DE102022115364A1 - FATP2 in T cells as a target molecule for the treatment of autoimmune diseases - Google Patents

Abstract

The present invention relates to the role of the FATP2 protein (Fatty Acid Transport Protein 2) in T cells in the development of autoimmune diseases, in particular rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases such as ulcerative colitis, and other autoimmune diseases with T -cellular involvement. The present invention particularly relates to methods for identifying compounds that bind to FATP2 protein and to the use of FATP2 protein to screen and identify FATP2-interacting and FATP2-inhibiting compounds. The present invention further relates to pharmaceutical compositions for use in the treatment of autoimmune diseases, in particular pharmaceutical compositions comprising active ingredients that bind to and/or inhibit the FATP2 protein.

Description

Technical field of the invention

The present invention relates to the role of the FATP2 protein (Fatty Acid Transport Protein 2) in T cells in the development of autoimmune diseases, in particular rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, multiple sclerosis , and other autoimmune diseases with T-cell involvement. The present invention particularly relates to methods for identifying compounds that bind to FATP2 protein and to the use of FATP2 protein to screen and identify FATP2-interacting and FATP2-inhibiting compounds. The present invention further relates to pharmaceutical compositions for use in the treatment of autoimmune diseases, in particular pharmaceutical compositions comprising active ingredients that bind to and/or inhibit the FATP2 protein.

Background and state of the art

Autoimmune diseases affect approximately 5 to 8% of the population worldwide. They are associated with high morbidity and premature mortality; After cardiovascular and tumor diseases, they represent the third most common group of diseases. In autoimmune diseases, the immune system attacks the body's own healthy tissue because it can no longer be differentiated between "foreign" and "self". Systemic autoimmune diseases include rheumatic joint inflammation; They affect around 20,000 children as juvenile idiopathic arthritis (JIA) and around 1.5 million adults in Germany as rheumatoid arthritis. Both diseases are autoimmune diseases of unknown origin that cause intermittent chronic inflammation of the joints. In addition to the cells of the innate immune system such as neutrophils and monocytes, which trigger the inflammatory processes, cells of the adaptive immune system such as T cells also determine the chronic inflammatory reactions in the joints.

Despite increasing therapeutic options, autoimmune diseases cannot yet be cured. The significant involvement of T cells has been demonstrated in a variety of autoimmune diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, ulcerative colitis and multiple sclerosis. In particular, so-called “memory” T cells play a role. These are mainly found in tissue and are not always amenable to immunosuppression.

Their inflammatory potential cannot be controlled by regulatory T cells (Treg cells) within the inflamed joints. Nearly all T cells within the joints of patients with juvenile idiopathic arthritis exhibit the memory T cell phenotype.

The metabolism of T cells in patients with (childhood) rheumatism differs significantly from that of healthy individuals; There is a fundamental dysregulation of fatty acid metabolism in patients with childhood rheumatism, especially in the inflamed joint. This particularly affects the memory T cells, which make up up to 95% of the T cells in the inflamed joint.

The functions of memory T cells are determined by metabolic conditions, which can also be accompanied by a reprogramming of their functions (so-called “metabolic reprogramming”). For the necessary energy metabolism, T cells essentially use the three energy resources glucose, glutamine and fatty acids. The transport proteins for neither glutamine nor fatty acids have yet been clearly identified and defined.

The metabolic properties of memory T cells differ significantly from the properties of naive and effector T cells (Geltink et al. 2018). Resting naïve T cells utilize oxidative metabolism to efficiently produce energy; they absorb glucose at a very low rate to provide the energy necessary to maintain their operational metabolic functions. Naïve T cells use pyruvate from glucose breakdown to generate ATP via oxidative phosphorylation or fatty acid oxidation. Activated T cells, on the other hand, have to proliferate and therefore switch to an anabolic growth program so that they can carry out their effector functions. The predominant metabolic state of activated T cells (both CD4 and CD8 positive T cells) is aerobic glycolysis, which is characterized by the conversion of pyruvate from glucose breakdown to lactate, despite sufficient oxygen for one complete glucose oxides tion is available (Geltink et al. 2018). This process, known as the “Warburg effect” from previous studies in tumor biology, is a common feature of actively proliferating cells. After effector cells have completed their tasks, they either die or transform into memory T cells.

Memory T cells form the “immunological memory” of the immune system in that they respond to a repeated encounter with the antigen with a stronger immune reaction (“secondary immune response”). The signal transduction and metabolic pathways that regulate the generation of memory T cells are therefore of great interest. Mitochondrial fatty acid oxidation is necessary for the development of CD8+ memory T cells and is dependent on tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) (Pearce et al. 2009). Fatty acid oxidation generates acetyl coenzyme A (CoA), which can be further metabolized in the citric acid cycle, as well as FADH ₂ and NADH+H ⁺ , which can be used directly to generate adenosine triphosphate (ATP) via the electron transport chain. Free fatty acids are energy-rich molecules, and fatty acid oxidation may represent a preferred energy source for memory T cells (“Tmem”) because they rely on metabolic pathways dependent on oxidative phosphorylation. Additionally, greater mitochondrial content confers a bioenergetic advantage to memory T cells by enhancing the rapid immune response in response to re-infection. In contrast to effector T cells, which are particularly dependent on glycolysis, CD8-positive memory T cells therefore have a higher respiratory reserve capacity and rely primarily on fatty acid oxidation.

In tissue-resident CD8-positive memory T cells, genes encoding fatty acid-binding proteins (FABP4 and FABP5) are among the most highly upregulated genes, as is the gene encoding CD36, a so-called “lipid-scavenger cell -surface receptor” (Pan et al. 2017). In contrast, the data on CD4-positive T cells with regard to their fatty acid metabolism are still sparse. However, it has been shown that genetic deletion of ACC1, a rate-limiting enzyme in fatty acid biosynthesis, increases the formation of CD4-positive memory T cells, and that inhibition of ACC1 function increases the formation of memory T cells cells increased during parasite infection in mice (Endo et al. 2019).

Until now, nothing was known in the prior art about the uptake mechanism of fatty acids in T cells and the identity of transport molecules for fatty acids in T cells. The therapeutic options for autoimmune diseases are currently limited and a cure is not possible. In particular, no therapy option that targets the metabolism of immune cells is available.

Thus, an object underlying the present invention was to provide methods and means for identifying active ingredients, compounds and compositions, and their use in the treatment of autoimmune diseases.

The present application discloses the identification of a new molecular target molecule (“targets”) for the therapy of autoimmune diseases. In the context of the present invention, the fatty acid transport protein 2 (FATP2, Fatty Acid Transport Protein 2), encoded by the SLC27A2 gene, was identified as a fatty acid transporter in T cells, and it was demonstrated for the first time that its blockage impairs the function of the T cells. Influence cells in terms of their production of cytokines, the activation of their effector functions and their proliferation.

Expression of fatty acid transport protein-2 (FATP2) has so far been detected in the liver, small intestine, kidney, pancreas, and placenta (Falcon et al. 2010; Perez et al. 2020; Khan et al. 2020). The protein is encoded by the “Solute Carrier Family 27 Member 2” (SLC27A2) gene and has two main functions, the activation of long-chain fatty acids as a “Very Long-Chain Acyl-Coenzyme A (CoA)” synthetase (ACSVL), and the transport of coenzyme A-activated long-chain fatty acids as a fatty acid transport protein (Falcon et al. 2010; Khan et al. 2020; Melton et al. 2013).

The transport molecule FATP2 was developed by Falcon et al. (2010) has been linked to the development of non-alcoholic fatty liver disease in mice. Other studies suggest that the molecule may be partially responsible for the pathogenesis of diabetic kidney disease (Khan et al. 2020). It has also been shown that upregulation of the expression of the SLC27A2 gene or the FATP2 protein in differentiated thyroid carcinomas can be associated with increased proliferation and migration of tumor cells (Feng et al. 2021), and that the FATP2 protein in Tumors may be involved in the reprogramming of neutrophils (Veglia et al. 2019). WO2020/172510 (Wistar Institute) discloses methods for tumor therapy by inhibiting, blocking or downregulating FATP2 activity in MDSCs (myeloid-derived suppressor cells), a heterogeneous population of immature and pathologically activated myeloid cells that accumulate in large quantities in tumor patients and suppress proliferation of T cells and natural killer (NK) cells, and promote the growth of tumors through this immunosuppressive effect.

The present application discloses the SLC27A2 gene or the FATP2 protein for the first time as a target molecule and thus a new therapeutic approach for the development of therapeutics for the treatment of patients with autoimmune diseases.

Summary of the invention

For the first time and surprisingly, the inventors have identified FATP2, encoded by the SLC27A2 gene, a fatty acid transporter in T cells, especially in memory T cells, the blockage of which significantly influences the functions of these T cells (proliferation, production of cytokines). . This fundamental step in understanding T cell function offers for the first time the possibility of specifically influencing this T cell function and reprogramming it by changing metabolism.

Until now, the uptake receptor/transporter of free fatty acids, which, along with glucose, represent an essential source of energy, especially in inflamed tissue, was not known for T cells. The fatty acid transport protein identified as FATP2 is upregulated 10-fold in T cells in inflamed joints of patients, such as those with childhood rheumatism, compared to the blood of the same patients and healthy controls. It has also been shown that in memory T cells that are made to proliferate, FATP2 expression is upregulated 30-fold, and at the same time the cells' fatty acid uptake increases massively.

The present invention provides methods and means for identifying drugs, compounds and compositions for use in the treatment of autoimmune diseases, in particular for identifying highly effective drugs, compounds and compositions for use in the treatment of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic Inflammatory bowel diseases including ulcerative colitis and Crohn's disease, multiple sclerosis, and other autoimmune diseases with T-cell involvement.

In view of the prior art, it was an object of the present invention to provide a method for inhibiting or reducing fatty acid uptake into a T cell, activation and/or proliferation of a T cell.

A further object of the present invention was to provide a method for identifying an active ingredient that binds to and/or inhibits the FATP2 protein or a fragment thereof.

It was a further object of the present invention to provide a method of using a nucleic acid encoding the FATP2 protein, or a fragment thereof, or the FATP2 protein itself, or a fragment thereof, for the identification of an active ingredient FATP2, or a fragment thereof, binds to provide.

It was a further object of the present invention to provide active ingredients for use in the treatment of an autoimmune disease, particularly for use in the treatment of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases including ulcerative colitis and Crohn's disease, multiple sclerosis, and others Autoimmune diseases with T-cell involvement, based on the findings described above.

A further object of the present invention is to provide pharmaceutical compositions containing these agents and methods for preparing such pharmaceutical compositions based on the findings described above.

The described and other technical tasks are solved by the devices or methods according to the independent claims of the current invention. The dependent claims describe preferred embodiments. Value ranges that are limited by numerical values should always contain the stated limit values.

The invention and general advantageous refinements are explained in more detail below.

Description of the drawings

• 1 shows that T cells present in the synovial fluid of juvenile idiopathic arthritis (JIA) patients have increased fatty acid uptake that is associated with increased expression of FATP2/SLC27A2. The fatty acid receptor FATP2 is mainly expressed in CD4+ memory cells.
• 1A : Uptake of free fatty acids into CD4+ T cells from the blood or synovium of JIA patients. The measurement was carried out after incubating the cells for 15 minutes with Bodipye™FL C ₁₂ (2 µM) and subsequent flow cytometric analysis. The mean fluorescence intensity (MFI) is shown minus the respective FM0 control (ΔMFI). Data show mean expression in 4 patients ± standard error, **p ≤ 0.01.
• 1B : FATP2 protein expression in CD4+ T cells from the peripheral blood (PB) or synovium (SF) of JIA patients (flow cytometric analysis). A representative image is shown.
• 1C : Expression of SLC27A2 RNA in CD4+ T cells of the synovium of JIA patients compared to the blood of the same patients.
• 1D : Protein expression of FATP2 in CD4+ T cell subtypes (effector memory T cells (T _em ), tissue resident memory T cells (T _m ) and naive T cells (T _naive )) from the blood or synovium of JIA Patients (flow cytometric analysis). The mean fluorescence intensity (MFI) is shown minus the respective FM0 control (ΔMFI). Data show mean expression in 5 patients ± standard error, ***p ≤ 0.001. ****p <_ 0.0001.
• 1E : mRNA expression of FATP2 compared to other metabolic proteins in CD4+ memory T cells after 48 hours and 72 hours of stimulation with anti-CD3 and anti-CD28 antibodies and in unstimulated CD4+ memory T cells. The CD4+ memory T cells were isolated from the blood of healthy controls using magnetic cell separation. Data show the mean expression of 4 experiments with different donors ± standard error, **p ≤0.01, ***p ≤0.001.
• 2 shows that the FATP2 inhibitor Lipofermata inhibits fatty acid uptake in CD4+ T cells, and inhibits IFN-γ expression and proliferation without inducing apoptosis.
• 2A : Uptake of free fatty acids in CD4+ T cells after addition of Lipofermata in increasing concentrations (0, 2, 5, 7.5 and 10 µM). For this purpose, the cells were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours. The measurement was carried out after incubating the cells for 15 minutes with Bodipye™ FL C ₁₂ (2 µM) and subsequent flow cytometric analysis. The mean fluorescence intensity (MFI) is shown minus the respective FM0 control (ΔMFI). Data show mean expression measured in 3 experiments ± standard error.
• 2 B : Exemplary histogram of fatty acid uptake in CD4+ T cells without the addition of Lipofermata (right) and after the addition of Lipofermata (left).
• 2C : Percentage number of IFN-γ positive CD4+ T cells after addition of Lipofermata in increasing concentrations (0, 2, 5, 7.5 and 10 µM). For this purpose, the cells were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours. The determination of IFN-γ positive cells was carried out using flow cytometry after 5 hours of restimulation with PMA and ionomycin in the presence of GolgiPlug™. Data show mean expression measured in 5-6 experiments ± standard error, *p ≤ 0.01.
• 2D : Exemplary dot diagram of IFN-γ expression without the addition of Lipofermata (left, control) and after the addition of Lipofermata (right).
• 2E : Percentage of proliferated CD4+ T cells after addition of Lipofermata in increasing concentrations (0, 2, 5, 7.5 and 10 µM). For this purpose, the cells were stained with “cell proliferation dye eFlour™” and stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours. Proliferated cells were determined using flow cytometry. Data show mean expression measured in 5 experiments ± standard error, *p ≤ 0.01.
• 2F : Exemplary dot diagram of cell proliferation without the addition of Lipofermata (left, control) and after the addition of Lipofermata (right).
• 2G : Exemplary dot diagram of dead cells with Lipofermata treatment (right) and without (left, control). The cells were treated with anti-CD3 and anti-CD28 antibodies for 72 hours stimulated and stained with propidium iodide (PI) for flow cytometric live/dead differentiation.

Detailed description of the invention

Before the invention is described in detail, it should be noted that this invention is not limited to specific components of the devices described or steps of the methods described, since these methods or devices can vary. It should also be noted that the terminology used herein is used solely for the purpose of specific embodiments described and is not intentionally limited.

It should be noted that in the specification and in the appended claims, the simple form such as "a" or "the" includes a singular and/or plural subject, unless the context clearly dictates otherwise. In the event that a parameter range has been specified, the limiting numerical values are included as limit values for the disclosed or claimed numerical range.

It should also be noted that the embodiments disclosed herein are not to be construed as individual embodiments that would not relate to one another. Features discussed in connection with one embodiment are also intended to be disclosed in connection with other embodiments shown herein. If in one case a particular feature is disclosed not with one embodiment but with another, those skilled in the art will understand that this does not necessarily mean that that feature should not be disclosed with the other embodiment. The person skilled in the art will understand that it is the principle of this application to disclose the relevant feature also for the other embodiment, but that this has not been done for reasons of clarity and to keep the specification to a manageable extent.

Further, the contents of the prior art documents referenced herein are incorporated by reference. This is particularly true for prior art documents that disclose standard or routine procedures. In this case, the primary purpose of incorporating by reference is to enable sufficient disclosure and avoid lengthy repetition.

1. (i) Hemmen oder Reduzieren der SLC27A2-Genexpression der T-Zelle,
2. (ii) Hemmen oder Reduzieren der Aktivität des Fettsäure-Transportproteins-2 (FATP2), und/oder
3. (iii) Fördern des Abbaus des FATP2-Proteins.

According to a first aspect, the present invention relates to a method for inhibiting or reducing fatty acid uptake into a T cell, the activation and/or the proliferation of a T cell, the method comprising at least one step selected from the group consisting of

1. (i) inhibiting or reducing T cell SLC27A2 gene expression,
2. (ii) inhibiting or reducing the activity of fatty acid transport protein-2 (FATP2), and/or
3. (iii) Promote the degradation of FATP2 protein.

As used herein, the term “T cells” refers to cells including T precursor cells, T lymphocytes, cytotoxic (possibly CD8-positive) T cells, (possibly CD4-positive) T “helpers” -cells (Th cells), Thl cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, Tfh cells, memory T cells, regulatory T cells (Treg), Suppressor T cells (Tsup), natural killer T cells (NKT cells), naive T cells, activated T cells, mucosa-associated invariant T cells, alpha-beta T cells, gamma Delta T cells, thymocytes, double-positive (CD4/CD8-positive) thymocytes, primary T cells, mature immunocompetent T cells, autoreactive T cells, peripheral T cells, synovial T cells, and T cells -lines.

The said inhibition or reduction of SLC27A2 gene expression can, for example, be an SLC27A2 gene “knock-down”, a “knock-out”, a conditional “gene knock-out”, a gene change or mutation in the sense of an insertion, deletion and /or substitution, a gene modification using a gene editing system, RNA interference, siRNA and/or antisense RNA.

As used herein, the term “gene editing system” or “gene editing systems” refers to molecular biology techniques for targeted modification of DNA, for example the SLC27A2 gene. The classic gene editing systems include, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas method, the CRISPR/Cpfl system and so-called meganucleases. The specific recognition of DNA occurs in zinc finger nuclease, TALEN and meganuclease by a specific protein part, while in CRISPR systems it is mediated by a specific RNA (Zhu and Zhu, 2022).

Zinc finger nucleases (ZFNs) are artificially produced restriction enzymes. They contain a zinc finger domain that binds to DNA and a nuclease domain that cuts the DNA. The zinc finger domain can be engineered to recognize a specific DNA sequence.

TALENs are fusion proteins composed of a TAL effector DNA-binding domain and an endonuclease domain. The sequence-specific binding occurs through the DNA-binding domain introduced during protein design, after which a sequence-specific cut is carried out by the endonuclease.

The CRISPR/Cas method (“Clustered Regularly Interspaced Short Palindromic Repeats”, grouped short palindromic repeats at regular intervals and CRISPRassociated, CRISPR-associated protein) is a molecular biological method for specifically cutting and modifying DNA. Genes can be inserted, removed or switched off using the CRISPR/Cas system, as well as nucleotides in a gene can be changed. The DNA-cutting Cas enzyme binds a specific RNA sequence. This RNA sequence is followed by another RNA sequence that can bind to a DNA with a complementary sequence via base pairing. The RNA serves as a bridge between Cas and the DNA to be cut. By complexing Cas, RNA and DNA, the DNA-cutting Cas enzyme is brought into close proximity to the bound DNA, whereupon the enzyme cuts the (indirectly bound) DNA. If DNA is inserted into the interface, another DNA is added which has overlapping sequences for one of the two ends of the interface at both ends. The DNA to be inserted is connected to the ends of the interface through the cell's own DNA repair (Nidhi et al. 2021).

The endonucleases mentioned are used to introduce targeted changes in the genome of individual cells or complex organisms. The enzymes cut double-stranded DNA at a predetermined target sequence, creating double-strand breaks. The double-strand breaks in turn activate DNA repair processes in the cell, such as “non-homologous end-joining” (NHEJ) or homologous repair, which is also known as “homology directed repair” (HDR). While NHEJ is used to specifically inactivate genes, HDR can be used to specifically insert defined mutations or entire DNA sections into the genome. Genome editing can be used to specifically destroy a gene (gene “knockout”), to introduce a gene at a specific location in the genome (gene “knockin”), or to introduce a point mutation in a gene.

The so-called “base editing” is a new precise method of genome editing, which consists of changing individual bases in the DNA sequence). Here, a mutated form of the Cas9 nuclease, which can no longer cut DNA, is coupled with a deaminase in the form of a fusion protein. This fusion protein is able to specifically recognize a desired DNA sequence using an sgRNA (“single-guide” RNA) and to change a base through deamination. In the case of fusion with cytidine deaminase, the cytidine is converted into uracil, which is replaced with thymidine through DNA repair and replication. This mutates the base pair C-G to T-A. Alternatively, Cas9 can be coupled with an adenosine deaminase so that the adenosine is converted to inosine, which is replaced with guanosine after DNA repair and replication. In this case, the base pair A-T is converted to G-C (Zhu and Zhu, 2022).

RNA interference (RNAi, RNA silencing) is a natural mechanism in eukaryotic cells that specifically switches off genes in the cell nucleus. It allows so-called “gene silencing”. RNA interference is based on an interaction of short pieces of RNA with the mRNA involving several enzyme complexes. As a result of the activity of these enzyme complexes, the mRNA is split into several fragments, destroying the encoded information and preventing translation into a protein.

The so-called “small interfering RNA” (siRNA) are short RNA fragments that lead to the selective degradation of the complementary mRNA in the organism. In this way, they specifically prevent gene expression and the formation of proteins.

Antisense RNA is a single-stranded RNA that is complementary to a protein-coding mRNA. Almost all antisense RNAs have secondary structures such as “stem-loops” and sometimes also more complex tertiary structures such as “pseudoknots” between these secondary structures. These structural elements determine the rate of degradation by intracellular ribonucleases as well as the rate at which the antisense RNA pairs with the complementary mRNA. Mating stops the translation of the gene.

Said inhibition or reduction of FATP2 (fatty acid transport protein 2) protein activity may include the use of an agent that binds to the FATP2 protein and/or inhibits or reduces its activity.

Preferably, said T cell is a synovial T cell, particularly preferably a synovial memory T cell.

The FATP2 protein can be a mammalian, non-primate, primate and in particular a human FATP2 protein or a fragment thereof.

According to a second aspect, the present invention relates to a method for identifying an active ingredient that binds to the FATP2 protein of a T cell, or a fragment thereof, and/or the activity of the FATP2 protein of a T cell, or a fragment of it, inhibited or reduced.

1. (i) Bereitstellen des FATP2-Proteins oder eines Fragments davon,
2. (ii) Zugabe von mindestens einem Wirkstoff, der auf Bindung an das FATP2-Protein oder ein Fragment davon untersucht werden soll, und
3. (iii) Identifizierung des mindestens einen Wirkstoffs, der an das FATP2-Protein oder ein Fragment davon gebunden hat.

The procedure includes at least the following steps:

1. (i) providing the FATP2 protein or a fragment thereof,
2. (ii) adding at least one active ingredient to be tested for binding to the FATP2 protein or a fragment thereof, and
3. (iii) identifying the at least one active ingredient that has bound to the FATP2 protein or a fragment thereof.

Preferably, the active ingredient to be screened and identified according to the present invention is a FATP2 inhibitor or FATP2 antagonist, an agent that inhibits or reduces the activity of the FATP2 protein, or a fragment thereof.

The active ingredient according to the present invention can be selected from the group consisting of a low molecular weight compound, a peptide, in particular a natural or synthetic peptide or peptide derivative, and a biologic or biological active ingredient.

In the context of the present invention, the term "low molecular weight compound", "small molecule" ("smol") or "chemical drug" refers to an organic compound of low molecular weight (<10,000 Daltons, especially <1,000 Daltons), often with a size on the order of 1 nm. Many drugs are small molecules. Such small molecules can regulate a biological process. Small molecules may be able to inhibit a specific function of a protein. In the field of pharmacology, the term “small molecule” refers specifically to molecules that bind to specific biological macromolecules and act as an effector by altering the activity or function of a target. For example, acetylsalicylic acid (ASA) is considered a low molecular weight compound that measures 180 Daltons and consists of 21 atoms. Such low molecular weight compounds often have little ability to trigger an immune response and remain relatively stable over time.

The low molecular weight compound according to the present invention may contain, for example, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkylene, arylene, amino, halogen, carboxylate derivative, cycloalkyl, among other chemical backbones, substituents, groups or residues. , carbonyl derivative, heterocycloalkyl, heteroaryl, heteroarylene, sulfonate, sulfate, phosphonate, phosphate, phosphine, phosphine oxide groups.

The “biologic,” “biologic drug,” “biologic therapeutic,” “biopharmaceutical,” or “biologic agent” according to the present invention is preferably an antibody, or an antigen-binding fragment thereof, or an antigen-binding derivative thereof, or an antibody-like molecule or protein, or an aptamer, or a nucleic acid.

In a preferred embodiment of the method for identifying an active ingredient that binds to the FATP2 protein or a fragment thereof and/or inhibits or reduces the activity of the FATP2 protein or a fragment thereof, the active ingredient is a member of a “library” of connections.

The “library” (mixture) of compounds can e.g. B. include low molecular weight compounds, natural or synthetic peptides or peptide derivatives, or biologics or biological active ingredients or biological compounds.

In the context of the present invention, the term “(combinatorial) compound library” or “library of compounds” refers to collections of chemical compounds, small molecules, natural or synthetic peptides or peptide derivatives, or macromolecules such as proteins or other biologics each containing a large number of related chemical, peptidic and biological species of molecules that can be used together in particular screening assays or identification steps.

Methods for producing molecular libraries of low molecular weight chemical compounds (“compound libraries”) and for high-throughput testing (“high-throughput screening”) of the compounds for interaction with the target molecule are described in the prior art (for example, Volochnyuk et al . 2019). These methods also include so-called “focus libraries”, highly annotated and pre-selected chemical molecule libraries (Wassermann et al. 2014), DNA-encoded libraries of chemical compounds (Martin et al. 2020), and chemoinformatics-based virtual molecule libraries (Saldivar -Gonzalez et al. 2020). The use of so-called “phage display” technologies to identify suitable “small molecule” active ingredients has been described, for example, by Takakusagi et al., 2020. Numerous other peptide and antibody “display” technologies such as “Bacterial Display ", "Yeast Surface Display" and "Mammalian Surface Display" as well as "Ribosome Display" are described in Valldorf et al., 2022.

Process for the production of molecular libraries, their immobilization and their high-throughput testing of biological molecules, for example peptides, peptide derivatives, proteins, antibodies, antigen-binding antibody fragments, antigen-binding antibodies Derivatives, or antibody-like molecules, are also described in the prior art (for peptide libraries, for example, in Bozovicar and Bratkovic 2019; Schwaar et al. 2019; for antibody libraries in Lin and Lerner 2021).

In a preferred embodiment of the method for identifying an active ingredient that binds to the FATP2 protein or a fragment thereof and/or inhibits or reduces the activity of the FATP2 protein or a fragment thereof, the biologic is an antibody, an antigen-binding one Fragment thereof, an antigen-binding derivative thereof, an antibody-like molecule or protein, an aptamer, or a nucleic acid.

In a preferred embodiment of the method for identifying an active ingredient that binds to the FATP2 protein or a fragment thereof and/or inhibits or reduces the activity of the FATP2 protein or a fragment thereof, the FATP2 protein is bound to a solid phase or is in solution.

According to a third aspect, the present invention relates to the use of a nucleic acid encoding the FATP2 protein or a fragment thereof, or the use of the FATP2 protein or a fragment thereof, in a method for identifying an active ingredient that binds to the FATP2 protein or a fragment thereof, and/or inhibits or reduces the activity of the FATP2 protein or a fragment thereof.

To express the FATP2 protein or a fragment thereof, a nucleic acid which encodes the FATP2 protein or a fragment thereof is cloned into a suitable expression vector, e.g. a suitable expression plasmid, as described (Green and Sambrook 2012). The recombinant expression protein is introduced by transfection into a cell suitable for the expression of the FATP2 protein or a fragment thereof, the cell is propagated in cell culture with a suitable cell culture medium, and the expressed protein is purified from the cells and/or the cell culture medium.

As used herein, the term “transfection” refers to any method of intentionally introducing a foreign nucleic acid into a eukaryotic cell. Various types of nucleic acids can be used for transfection into eukaryotic cells, in particular deoxyribonucleic acid (DNA), ribonucleic acid (RNA), as well as small, non-coding RNAs such as siRNA, shRNA, and miRNA.

With regard to transfection, a distinction is made between stable and transient transfection. In stable transfection, long-term expression of the transgene is achieved by integrating the nucleic acid introduced into the cell into the cellular genome, while transient transfection, in which the expression of the transgene only occurs temporarily, does not require integration of the nucleic acid into the cellular genome ( Fus-Kujawa et al. 2021).

The selection of the optimal transfection method depends on various factors, in particular the type and origin of the target or production cell as well as the type of nucleic acid introduced. For the introduction of foreign (modified homologous and/or heterologous) nucleic acid encoding the desired transgene(s) into eukaryotic cells, physical, chemical and viral vector-based transfection methods can be used. Physical transfection methods include, for example, electroporation, sonoporation, magnetofection, microinjection and biolistic methods. Chemical transfection methods include the calcium phosphate method, the use of dendrimers, cationic polymers such as diethylaminoethyl dextran (DEAE-dextran), nanoparticles, non-liposomal nanoparticles, and liposomal transfection. When transfecting using viral vectors (so-called “transduction”), genetically modified retroviruses and lentiviruses, adenoviruses and adeno-associated viruses (AAV) are used in particular (Fus-Kujawa et al. 2021).

According to a fourth aspect, the present invention relates to an active ingredient obtained by said method for identifying an active ingredient that binds to the FATP2 protein of a T cell or a fragment thereof and/or the activity of the FATP2 protein of a T cell, or a fragment thereof, inhibited or reduced, or obtained by one of the above-described embodiments of said method.

Furthermore, the present invention relates to an active ingredient that binds to the FATP2 protein or a fragment thereof in a T cell, and/or inhibits or reduces the activity of the FATP2 protein, or a fragment thereof, and/or the degradation of the FATP2 protein promotes.

Furthermore, the present invention relates to an active ingredient which inhibits or reduces the expression of the SLC27A2 gene in a T cell, preferably where the T cell is a synovial T cell.

In a preferred embodiment, the present invention relates to an active ingredient, wherein the active ingredient is a low molecular weight compound (smol), a peptide or peptide derivative, or a biologic, preferably wherein the biologic is an antibody or an antigen-binding fragment thereof, or an antigen -binding derivative thereof, or an antibody-like protein, or an aptamer or a nucleic acid.

In a preferred embodiment, the active ingredient binds specifically to the FATP2 protein or a fragment thereof with a high or particularly high affinity and/or avidity. In a preferred embodiment, the active ingredient, when bound to FATP2, reduces or inhibits FATP2 activity.

The term “specifically bind” as used herein means that the active ingredient has a dissociation constant KD with respect to its binding to the FATP2 protein molecule or an epitope thereof of at most about 100 µM. In one embodiment, the KD is about 100 µM or lower, about 50 µM or lower, about 30 µM or lower, about 20 µM or lower, about 10 µM or lower, about 5 µM or lower, about 1 µM or lower, about 900 nM or lower, about 800 nM or lower, about 700 nM or lower, about 600 nM or lower, about 500 nM or lower, about 400 nM or lower, about 300 nM or lower, about 200 nM or lower, about 100 nM or lower, about 90 nM or lower, about 80 nM or lower, about 70 nM or lower, about 60 nM or lower, about 50 nM or lower, about 40 nM or lower, about 30 nM or lower, about 20 nM or lower, or about 10 nM or lower, about 1 nM or lower, about 900 pM or lower, about 800 pM or lower, about 700 pM or lower, about 600 pM or lower, about 500 pM or lower, about 400 pM or lower, about 300 pM or lower, about 200 pM or lower, about 100 pM or lower, about 90 pM or lower, about 80 pM or lower, about 70 pM or lower, about 60 pM or lower, about 50 pM or lower, about 40 pM or lower, about 30 pM or lower, about 20 pM or lower, or about 10 pM or lower, or about 1 pM or lower.

According to a fifth aspect, the present invention relates to an antibody, or an antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein, which specifically binds to the FATP2 protein, preferably to the FATP2 protein in a T cell .

In a preferred embodiment, the present invention relates to said antibody, or antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein, wherein the antibody, or antigen-binding fragment or derivative thereof, or antibody-like protein inhibits FATP2 activity , i.e. acts as an inhibitor or antagonist of FATP2.

As used herein, the term “antibody” refers to a protein consisting of one or more polypeptide chains encoded by immunoglobulin genes or fragments of immunoglobulin genes or cDNAs derived therefrom. These immunoglobulin genes include the constant region light chain kappa, lambda and heavy chain alpha, delta, epsilon, gamma and mu genes, as well as any of the many different variable region genes.

The basic structural unit of the immunoglobulin (antibody) is usually a tetramer, which consists of two identical pairs of polypeptide chains, the light chains (L, with a molecular weight of about 25 kDa) and the heavy chains (H, with a molecular weight of about 50- 70 kDa). Each heavy chain consists of a heavy chain variable region (abbreviated as VH or _VH ) and a heavy chain constant region (abbreviated as CH or _CH ). The heavy chain constant region consists of three domains, namely CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated as VL or _VL ) and a light chain constant region (abbreviated as CL or _CL ). The VH and VL regions can be further subdivided into regions of hypervariability, also referred to as complementarity determining regions (CDR), interspersed with regions that are more conserved, referred to as framework regions (FR). Each VH and VL region consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains form a binding domain that interacts with an antigen.

The CDRs are most important for binding the antibody or the antigen-binding part of it. The FRs can be replaced with other sequences as long as the three-dimensional structure required for antigen binding is preserved.

The term “antigen-binding part” of the (monoclonal) antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen in its native form. Examples of antigen-binding portions of the antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains, an F(ab')2 fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bridge at the hinge region, an Fd fragment consisting of the VH and CHl domains, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and a dAb fragment consisting of a VH domain and an isolated complementarity determining region (CDR).

The antibody, antibody fragment or antibody derivative thereof according to the present invention may be a monoclonal antibody. The antibody can be of the IgA, IgD, IgE, IgG or IgM isotype.

As used herein, the term “monoclonal antibody (mAb)” refers to an antibody composition having a homogeneous antibody population, i.e. a homogeneous population consisting of a whole immunoglobulin or a fragment or derivative thereof. Such an antibody is particularly preferably selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or a fragment or derivative thereof.

As used herein, the term "fragment" refers to fragments of such antibody that retain their target binding capacities, e.g., a CDR (complementarity determining region), a hypervariable region, a variable domain (Fv), an IgG heavy chain (consisting of VH -, CH1, hinge, CH2 and CH3 regions), an IgG light chain (consisting of VL and CL regions) and/or a Fab and/or F(ab) ₂ .

As used herein, the term “derivative” refers to protein constructs that are structurally different from, but still share some structural relationship to, the common antibody concept, e.g. B. scFv, Fab and/or F(ab)2, as well as bi-, tri- or higher-specific antibody constructs. All of these elements are explained below.

Other antibody derivatives known to those skilled in the art are diabodies, camelid antibodies, domain antibodies, two-chain bivalent homodimers consisting of scFvs, IgAs (two IgG structures linked by a J chain and a secretory component), shark Antibodies, antibodies consisting of New World primate scaffold plus non-New World primate CDR, dimerized constructs comprising CH3+VL+VH, other scaffold protein formats comprising CDRs, and antibody conjugates.

As used herein, the term “antibody-like protein” refers to a protein that has been modified (e.g., by mutagenesis of Ig loops) to specifically bind to a target molecule. Typically, such an antibody-like protein comprises at least one variable peptide loop bound to a protein backbone at both ends. This double structural constraint increases the binding affinity of the antibody-like protein to a level comparable to that of an antibody. The length of the variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein can be any protein with good solubility properties.

Preferably the scaffolding protein is a small globular protein. Antibody-like proteins include, without limitation, affibodies, anticalins and designed ankyrin proteins and affilin proteins. Antibody-like proteins can be derived from large libraries of mutants, e.g. B. by panning from large phage display libraries, and can be isolated in analogy to regular antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins. Antibody-like proteins have been described, for example, in Binz et al. (2005) and Hosse et al. (2006).

As used herein, the term "Fab" refers to an IgG fragment comprising the antigen binding region, the fragment being composed of a constant and a variable domain of each of the heavy and light chains of the antibody.

As used herein, the term “F(ab) ₂ ” refers to an IgG fragment consisting of two Fab fragments linked together by disulfide bonds.

The term “scFv” as used herein refers to a single chain variable fragment that is a fusion of the variable regions of the heavy and light chains of immunoglobulins linked together by a short linker, usually serine (S) and/or glycine (G) includes residues. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker peptide.

Modified antibody formats include, for example, bi- or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates and the like.

IgG, scFv, Fab and/or F(ab)2 are antibody formats well known to those skilled in the art. Detailed explanations and techniques can be found in relevant textbooks.

According to preferred embodiments of the present invention, the antibody or the antigen-binding fragment thereof or the antigen-binding derivative thereof is a murine, a chimeric, a humanized or a human antibody or an antigen-binding fragment or an antigen-binding derivative thereof.

Mouse-derived monoclonal antibodies (mAbs) can cause undesirable immunological side effects because they contain a protein from another species that can induce an immune response. To overcome this problem, antibody humanization and maturation methods have been developed to generate antibody molecules with minimal immunogenicity when used in humans, while ideally maintaining the specificity and affinity of the non-human parental antibody. With these methods z. B. the framework regions of a mouse mAb are replaced by corresponding human framework regions (so-called CDR grafting). WO200907861 discloses the generation of humanized forms of mouse antibodies by linking the CDR regions of non-human antibodies to human constant regions using recombinant DNA technology. US6548640 describes CDR transplant techniques, and US5859205 describes the production of humanized antibodies.

As used herein, the term “humanized antibody” refers to an antibody, a fragment or a derivative thereof, in which at least a portion of the constant regions and/or the framework regions and optionally a portion of the CDR regions of the antibody are derived from human immunoglobulin sequences or is adapted to this.

According to a sixth aspect, the present invention relates to an active ingredient as described above, or an antibody, an antigen-binding fragment or an antigen-binding derivative thereof, or an antibody-like protein as described above, for use in the treatment of an autoimmune disease.

The autoimmune disease is preferably rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases including ulcerative colitis and Crohn's disease, multiple sclerosis, and other autoimmune diseases with T-cell involvement.

Furthermore, the present invention relates to a pharmaceutical composition comprising the active ingredient as described above, or the antibody, the antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein as described above, and one or more pharmaceutically acceptable excipients Use in the treatment of an autoimmune disease, preferably wherein said autoimmune disease is selected from the group consisting of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases including ulcerative colitis and Crohn's disease, multiple sclerosis, and other autoimmune diseases with T-cell involvement.

In a preferred embodiment of the present invention, the said pharmaceutically acceptable excipient(s) is/are selected from the group consisting of pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, Lubricants, disinfectants, adsorbents and/or preservatives.

The said pharmaceutical composition can be administered in the form of powder, tablets, pills, capsules or beads. In aqueous form, the pharmaceutical formulation may be ready for administration, while in lyophilized form the formulation must be converted into a liquid form before administration, e.g. B. by adding water for injections containing a preservative such as. E.g., but not limited to, benzyl alcohol, antioxidants such as vitamin A, vitamin E, vitamin C, retinyl palmitate and selenium, the amino acids cysteine and methionine, citric acid and sodium citrate, synthetic preservatives such as the parabens methylparaben and propylparaben may or may not contain.

The pharmaceutical formulation may further contain one or more stabilizers, e.g. B. can be an amino acid, a sugar polyol, a disaccharide and / or a polysaccharide. The pharmaceutical formulation may further contain one or more surfactants, one or more isotonizing agents and/or one or more metal ion chelators and/or one or more preservatives.

The pharmaceutical formulation as described herein may be suitable for at least oral, parenteral, intravenous, intramuscular or subcutaneous administration. Alternatively, the active ingredient or antibody according to the present invention can be provided in a sustained release formulation which allows the sustained release of the active ingredient over a certain period of time.

Furthermore, primary packaging, such as. B. a prefilled syringe or pen, a vial or an infusion bag, comprising said pharmaceutical formulation according to this aspect of the invention.

The prefilled syringe or pen may contain the formulation either in freeze-dried form (which must then be dissolved, for example with water for injections, before administration) or in aqueous form. The syringe or pen is often a single-use, disposable device and can have a volume of between 0.1 and 20 ml. However, the syringe or pen can also be a reusable syringe or a multi-dose pen.

Furthermore, the present invention relates to the use of an active ingredient that binds to the FATP2 protein in a method for treating an autoimmune disease, preferably wherein said autoimmune disease is selected from the group consisting of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases including Ulcerative colitis and Crohn's disease, multiple sclerosis, and other autoimmune diseases with T-cell involvement. Preferably, the active ingredient, when bound to FATP2, inhibits FATP2 activity.

The present invention further relates to the use of an active ingredient that binds to the FATP2 protein for the production of a medicament for treating an autoimmune disease, the autoimmune disease preferably being selected from the group consisting of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel disease including ulcerative colitis and Crohn's disease, multiple sclerosis, and other autoimmune diseases with T-cell involvement. Preferably, the active ingredient, when bound to FATP2, inhibits FATP2 activity.

The present invention further relates to a method for treating or preventing an autoimmune disease, the method comprising administering an active ingredient that binds to and/or inhibits the FATP2 protein in a therapeutically effective dose or amount to a human or animal subject.

As used herein, the term "effective dose" or "effective amount" means a dose or amount of the active ingredient necessary, in terms of dosages and periods of administration, to achieve the desired therapeutic result in a patient. Effective amounts may vary depending on factors such as the disease state, the patient's age, gender and/or weight, the pharmaceutical formulation, the subtype of disease being treated, and the like, but may nevertheless be routinely determined by one skilled in the art.

According to a seventh aspect, the present invention relates to a method for producing an active ingredient according to the method for identifying said active ingredient that binds to the FATP2 protein or a fragment thereof and/or the activity of the FATP2 protein or a fragment thereof, inhibits or reduces, as described above, further comprising the purification of said active ingredient.

1. (i) das Verfahren zur Identifizierung des besagten Wirkstoffs, der an das FATP2-Protein oder ein Fragment davon bindet, und/oder die Aktivität des FATP2-Proteins, oder eines Fragments davon, hemmt oder reduziert, wie oben beschrieben, und ferner
2. (ii) das Mischen des identifizierten Wirkstoffs mit einem pharmazeutisch akzeptablen Träger.

The present invention further relates to a method for producing a pharmaceutical composition, comprising

1. (i) the method for identifying said active ingredient that binds to the FATP2 protein or a fragment thereof and/or inhibits or reduces the activity of the FATP2 protein or a fragment thereof, as described above, and further
2. (ii) mixing the identified active ingredient with a pharmaceutically acceptable carrier.

1. (i) dem Wirkstoff, der an das FATP2-Protein oder ein Fragment davon in einer T-Zelle bindet, und/oder die Aktivität des FATP2-Proteins, oder eines Fragments davon, in einer T-Zelle, hemmt oder reduziert, wie oben beschrieben, oder dem Antikörper oder antigenbindenden Fragment oder antigen-bindenden Derivat davon oder dem antikörperähnlichen Protein wie oben beschrieben, oder der pharmazeutischen Zusammensetzung, umfassend den Wirkstoff wie oben beschrieben, oder den Antikörper, das antigen-bindende Fragment oder antigen-bindende Derivat davon, oder ein antikörperähnliches Protein, wie oben beschrieben, und einen oder mehrere pharmazeutisch verträgliche Hilfsstoffe, und
2. (ii) einer oder mehreren weiteren therapeutisch aktiven Verbindungen.

According to an eighth aspect, the present invention relates to a composition comprising a combination of

1. (i) the active ingredient that binds to the FATP2 protein or a fragment thereof in a T cell and/or inhibits or reduces the activity of the FATP2 protein or a fragment thereof in a T cell, as above described, or the antibody or antigen-binding fragment or antigen-binding derivative thereof or the antibody-like protein as described above, or the pharmaceutical composition comprising the active ingredient as described above, or the antibody, the antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein as described above, and one or more pharmaceutically acceptable excipients, and
2. (ii) one or more further therapeutically active compounds.

1. (i) die pharmazeutische Zusammensetzung wie oben beschrieben,
2. (ii) eine Vorrichtung zur Verabreichung der besagten Zusammensetzung, und
3. (iii) optional eine Gebrauchsanweisung.

According to a ninth aspect, the present invention relates to a therapeutic kit comprising:

1. (i) the pharmaceutical composition as described above,
2. (ii) a device for administering said composition, and
3. (iii) optional instructions for use.

Examples

The present invention is explained in more detail by the examples and drawings shown and discussed below. It should be noted that the examples and drawings are only descriptive and are not intended to limit the invention in any way.

The invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments may be understood and practiced by those skilled in the art in practicing the claimed invention from a study of the drawings, the disclosure and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually distinct dependent claims does not mean that a combination of these measures cannot be used to advantage. Any reference symbols in the claims are not to be understood as a limitation of the scope of application.

All amino acid sequences disclosed herein are presented from N-terminus to C-terminus; all nucleic acid sequences disclosed here are shown 5'->3'.

Example 1: Increased fatty acid uptake by T cells in the synovial fluid of JIA patients using the transport protein FATP2

The inventors were able to show that T cells found in the synovial fluid of juvenile idiopathic arthritis (JIA) patients exhibit increased uptake of fatty acids, which is accompanied by increased expression of FATP2/SLC27A2. The fatty acid receptor FATP2 is mainly expressed in CD4+ memory cells ( 1 ).

Example 1A: Uptake of free fatty acids in CD4-positive T cells from the blood or synovium of JIA patients

The uptake of free fatty acids into CD4+ T cells from the blood or synovium of juvenile idiopathic arthritis (JIA) patients was determined ( 1A) . The measurement was carried out after incubating the cells for 15 minutes with Bodipye™FL C ₁₂ (2 µM) and subsequent flow cytometric analysis. The mean fluorescence intensity (MFI) is shown minus the respective FM0 control (ΔMFI). Data show mean expression in four patients ± standard error, **p ≤ 0.01.

Data show that uptake of free fatty acids by CD4-positive T cells is dramatically increased in the synovial fluid of JIA patients compared to peripheral blood. The data also suggests that fatty acid oxidation plays an important role in memory T cells to carry out their functions.

This confirms an earlier publication by Andreas Radbruch's working group, which shows that T cells in the synovial fluid depend on fatty acids in order to proliferate (Hradilkova et al. 2019). However, Andreas Radbruch's working group suggested that this dependence was dependent on the transcription factor TWISTl, which is predominantly expressed in PD1-positive T cells. However, TWIST 1 was not found to be upregulated in normal CD4-positive effector cells in the synovial fluid, and expression in synovial fluid regulatory T cells (Tregs) was undetectable (RNAseq single-cell data, Bas Vastert, UMC Utrecht, personal communication). In addition, the expression of CD36, another receptor for lipid uptake, is drastically reduced in synovial fluid regulatory T cells (Tregs) as well as synovial fluid T cells (data not shown) compared to peripheral blood, which is in contrast published data for murine CD8-positive resting memory T cells ( Pan, Tian et al., 2017, Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism, Nature Vol. 543, p. 252-256 ). This is also interesting because CD36 is usually upregulated in Treg cells in hypoxic environments (such as in tumors) with increased glycolysis and lactate concentration (around 5 mmol/liter, which is approximately the same as the concentration found in the synovial fluid of JIA -patients), where it is critical for the suppression of CD8-positive T cells.

Instead, the inventors surprisingly found a specific upregulation of fatty acid transporter 2 (FATP2, also called SLC27A2) in CD4-positive T effector cells in the synovial fluid and even more so in CD4-positive Treg cells in the synovial fluid compared to the peripheral blood healthy children, healthy adults and children with JIA ( 2 ), which may be responsible for the effect the inventors observed regarding fatty acid transport.

Example 1B: FATP2 protein expression in CD4-positive T cells from the blood or synovium of JIA patients

FATP2 protein expression in CD4+ T cells from the peripheral blood (PB) or synovium (SF) of JIA patients was determined using flow cytometric analysis ( 1B) . A representative image is shown. In addition, the expression of SLC27A2 RNA was determined in CD4+ T cells of the synovium of JIA patients compared to the blood of the same patients ( 1C) .

Furthermore, the protein expression of FATP2 was measured in CD4+ T cell subtypes (effector memory T cells (T _ern ), tissue-resident memory T cells (T _rm ) and naive T cells (T _naïve )) from the blood or synovium of JIA patients determined by flow cytometric analysis ( 1D) . The mean fluorescence intensity (MFI) is shown minus the respective FM0 control (ΔMFI). Data show mean expression in five patients ± standard error, ***p ≤ 0.001. ****p < 0.0001.

Example 1C: SLC27A2 mRNA expression in stimulated and unstimulated CD4-positive memory T cells from the blood of healthy controls

The mRNA expression of SLC27A2/FATP2 compared to other metabolic proteins in CD4+ memory T cells after 48 h and 72 h stimulation with anti-CD3 and anti-CD28 antibodies and in unstimulated CD4+ memory T cells cells was determined ( 1 E) . The CD4+ memory T cells were isolated from the blood of healthy control subjects using magnetic cell separation. Data show the mean expression of four experiments with different donors ± standard error, **p ≤ 0.01, ***p ≤ 0.001.

Data demonstrate that SLC27A2/FATP2 is highly upregulated in stimulated memory T cells compared to other metabolic proteins. SLC27A2/FATP2 mRNA expression was dramatically higher in MACS-isolated human naïve CD4+ and CD45R0 CD4+ memory T cells from peripheral blood of healthy donors after stimulation than without stimulation. The data suggest a specific role for this transport protein in the activation of memory T cells.

Example 2: Blockade of fatty acid uptake in memory T cells reduces IFNγ production and proliferation of the cells

Some small molecule antagonists of FATP2 have already been described that inhibit fatty acid uptake in cells, for example Lipofermata (Adeshakin et al. 2021) and Grassofermata (Saini et al. 2015). The inventors were able to show that the FATP2 inhibitor Lipofermata inhibits fatty acid uptake in CD4+ T cells and inhibits IFN-γ expression and proliferation of T cells without inducing apoptosis ( 2 ). With the help of Lipofermata, the function of FATP2 in T cells could be clearly confirmed.

Using Lipofermata (5-Bromo-5'-phenyl-spiro[3H-indole-3,2'(3'H)-[1,3,4]thiadiazol]-2(1H)-one) the fatty acid absorption in the Memory T cells are effectively blocked and the proliferation and production of inflammatory mediators are inhibited. This absorption route is therefore of central importance for the metabolism of T cells, which play an essential role in the pathogenesis of childhood rheumatism and other autoimmune diseases. A targeted blockade of this signaling pathway opens up the use of a completely new mechanism of action in the therapy of autoimmune diseases.

The uptake of free fatty acids in CD4+ T cells after addition of Lipofermata in increasing concentrations of 0, 2, 5, 7.5 and 10 µM was examined ( 2A) . For this purpose, the cells were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours. The measurement was carried out after incubating the cells for 15 minutes with Bodipye™FL C ₁₂ (2 µM) and subsequent flow cytometric analysis. The mean fluorescence intensity (MFI) is shown minus the respective FM0 control (ΔMFI). Data show mean expression measured in three experiments ± standard error.

2 B shows an exemplary histogram of fatty acid uptake in CD4+ T cells without the addition of Lipofermata (right) and after the addition of Lipofermata (left).

In addition, the percentage number of IFN-γ-positive CD4+ T cells was examined after addition of Lipofermata in increasing concentrations of 0, 2, 5, 7.5 and 10 µM ( 2C) . For this purpose, the cells were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours. The determination of IFN-γ-positive cells was carried out after 5 hours of restimulation with PMA and ionomycin in the presence of GolgiPlug™ using flow cytometry. Data show mean expression measured in 5-6 experiments ± standard error, *p ≤ 0.01.

2D shows an exemplary dot diagram of IFN-γ expression without the addition of Lipofermata (left) and after the addition of Lipofermata (right). Furthermore, the percentage of proliferated CD4+ T cells was determined after adding Lipofermata in increasing concentrations of 0, 2, 5, 7.5 and 10 µM ( 2 E) . For this purpose, the cells were stained with “Cell proliferation dye eFlour™” and stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours. Proliferated cells were determined using flow cytometry. Data show mean expression measured in five experiments ± standard error, *p ≤ 0.01.

2 F shows an exemplary dot diagram of cell proliferation without the addition of Lipofermata (left) and after the addition of Lipofermata (right).

2G shows an exemplary dot diagram of the dead cells with Lipofermata treatment (right) and without (left). For this purpose, the cells were stimulated for 72 hours with anti-CD3 and anti-CD28 antibodies and stained with propidium iodide (PI) for flow cytometric live/dead differentiation.

In summary, the present data show a drastically increased uptake of free fatty acids in T cells in the synovial fluid of JIA patients, which could be related to the specific upregulation of SLC27A2-encoded FATP2, and which resulted in a change in its metabolism and thus its function cells. It should be noted that virtually all effector T cells in the synovium represent the memory phenotype. The increased expression of SLC27A2-encoded FATP2 could be blocked by a specific inhibitor (Lipofermata, Veglia et al. 2019). To test the effect of this inhibitor on T cells, CD4+ T cells were isolated from the blood of healthy donors and stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of lipofermata. It was shown that Lipofermata practically completely blocked T cell proliferation and reduced IFN-γ production in a concentration-dependent manner without inducing apoptosis.

Example 3: Screening for active ingredients that bind to and/or inhibit the transport protein FATP2

Screening experiments enable the identification and validation of small molecule therapeutic compounds, peptides and/or biologics that bind to and/or inhibit the activity of the FATP2 protein.

DNA-encrypted substance libraries are generated and screened as described (Kunig et al. 2018). Furthermore, phage display technologies (Takakusagi et al. 2020), cell surface display or ribosome display technologies (Valldorf et al. 2022) and/or combinatorial peptide libraries (Bozovicar and Bratkovic 2019) are used. For this purpose, recombinant FATP2 protein or fragments thereof, which can carry a tag for marking, identification or purification, e.g. a His tag or a FLAG tag, are expressed in bacterial expression systems such as L. coli, or in insect cells or mammalian cells.

The purified FATP2 protein is incubated with substances from the substance library and isolated by immunoprecipitation. The compounds bound to the FATP2 protein are identified, for example, by Sanger sequencing of the DNA barcodes. The identified agents and compounds are then tested in appropriate bioassays for their effect on the function of FATP2, its fatty acid transport activity, as well as IFN-γ production and the proliferation of memory T cells. Experimental mouse in vivo models are also used for this purpose.

For the identification and validation of small-molecule therapeutic compounds, peptides and/or biologics that have an effect on FATP2 activity or its expression, an in vitro fluorochrome reporter system based on human cells is being established, for example the expression of eGFP -FATP2 fusion protein or a luciferase-based reporter system is used to screen compound libraries in 384- to 1,536-well assays to identify compounds that reduce eGFP fluorescence or luciferase levels as readout. The expression of these human FATP2 fusion reporter constructs in the cells mentioned can e.g. B. by transfection and selection via resistance gene cassettes or by viral transduction. Human cell lines such as 293T cells are used for these assays. In parallel to this screening, cytotoxicity assays are performed to exclude compounds that exert an effect on reporter fluorescence or activity due to nonspecific toxicity or induction of apoptosis.

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• Pearce EL et al. (2009). Enhancing CD8 T cell memory by modulating fatty acid metabolism. Nature Vol. 460 No. 7251, p. 103-107 .
• Perez VM et al. (2020). Deletion of fatty acid transport protein 2 (FATP2) in the mouse liver changes the metabolic landscape by increasing the expression of PPARcc-regulated genes. J. Biol. Chem. Vol. 295 No. 17, p. 5737-5750 .
• Saini N. et al. (2015). Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity. Biochem Biophys Res Commun. Vol. 465 No. 3, p. 534-541 .
• Saldivar-Gonzalez FI et al. (2020). Chemoinformatics-based enumeration of chemical libraries: a tutorial. Journal of Cheminformatics Vol. 12 No. 64 . https://doi.org/10.1186/s13321-020-00466-z.
• Schwaar T. et al. (2019). Effcient screening of combinatorial peptide libraries by spatially ordered beads immobilized on conventional glass slides. High-Throughput Vol. 8 No. 11. doi:10.3390/ht8020011.
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• Valldorf B. et al., (2022). Antibody display technologies: selecting the cream of the crop. Biol. Chem. Vol. 403 No. 5-6, p. 455-477 .
• Veglia F. et al. (2019). Fatty acid transporter 2 reprograms neutrophils in cancer. Nature Vol. 569 No. 7754, p. 73-78 .
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• Zhu G. and Zhu H. (2022). Modified gene editing systems: various bioengineering tools and crop improvement. Front. Plan Be. Vol. 13, p. 847169 . doi: 10.3389/fpls.2022.847169.

Sequences:

• Human FATP2, nucleotide and amino acid sequences, respectively
• (SLC27A2, Solute Carrier Family 27 Member 2, Homo sapiens, human;
• NCBI Entrez Gene No. 11001)

SEQ ID No. 1, nucleotide sequence entry containing the human FATP2 sequence:

SEQ ID No. 2, coding nucleotide sequence of human FATP2 (nucl. 205 - 2067 of SEQ ID No. 1):

SEQ ID NO. 3, amino acid sequence of human FATP2:

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2020/172510 [0014]
• WO 200907861 [0089]
• US 6548640 [0089]
• US 5859205 [0089]

Non-patent literature cited

• Pan, Tian et al., 2017, Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism, Nature Vol. 543, p. 252-256 [0114]
• Binz H.K., Amstutz P., and Plückthun A. (2005). Engineering novel binding proteins from nonimmunoglobulin domains. Nature Biotechnology Vol. 23 No. 10, p. 1257-1268 [0132]
• Bozovicar K. and Bratkovic T. (2019). Evolving a peptide: Library platforms and diversification strategies. Int. J. Mol. Sci. Vol. 21 No. 215 [0132]
• Chen Y et al. (2020). Involvement of FATP2-mediated tubular lipid metabolic reprogramming in renal fibrogenesis. Cell Death and Disease Vol. 11, No. 994 [0132]
• Endo Y et al. (2019). ACC1 determines memory potential of individual CD4+ T cells by regulating de novo fatty acid biosynthesis. Nature Metabolism Vol. 1, p. 261-275 [0132]
• Falcon A. et al. (2010). FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase. Am J Physiol Endocrinol Metab Vol. 299, p. E384-E393 [0132]
• Feng K. et al. (2022). Upregulated SLC27A2/FATP2 in differentiated thyroid carcinoma promotes tumor proliferation and migration. J Clin Lab Anal. Vol. 36, p. e24148 [0132]
• Fus-Kujawa A. et al. (2021). An overview of methods and tools for transfection of eukaryotic cells in vitro. Front. Bioeng. Biotechnol. Vol. 9:701031 [0132]
• Hradilkova K. et al. (2019). Regulation of fatty acid oxidation by Twist 1 in the metabolic adaptation of T helper lymphocytes to chronic inflammation. Arthritis & Rheumatology Vol. 71 No. 10, p. 1756-1765 [0132]
• Hosse R.J., Rothe A., and Power B.E. (2006). A new generation of protein display scaffolds for molecular recognition. Protein Science Vol. 15, p. 14-27 [0132]
• Khan S et al. (2020). Fatty acid transport protein-2 regulates glycemic control and diabetic kidney disease progression. JCI Insight Vol. 5 No. 15, p. el36845 [0132]
• Kunig V. et al. (2018). DNA-encoded libraries - an efficient small molecule discovery technology for the biomedical sciences. Biol. Chem. Vol. 399 No. 7, p. 691-710 [0132]
• Lin C.-W. and Lerner R.A. (2021). Antibody libraries as tools to discover functional antibodies and receptor pleiotropism. Int. J. Mol. Be. Vol. 22 No. 4123 [0132]
• Melton E.M. et al. (2013). Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvll) reveals distinct patterns of trafficking of exogenous fatty acids. Biochem Biophys Res Commun. Vol. 440 No. 4, p. 743-748 [0132]
• Nidhi S et al. (2021). Novel CRISPR-Cas Systems: an updated review of the current achievements, applications, and future research perspectives. Int. J. Mol. Be. Vol. 22, p. 3327 [0132]
• Ohl K. et al. (2018). The transcription factor CREM drives an inflammatory phenotype of T cells in oligoarticular juvenile idiopathic arthritis. Pediatric Rheumatology Vol. 16 No. 39 [0132]
• Pearce E.L. et al. (2009). Enhancing CD8 T cell memory by modulating fatty acid metabolism. Nature Vol. 460 No. 7251, p. 103-107 [0132]
• Perez V.M. et al. (2020). Deletion of fatty acid transport protein 2 (FATP2) in the mouse liver changes the metabolic landscape by increasing the expression of PPARcc-regulated genes. J. Biol. Chem. Vol. 295 No. 17, p. 5737-5750 [0132]
• Saini N. et al. (2015). Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity. Biochem Biophys Res Commun. Vol. 465 No. 3, p. 534-541 [0132]
• Saldivar-Gonzalez F.I. et al. (2020). Chemoinformatics-based enumeration of chemical libraries: a tutorial. Journal of Cheminformatics Vol. 12 No. 64 [0132]
• Takakusagi T. et al. (2020). Phage display technology for target determination of small molecule therapeutics: an update. Expert Opinion on Drug Discovery Vol. 15 No. 10, p. 1199-1211 [0132]
• Valldorf B. et al., (2022). Antibody display technologies: selecting the cream of the crop. Biol. Chem. Vol. 403 No. 5-6, p. 455-477 [0132]
• Veglia F. et al. (2019). Fatty acid transporter 2 reprograms neutrophils in cancer. Nature Vol. 569 No. 7754, p. 73-78 [0132]
• Volochnyuk D.M. et al. (2019). Evolution of commercially available compounds for HTS. Drug Discovery Today Vol. 24 No. 2, p. 390-402 [0132]
• Aquarius A.M. et al. (2014). Composition and applications of focus libraries to phenotypic assays. Frontiers in Pharmacology Vol. 5 No. 164 [0132]
• Zhu G. and Zhu H. (2022). Modified gene editing systems: various bioengineering tools and crop improvement. Front. Plan Be. Vol. 13, p. 847169 [0132]

Claims (30)

Method for inhibiting or reducing fatty acid uptake into a T cell, activation and/or proliferation of a T cell, the method comprising at least one step selected from the group consisting of (i) inhibiting or reducing T cell SLC27A2 gene expression, (ii) inhibiting or reducing the activity of fatty acid transport protein-2 (FATP2), and/or (iii) Promote the degradation of FATP2 protein. Procedure according to Claim 1 , wherein the inhibition or reduction of SLC27A2 gene expression involves an SLC27A2 gene knock-down, knock-out, conditional gene knockout, a gene change in the sense of an insertion, deletion and / or substitution, a gene change using a gene editing system , RNA interference, siRNA and/or antisense RNA. Procedure according to Claim 1 , wherein inhibiting or reducing FATP2 activity involves the use of an agent that binds to the FATP2 protein. Procedure according to one of the Claims 1 until 3 , where said T cell is a synovial T cell. Method for identifying an active substance that binds to the FATP2 protein of a T cell, or a fragment thereof, and/or inhibits or reduces the activity of the FATP2 protein of a T cell, or a fragment thereof. Procedure according to Claim 5 , comprising at least the following steps: (i) providing the FATP2 protein or a fragment thereof, (ii) adding at least one active ingredient to be tested for binding to the FATP2 protein or a fragment thereof, and (iii) identification of at least one active ingredient that has bound to the FATP2 protein or a fragment thereof. Procedure according to one of the Claims 5 and 6 , where the active ingredient is a FATP2 inhibitor. Procedure according to one of the Claims 5 until 7 , where the active ingredient is a member of a library of compounds. Procedure according to one of the Claims 5 until 8th , where the active ingredient is selected from the group consisting of a low molecular weight compound, a peptide and a biologic. Procedure according to Claim 9 , wherein the biologic is an antibody, an antigen-binding fragment thereof, an antigen-binding derivative thereof, an antibody-like molecule or an aptamer. Procedure according to one of the Claims 5 until 10 , where the FATP2 protein is bound to a solid phase or is in solution. Use of a nucleic acid encoding the FATP2 protein or a fragment thereof, or the FATP2 protein or a fragment thereof, in a method for identifying an active ingredient that binds to the FATP2 protein or a fragment thereof, according to one of the Claims 5 until 11 . Active ingredient obtained by the process according to one of the Claims 5 until 11 . Active ingredient that inhibits or reduces the expression of the SLC27A2 gene in a T cell, preferably wherein the T cell is a synovial T cell. Active substance that binds to the FATP2 protein or a fragment thereof in a T cell and/or inhibits or reduces the activity of the FATP2 protein or a fragment thereof and/or promotes the degradation of the FATP2 protein. Active ingredient according to one of the Claims 13 until 15 , where the active ingredient is a small molecule (“smol”), a peptide or a biologic, preferably where the biologic is a Antibody or a fragment thereof, a derivative thereof, an antibody-like protein, or an aptamer. Antibody, or antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein that specifically binds to the FATP2 protein in a T cell. Antibody, or antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein Claim 17 , wherein the antibody, or the antigen-binding fragment or derivative thereof, or the antibody-like protein, inhibits FATP2 activity. Active ingredient according to one of the Claims 13 until 16 or antibody, antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein, according to any of the Claims 17 and 18 , for use in the treatment of an autoimmune disease. Active ingredient or antibody, antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein, for use according to Claim 19 , wherein said autoimmune disease is selected from the group consisting of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases including ulcerative colitis and Crohn's disease, multiple sclerosis, and other autoimmune diseases with T-cell involvement. Use of an active ingredient that binds to the FATP2 protein in a method for treating an autoimmune disease, said autoimmune disease preferably being selected from the group consisting of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases including ulcerative colitis and Crohn's disease, Multiple sclerosis, and other autoimmune diseases with T-cell involvement. Use of an active ingredient that binds to the FATP2 protein for the production of a medicament for the treatment of an autoimmune disease, said autoimmune disease preferably being selected from the group consisting of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases including ulcerative colitis and Crohn's disease , multiple sclerosis, and other autoimmune diseases with T-cell involvement. Use of an active ingredient according to one of the Claims 21 and 22 , where the active ingredient, when bound to FATP2, inhibits FATP2 activity. A method for treating or preventing an autoimmune disease, the method comprising administering to a human or animal subject an agent that binds to and/or inhibits the FATP2 protein at a therapeutically effective dose. Process for producing an active ingredient according to a process according to one of Claims 5 until 11 , further comprising the purification of the active ingredient. Pharmaceutical composition comprising the active ingredient according to one of Claims 13 until 16 or the antibody, the antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein, according to any of the Claims 17 and 18 , and one or more pharmaceutically acceptable excipients, for use in the treatment of an autoimmune disease, preferably wherein said autoimmune disease is selected from the group consisting of rheumatism, rheumatoid arthritis, juvenile idiopathic arthritis, chronic inflammatory bowel diseases including ulcerative colitis and Crohn's disease, multiple sclerosis, and other autoimmune diseases with T-cell involvement. Pharmaceutical composition according to Claim 26 , wherein the excipients are selected from the group consisting of pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, lubricants, disinfectants, adsorbents and/or preservatives. A process for producing a pharmaceutical composition, comprising (i) the process according to one of Claims 5 until 11 , and further (ii) mixing the identified active ingredient with a pharmaceutically acceptable carrier. A composition comprising a combination of (i) the active ingredient that binds to the FATP2 protein in a T cell according to one of the Claims 13 until 16 , the antibody or antigen-binding fragment or antigen-binding derivative thereof or the antibody-like protein according to one of the Claims 17 and 18 , or the pharmaceutical composition according to one of the Claims 26 and 27 , and (ii) one or more other therapeutically active compounds. Therapeutic kit comprising: (i) the pharmaceutical composition according to one of Claims 26 , 27 or 29 , (ii) a device for administering the composition, and (iii) optionally instructions for use.

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