TARGET MOLECULE SPECIFIC CAR T-Reg CELLS SUITABLE FOR USE IN SINGLE-GENE DISEASES CHARACTERIZED BY INFLAMMATION SUCH AS FIBRODYSPLASIA OSSIFICANS PROGRESSIVA AND FAMILIAL MEDITERRANEAN FEVER (FMF)

Technical Field

The present invention relates to single gene diseases (monogenic autoinflammatory diseases) that progress with inflammation.

In particular, the present invention relates to the use of target molecule specific CAR T- reg cells suitable for the use of the treatment of single gene diseases that progress with inflammation, for example, the use of activin-A specific CAR T-reg cells in Fibrodysplasia Ossificans Progressive (FOP) and pyrin specific CAR T-reg (regulatory T lymphocyte) cells in FMF disease and other autoinflammatory single gene diseases with overexpression/activity of receptors and the preparation of product for their treatment and the treatment.

State of the Art

Fibrodysplasia ossificans progressiva disease is a disorder characterized by ACVR1 (ALK2) gene defect. In this disease, the ALK2 gene is over-activated, resulting in a clinical picture manifested by ossification in tissues, and it is a disease characterized by severe deformity and early death. However, due to the fact that it is an extremely rare disease (with a ratio of 1/2,000,000), much research has not been done to date.

Physiopathology: The ACVR1 gene encodes the activin receptor type 1 protein, which is a member of the BMP (bone morphogenetic protein) family and plays a role in the cell signaling pathway. Repetitive heterozygous missense mutations in the glycine-serine (GS) activation domain of this protein lead to BMP Type I receptor destabilization and increased intracellular signal communication, making FOP patients sensitive to the activin molecule triggered by inflammation, and translational osteogenic molecule synthesis begins in DNA with the activation of mutational receptor SMAD (1 / 5 / 8) and SMAD (4) stimulated by activin; the system triggered by the inclusion of the mTOR pathway causes tissue mesenchymal stem cells to rapidly develop osteoblasts and abnormal osteoclasts, causing ectopic chondrogenesis, osteogenesis and joint fusion in fibrodysplasia ossificans progressive disease.

The current treatment in fibrodysplasia ossificans progressiva disease appears as supportive treatment. Glucocorticoid, nonsteroidal and anti-inflammatory drugs, cyclooxygenase 2 inhibitors, leukotriene inhibitors are used in acute attacks with large joint involvement and mast cell stabilizers are thought to be effective in the long term.

One of the studies in the state of the art is the European Patent document numbered EP3191512B1. This document discloses methods for treating Fibrodysplasia Ossificans Progressiva (FOP). The disclosed method comprises administering an effective regimen of an activin receptor type 2A (ACVR2A) and/or an activin receptor type 2B (ACVR2B) antagonist or an activin receptor type 1 (ACVR1) antagonist.

However, in the known state of the art, there are not sufficient double-blind, randomized, placebo-controlled studies proving the effectiveness of any treatment method, and current treatments are only treatment regimens that can reduce the symptoms of the disease and cause side effects at the same rate. In addition, in the ongoing studies for the treatment of this disease, one of the four points that play a role in the pathology of this disease is targeted: a) inflammation b) activin levels increased by inflammation c) control of abnormal activin receptor d) blocking of processes stimulated by activin receptors. A treatment method that can be effective at more than one point at the same time has not been designed yet. Furthermore, it is not possible to say that these treatments are ideal regimens due to the need for continuous drug use in the long term and possible side effects. Information on the status of these treatments is given in Table 1 .

Table 1

Therefore, due to the inadequacy of the existing solutions on the subject made it necessary to make an improvement in the related technical field to prevent the problems related with the field and to eliminate them. Object of the Invention

The present invention relates to use of target molecule specific CAR T-reg cells suitable for the use of the treatment of single gene diseases that progress with inflammation, for example, the use of activin-A specific CAR T-reg cells in Fibrodysplasia Ossificans Progressive (FOP) and pyrin-specific CAR T-reg (regulatory T lymphocyte) cells in FMF disease and other autoinflammatory single gene diseases with overexpression/activity of receptors and the preparation of product for their treatment and administering the treatment, that meets the requirements mentioned above, eliminates all disadvantages and provides some additional advantages.

The primary purpose of the present invention is; reducing the amount of free activin-A, blocking the inflammation that causes activin secretion with anti-inflammatory cytokines secreted from T-reg cells, inhibition of macrophages and mast cells that play a role in inflammation by T-reg cells, and T-reg-mediated inhibition of osteoclasts, which is an important pathway during osteogenesis. It is the development of a four-point and simultaneous agent, and the treatment of single gene diseases with inflammation with it.

Another objective of the invention is the ability of T-reg cells to reduce their numbers with decreased stimulation after inflammation and/or blockade of activin-A/pyrin molecules through T-reg cells (self-dosing effect/ smart therapy).

Another objective of the invention is to provide ease of use due to its long-term effectiveness and to make intervention in the treatment by stopping the side effects of the treatment by blocking the current treatment over the switch control (on-off gene) mechanism at any time (in case of excessive side effects). It will be possible to stop the applied smart therapy in undesirable situations by the control gene, which is also a feature of smart therapy.

To achieve the objective described above, the invention is target molecule specific CAR T-reg cells suitable for use in the treatment of single gene diseases that progress with inflammation selected from Fibrodysplasia Ossificans Progressive (FOP) and Familial Mediterranean Fever (FMF) and is characterized by being activin- A specific CAR T-reg cells or pyrin-specific CAR T-reg cells or target molecule-specific CAR T-reg cells.

The structural and characteristic features of the invention and all its advantages will be understood more clearly by means of the figures provided below and the detailed explanation written with reference to these figures, and therefore the evaluation should be made by taking these figures and detailed explanation into account. Figures to Help Understand the Invention

Figure 1 - CAR T-reg Transgene Vector Design Map

Figure 2 - Schematic view of CAR T-reg

Figure 3 - CAR T-reg Production Diagram

Figure 4 - Target Molecule Specific CAR T-reg Action Mechanism

Detailed Description of the Invention

This detailed explanation describes the present invention only for a better understanding of the subject and without causing any limiting effect.

Target molecule specific CAR T-reg cells suitable for use in the treatment of Fibrodysplasia Ossificans Progressive (FOP) or Familial Mediterranean Fever (FMF), selected from single gene diseases that progress with inflammation, are basically activin-A specific CAR T-reg cells or pyrin-specific CAR T-reg cells. Here, activin-A specific CAR T-reg cells are preferred for use in the treatment of Fibrodysplasia Ossificans Progressive (FOP), and pyrin-specific CAR T-reg cells are preferred for use in the treatment of Familial Mediterranean Fever (FMF). This activin-A specific CAR T- reg cells or pyrin specific CAR T-reg cells are cells expressing the switch I control gene at the same time. Figure 1 shows the CAR T-reg transgene vector design map, and Figure 2 shows the schematic view of CAR T-reg.

A method for obtaining target molecule specific CAR T-reg cells suitable for use in the treatment of Fibrodysplasia Ossificans Progressive (FOP) or Familial Mediterranean Fever (FMF), selected from single gene diseases that progress with inflammation, basically activin-A specific CAR T-reg cells or pyrin-specific CAR T-reg cells; the method comprises the following process steps in its most basic form: a. Design/production of transgene CAR plasmid containing antibody or receptor specific to the target molecule, b. Production of lentivirus/ retrovirus/ adenovirus/ AAV/ transposon/ electroporation encoding the transgene, c. Isolating the T-reg cell population with CD3, CD4, CD25 or FOXP3 or other expressions representing the T-reg cell population from patient blood or apheresis sample; d. Transduction of the T-reg cell population (obtaining CAR T-reg cells) with the CAR construct containing activing-A or pyrin or target molecule receptor or antibody; e. Expansion of the resulting activin-A or pyrin or target molecule positive CAR T-reg cells; f. Freezing the prepared activin-A or pyrin or target molecule positive CAR T-reg cells preferably at -196 <C and storing them ready for in fusion.

The CAR T-reg Production Schematic is given in Figure 3. Prepared cells are suitable for direct infusion into the patient

In an exemplary embodiment of the invention:

At the “A” process step, transgene CAR plasmids containing antibodies or receptors specific to the target molecule used in therapy are designed/produced according to the map shown in Figure 1.

The "B" process step includes the process step of generating a lentivirus/retrovirus/ adenovirus/ AAV/ transposon/ electroporation encoding/transferring the transgene.

At the C” process step, T-reg cells with CD3, CD4, CD25 or FOXP3 or other expressions representing the T-reg cell population are isolated from patient blood or apheresis samples. This process can be done by automated means or manual methods.

At the "D" process step, isolated T-reg cells are transducted by the carrier systems described above (lentivirus/retrovirus/ adenovirus/ AAV/ transposon/ electroporation) to produce activin or pyrin or target molecule specific CAR expression by automated or manual methods. In short, the target molecule specific CAR - T- reg is produced. Preferably, expression profile and quality control tests are completed here.

At the “E” process step, the produced activin or pyrin or the target molecule specific CAR T-reg cells are taken into the reproduction process to reach the target number. This figure is in the dose range of 0.5 - 8.0 x 10 ⁶ / kg. This step preferably includes repeating the expression profiling and quality control tests. At the “F” process step, the prepared activin-A or pyrin or the target molecule positive CAR T-reg cells are stored by freezing, preferably at -196 <C ready for infusion, or transferred to the relevant center at +4 <C for dir ect use.

By means of the specific CAR T-reg cells developed with the invention, effect is provided simultaneously at four points, namely, reducing the amount of free activin (shot 1 ), blocking inflammation that causes activin-A secretion (shot 2), inhibiting mast and macrophage cells that also play a role in inflammation (shot 3), and inhibiting osteoclasts, which is an important pathway during osteogenesis (shot 4). The mechanism of action of activin-A or pyrin or target molecule specific CAR T-reg is shown in Figure 4. With this feature, unlike all formulations, agents and methods in the known state of the art, it has a four-shot effect. In addition, in the absence of activin-A and inflammation, activin-A/target specific CAR T-reg cells are deactivated, their number decreases, and almost performs self-dosing. With this feature, it is of the nature of a smart treatment. Other important features of these developed cells are their ease of use as they require an application for a minimum of 6 months and a maximum of 3 years, the possibility to block the current treatment at any time via the switch control (on - off gene) mechanism, and the ability to make intervention in the disease with different mechanisms by stopping the side effects of the treatment.

In order to reduce the amount of activin-A, a high amount of expression (presentation) of the original highly active activin receptor or antibody will be provided on the regulatory T lymphocytes of the patient by gene engineering method (lentivirus/retrovirus/ adenovirus/ AAV/ transposon/ electroporation). This method is known today as CAR T cell therapy (T cell expressing chimeric antigen receptor). In this CAR T cell model, the high affinity activin-A/ pyrin/target molecule will bind to the membrane with its receptor/antibody CD8 or a different anchor protein, continue with transmembrane CD8 or a different molecule, and CD3 zeta chain is added in the last step, with or without integrating T cell co-simulator molecules to the intracytoplasmic region or (see Figures 1 and 2). This way, T lymphocyte carrying a brand new chimeric receptor is synthesized (regulatory T lymphocyte). The T lymphocytes to be used in this transgene transfer will be regulatory T lymphocytes (no normal T lymphocytes are used, but T lymphocytes with all expressions expressing CD3, CD4, FOXP3, and CD25 or representing other T-reg cells are used). In this model, when activin-A is secreted, the activity molecules will be blocked by a large number of T-regs carrying a high - affinity activing-A receptor/antibody (shot 1 ), as a result of this blocking, T-reg cells will multiply, and if the amount of activin-A decreases by binding more activin-A receptors, the number of T lymphocytes carrying the activin-A receptor will tend to decrease (selfdosing feature/smart therapy).

Regulatory T lymphocytes stimulated by activin-A will block the inflammation signal by means of cytokines such as IL-10, (shot 2) and rapidly stop the inflammation reaction by targeting and suppressing the mast and macrophage cells that cause it (shot 3). Since this reaction is especially specific to self-reactive cells (T-reg property), it does not create a tendency to infection while anti-inflammation is induced and T-reg cells cease to function with the termination of inflammation (self-dosing feature/smart therapy).

FOP research indicates that abnormal multinuclear large osteoclast development in patients may be significant in heterotopic osteogenesis. T-reg cells are expected to control the process of heterotropic osteogenesis by inhibiting osteoclasts (shot 4).

CAR T Cell therapies conducted in ALL and NHL show that CAR T cells can remain circulating for minimum 6 months and maximum 3 years. This means that the disease can be controlled with the administering of the current recommended treatment at most twice a year and at least once in 3 years.

The most important side effects of CAR T Cell therapies are CRS, neurotoxicity and hemophagocytic syndrome. The underlying cause of this syndrome is T-reg external T Cell activation. Due to the reason that the cells used here are T-reg, these side effects are not expected; as can be seen from the formula given above, there is a switch control gene in the T - reg cells produced for this disease; specific CAR T-reg cells are destroyed by a specific agent that will turn the control switch on and off (e.g. CAR T-reg cells expressing EGFRt are destroyed by a single dose injection of EGFR antibodies using EGFRt expression as switch control).