serafin, and Kathleen M Caron. 2023. “Clinical Potential of Adrenomedullin Signaling in the Cardiovascular System.” Circulation Research 132 (9): 1185-1202. https: / / doi.org / 10.1161 / CIRCRESAHA.123.321673.

[0845] Blet, Alice, Benjamin Deniau, Karine Santos, Dirk P.T. van Lier, Feriel Azibani, Xavier Wittebole, Benjamin G. Chousterman, et al. 2021. “Monitoring Circulating Dipeptidyl Peptidase 3 (DPP3) Predicts Improvement of Organ Failure and Survival in Sepsis: A Prospective Observational Multinational Study.” Critical Care 25 (1): 1-10. https: / / doi.org / 10.1186 / sl3054-021-03471-2.

[0846] Caironi, Pietro, Roberto Latini, Joachim Struck, Oliver Hartmann, Andreas Bergmann, Giuseppe Maggio, Marco Cavana, et al...

Claims

T75369WO BOEHMERT & BOEHMERTCLAIMS1. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient wherein the level of Pro-Adrenomedullin or a fragment thereof in a sample of bodily fluid obtained from said patient is above a threshold.

2. A method of therapy guidance or therapy stratification or therapy monitoring of a treatment of a patient with a DPP3 binder, the method comprising:• providing a sample of bodily fluid of said patient,• determining the level of Pro-Adrenomedullin or a fragment thereof in said sample, and• comparing said level of Pro-Adrenomedullin or a fragment thereof to a threshold, wherein the level of Pro-Adrenomedullin or a fragment thereof in said sample being above said threshold is indicative that said patient requires treatment with a DPP3 binder or wherein if said level of Pro-Adrenomedullin or a fragment thereof in said sample is above said threshold, said patient is treated with a DPP3 binder.

3. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1, or method according to claim 2, wherein said Pro-Adrenomedullin or a fragment thereof is selected from the group comprising Pro-Adrenomedullin (SEQ ID No. 33), PAMP (SEQ ID NO: 34), PAMP-Gly (SEQ ID NO: 35), MR-proADM (SEQ ID NO: 38), ADM-Gly (SEQ ID NO: 37), mature ADM (SEQ ID NO: 36) and CT- proADM (SEQ ID NO: 39).

4. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or 3, or method according to claim 2 or 3, wherein said fragment of Pro-Adrenomedullin is MR-proADM and the threshold of the level of MR-proADM is between 0.5 and 2 nmol / L, more preferred between 0.6 and 1.5 nmol / L, even more preferred between 0.7 and 1 nmol / L, most preferred said threshold is 0.8 nmol / L, or said threshold of the level of MR-proADM is an x-fold of the median level of MR-proADM in a healthy population, particularly the threshold level of MR-proADM is in the range between 1.2-fold and 4.9-fold, more preferred between 1.5-fold and 3.7-fold, even moreT75369WO BOEHMERT & BOEHMERT preferred between 1.7-fold and 2.4-fold, most preferred said threshold is 2.0-fold of the median of the level of MR-proADM in a healthy population.

5. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or 3, or method according to claim 2 or 3, wherein said fragment of Pro-Adrenomedullin is mature PAMP and the threshold of the level of mature PAMP is between 0.7 and 1.2 pmol / L, more preferred between 0.5 and 1.0 pmol / L, most preferred said threshold is 0.9 pmol / L, or said threshold level of mature PAMP, and / or the threshold of the level of mature PAMP is an x-fold of the mean level of mature PAMP in a healthy population, particularly the threshold level of mature PAMP is in the range between the 1.4-fold and 2.4-fold, more particular in the range between 1.6-fold and 2.0-fold, most particular said threshold is the 1.8-fold of the mean of the level of mature PAMP in a healthy population.

6. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or 3, or method according to claim 2 or 3, wherein said fragment of Pro-Adrenomedullin is PAMP-Gly and the threshold of the level of PAMP-Gly is between 1.5 and 2.4 pmol / L, even more preferred between 1.8 and 2.2 pmol / L, most preferred said threshold is 2 pmol / L, or said threshold level of PAMP-Gly, and / or the threshold of the level of PAMP-Gly is an x-fold of the mean level of PAMP-Gly in a healthy population, particularly the threshold level of PAMP-Gly is in the range between the 1.3-fold and 2.1-fold, more particular in the range between 1.6-fold and 1.9-fold, most particular said threshold is the 1.7-fold of the mean of the level of PAMP-Gly in a healthy population.

7. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or 3, or method according to claim 2 or 3, wherein said fragment of Pro-Adrenomedullin is ADM-Gly and the threshold of the level of ADM-Gly is between 25 and 125 pg / ml, more preferred between 30 and 100 pg / ml, even more preferred between 35 and 75 pg / ml, most preferred said threshold is 40 pg / ml, said threshold level of ADM-Gly, and / or the threshold of the level of ADM-Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly the threshold level of ADM-Gly is in the range between the 0.9-fold and 4.6-fold, more particular in the range between the 1.1-fold and 3.7-fold, more particular in the range between 1.3-fold and 2.8-fold, most particular said threshold is the 1.5-fold of the median of the level of ADM-Gly in a healthy population,T75369WO BOEHMERT & BOEHMERT or said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly, and the threshold of the level of ADM-Gly is between 40 and 175 pg / ml, more preferred between 40 and 125 pg / ml, most preferred 40 pg / ml, or wherein said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro-Adrenomedullin is ADM-Gly and said threshold level of ADM-Gly is an x-fold of the median level of ADM-Gly in a healthy population, particularly the threshold level of ADM-Gly is in the range between 1.5-fold and 6.5-fold, more preferred between 1.5-fold and 4.6-fold, most preferred said threshold is 1.5-fold of the median of the level of ADM-Gly in a healthy population.

8. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or 3, or method according to claim 2 or 3, wherein said fragment of Pro-Adrenomedullin is mature ADM and the threshold of the level of mature ADM is between 35 and 125 pg / ml, more preferred between 40 and 100 pg / ml, even more preferred between 50 and 90 pg / ml, most preferred said threshold is 70 pg / ml, or said threshold level of mature ADM, and / or the threshold of the level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, particularly in the range between the 2.6-fold and 9.1-fold, more particular in the range between the 2.9- fold and 7.3-fold, more particular in the range between 3.6-fold and 6.6-fold, most particular said threshold is the 5.1 -fold of the median of the level of mature ADM in a healthy population, or or said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro-Adrenomedullin is mature ADM and said threshold level of mature ADM, and the threshold of the level of ADM-Gly is between 25 and 150 pg / ml, more preferred between 30 and 125 pg / ml, most preferred 30 pg / ml, or wherein said patient is a cancer patient receiving chimeric antigen receptor T-cell therapy (CAR T-cell therapy), said fragment of Pro-Adrenomedullin is mature ADM and said threshold level of mature ADM is an x-fold of the median level of mature ADM in a healthy population, particularly in the range between 1.8-fold and 10.9-fold, more preferred between 2.2-fold and 9.1-fold, most preferred said threshold is 2.2-fold of the median of the level of mature ADM in a healthy population.

9. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or 3, or method according to claim 2 or 3, wherein said fragmentT75369WO BOEHMERT & BOEHMERT of Pro-Adrenomedullin is CT-proADM and the threshold of the level of CT-proADM is between 75 and 350 pmol / L, more preferred between 100 and 250 pmol / L, even more preferred between 125 and 200 pmol / L, most preferred said threshold is 150 pmol / L, or said threshold level of CT-proADM, and / or the threshold of the level of CT-proADM is an x-fold of the median level of CT-proADM in a healthy population, particularly the threshold level of CT-proADM is in the range between the 1.0-fold and 4.5-fold, more particular in the range between the 1.3-fold and 3.2-fold, more particular in the range between 1.6-fold and 2.6-fold, most particular said threshold is the 1.9-fold of the median of the level of CT-proADM in a healthy population.

10. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or any one of claims 3 to 9, or method according to any one of claims 2 to 9, wherein said sample is a bodily fluid sample selected from the group comprising whole blood, plasma and serum.

11. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or any one of claims 3 to 10, or method according to any one of claims 2 to 10, wherein said patient is a patient suffering from a severe infectious disease, inflammation, sepsis, shock, pulmonary diseases including ARDS, cardiovascular diseases including acute myocardial infarction, acute or chronic heart failure, organ dysfunction, a hepatic disease, a renal disease, an infectious disease, an autoimmune disease including ulcerative colitis, arthritis other chronic inflammatory diseases, other chronic inflammatory diseases, age-related macular degeneration, diabetic macular edema, a psychiatric disease, a neurodegenerative disease, or cancer, in particular wherein the patient is a cancer patient receiving immune effector cell therapy (IECT), in particular chimeric antigen receptor T-cell therapy (CAR T-cell therapy), and / or wherein said patient suffers from organ dysfunction, in particular kidney dysfunction, lung dysfunction, heart dysfunction, or liver dysfunction, and / or wherein said patient suffers from shock, in particular selected from the group comprising cardiogenic shock, septic shock and anaphylactic shock.

12. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or any one of claims 3 to 11, or method according to any one of claims 2 to 11, wherein the DPP3 binder is selected from the group comprising smallT75369WO BOEHMERT & BOEHMERT molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non-Ig scaffolds.

13. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or any one of claims 3 to 12, or method according to any one of claims 2 to 12, wherein said DPP3 binder is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised in SEQ ID No. 1 :MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSP EAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNY KSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLR HLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPY YEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKA KAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYR DPFGSRGEFEGFVAVVNKAMSAKFERLASAEQLLKELPWPPTFEKDKFLTPDF TSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLE EDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPET GEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADA EDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGL VTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRAL YEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAA GLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA, particularly wherein said DPP3 binder is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds an epitope of at least 4 or 5 amino acids in length comprised inSEQ ID No. 2: CETVINPETGEQIQSWYRSGE, particularly SEQ ID NO: 3: INPET G.

14. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or any one of claims 3 to 13, or method according to any one of claims 2 to 13, wherein said binder is a monoclonal antibody or monoclonal antibody fragment,T75369WO BOEHMERT & BOEHMERT particularly wherein said monoclonal antibody or monoclonal antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity determining regions (CDRs) in the heavy chain comprise the sequences:CDR1: SEQ ID NO: 6: GFSLSTSGMSCDR2: SEQ ID NO: 7: IWWNDNKCDR3: SEQ ID NO: 8: ARNYSYDY and wherein the complementarity determining regions (CDRs) in the light chain comprise the sequences:CDR1: SEQ ID NO: 9: RSLVHSIGSTYCDR2: KVSCDR3: SEQ ID NO: 10: SQSTHVPWT, more particularly wherein said monoclonal antibody or monoclonal antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment, even more particularly wherein said monoclonal antibody or monoclonal antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence:SEQ ID NO: 11:MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLS TSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQV VLTMTNMDPVDTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG, and wherein the light chain comprises the sequence:SEQ ID NO: 12:T75369WO BOEHMERT & BOEHMERTMETDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSI GSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA EDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLS SPVTKSFNRGEC, even more particularly wherein said monoclonal antibody or monoclonal antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence:SEQ ID NO: 68:QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHI WWNDNI<SYNPALI<SRLTITRDTSI<NQVVLTMTNMDPVDTGTYYCARNYS YDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, and wherein the light chain comprises the sequence:SEQ ID NO: 69:DIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLI YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFG GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC.

15. DPP3 binder for use in therapy or prevention of endothelial dysfunction in a patient according to claim 1 or any one of claims 3 to 14, or method according to any one of claims 2 to 14, wherein said DPP3 binder is a small molecule and is selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, or a derivative or analog of any of the aforementioned compounds, and benzimidazol derivatives (i.e. compounds having a benzimidazole scaffold).

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