Medical use of regulatory t cell activators

Abstract

The present invention provides a CD4 + Foxp3 + activator of regulatory T cells, wherein said disease is characterized by an inflammatory infiltrate of cytotoxic CD8 + T cells comprising epidermal cell apoptosis, melanocyte apoptosis in the epidermis, or epidermal appendage cell apoptosis. The present invention also provides a method of preparing a medicament suitable for treating a subject suffering from said inflammatory epidermal disease or preventing said inflammatory epidermal disease in a subject, said method comprising activating CD4 + Foxp3 + activator of regulatory T cells ex vivo. The present invention also provides a CD4 + Foxp3 + activator of regulatory T cells for use in treating or preventing said inflammatory epidermal disease. Furthermore, the present invention provides an ex vivo activated CD4 + Foxp3 + activator of regulatory T cells for use in treating or preventing said inflammatory epidermal disease.

Description

Technical Field

[0001] This invention relates to the prevention and treatment of inflammatory epidermal diseases, including those involving cytotoxic CD8+ cells that cause epidermal damage, particularly epidermal cell apoptosis. + Inflammatory autoimmune epidermal diseases associated with T-cell inflammatory infiltration, such as lichen planus. Background Technology

[0002] Autoimmunity is an immune response to the body's own healthy tissues, typically involving T and B lymphocytes, and can lead to serious diseases that significantly impact a patient's quality of life, making their treatment challenging for healthcare professionals. These diseases can be classified according to the affected body systems or regions, including, for example, the gastrointestinal system, musculoskeletal system, endocrine system, and skin system.

[0003] An example of an autoimmune disease of the skin system is lichen planus (LP). This is a relatively rare skin disease with an estimated incidence of 0.14–1.27% in the general population, and it typically occurs in middle-aged adults. The histopathological features of this disease include lesions on the skin, mucous membranes, and hair follicles and nail apparatus, in which different areas of the body are affected individually, simultaneously, or sequentially (see, for example, Vi...). i et al., Int. J. Mol. Sci. 2023, 24(3), 3038).

[0004] Research on this disease indicates that it is a T-cell-mediated disease, in which the pathogenic mechanism appears to be mediated by the release of Th1 cytokines by CD4+. + Th cell-driven, leading to Langerhans cell recruitment and CD8 cytotoxicity. + Clonal expansion of cells then leads to apoptosis of epidermal cells, particularly keratinocytes (Tziotzios et al., J. Am. Acad. Dermatol. (2018), 79(5): p807-818; Firth et al., Arch. Dermatol. Res. (2015) 307:333-339). It is also known that CD4+ levels in PBMCs of LP patients are higher than in healthy controls. + Increased regulatory T cells (Domingues et al. J. Transl. Med. (2016) 14:171; Firth et al., Arch.Dermatol. Res. (2015) 307:333-339). However, these regulatory T cells are unable to control CD8. +The cause of cell proliferation is unclear, and these cells are thought to be possibly dysfunctional (Zhou et al., Inflammation (2016)39: 1205-1215). Recent clinical approaches to treat this disease include using the anti-IL-17 antibody secukinumab, the IL-12 / 23-targeting IL-23 utekinumab, and the IL-23 inhibitor guselkumab to block the effects of these inflammatory cytokines.

[0005] In the past, depleted anti-CD4 antibodies have been investigated for the treatment of autoimmune diseases and cancer (e.g., in US 2010 / 015134A1 entitled "Therapy with CD4 binding peptides and radiation"). However, such treatments have not been applied to the treatment of lichen planus, and because they may induce CD4 binding... + The significant depletion of T cells and the resulting side effects mean that it is no longer routinely considered a treatment for autoimmune diseases.

[0006] Non-depleting anti-CD4 antibodies Clenoliximab and TRX1 have previously been suggested for the treatment of many autoimmune diseases (e.g., in U.S. Patent 2009 / 0226430A1 entitled “Recombinant Anti-CD4 Antibody for Human Treatment”, and in U.S. Patent 2010 / 0021460A1 entitled “Method of Treating Autoimmune Diseases Using CD4 Antibody”).

[0007] Clenoliximab is currently believed to mediate intracellular immunomodulatory effects through the following (1) to (3): (1) CD4-mediated apoptosis. + T cell depletion or complement-mediated mAB-coated CD4 + (2) T cell lysis; (3) receptor regulation resulting from the internalization or stripping of CD4 receptors from the cell surface; and (4) inhibition of T cell activation by antagonizing CD4-MHC class II interactions, which may lead to induced immune anergy (https: / / www.creativebiolabs.net / clenoliximab-overview.htm#:~:text=Clenoliximab%20can%20mediate%20in%20vivo,T%20cell%20activation%20by%20antagonism).

[0008] Similarly, TRX1 is currently believed to work by reducing the density of CD4 expressed on the surface of T cells capable of participating in immune responses, thereby reducing functional CD4.+ The number of effector T lymphocytes (https: / / www.creativebiolabs.net / Anti-CD4-Therapeutic-Antibody-32250.htm).

[0009] To the inventors' knowledge, these non-depleting anti-CD4 antibodies have not yet been used to treat lichen planus.

[0010] Other inflammatory epidermal diseases exhibit similar histopathology, including those containing cytotoxic CD8. + Inflammatory infiltration of cells and apoptosis of epidermal cells. Examples include inflammatory autoimmune epidermal diseases such as cutaneous or mucosal lupus erythematosus, allogeneic epidermal diseases such as cutaneous or mucosal graft-versus-host disease, and allergic dermatitis such as severe cutaneous drug reactions such as Stevens-Johnson syndrome.

[0011] In addition, there are additional autoimmune skin diseases, including CD8. + T cells attack cells within the epidermis or skin appendages. For example, alopecia areata is a condition associated with CD8+. + T cells attack hair follicle-related autoimmune diseases, and in some forms of vitiligo (a condition that causes loss of skin pigmentation), melanocytes in the epidermis are similarly attacked.

[0012] There is a need to continue developing new therapies for these diseases, especially those with relatively low prevalence, such as lichen planus, where research and treatment are limited. Summary of the Invention

[0013] Therefore, in a first aspect, the present invention provides a CD4 + FoxP3 + Regulatory T-cell activators for the treatment or prevention of inflammatory epidermal diseases in subjects, wherein the diseases are characterized by cytotoxic CD8+ cells that induce apoptosis of epidermal cells, melanocytes in the epidermis, or epidermal appendage cells. + Characterized by inflammatory infiltration of T cells.

[0014] In a second aspect, the present invention provides a method for preparing a medicament suitable for treating or preventing inflammatory epidermal diseases in subjects, said inflammatory epidermal diseases comprising cytotoxic CD8+ that induces apoptosis of epidermal cells, apoptosis of melanocytes in the epidermis, or apoptosis of epidermal appendage cells. + The method is characterized by inflammatory infiltration of T cells, and includes the use of CD4+. + FoxP3 + Regulatory T cell activators activate CD4 cells from subjects in vitro. + FoxP3+ Regulatory T cells.

[0015] Furthermore, in a third aspect, the present invention provides an activated CD4 + FoxP3 + Regulatory T cells for the treatment or prevention of inflammatory epidermal diseases in subjects, wherein the diseases are characterized by cytotoxic CD8+ cells that induce apoptosis of epidermal cells, melanocytes in the epidermis, or epidermal appendage cells. + Characterized by inflammatory infiltration of T cells, wherein the activated CD4 + FoxP3 + Regulatory T cells utilize CD4 + FoxP3 + Regulatory T cell activators activate CD4 cells from subjects in vitro. + FoxP3 + It is prepared from regulatory T cells.

[0016] The preferred features of the invention are set forth in the dependent claims.

[0017] In particular, the inventors have surprisingly discovered that regulatory T cells from patients with lichen planus can be activated by an activator, and once activated, they can control autoreactive CD8. + T cell proliferation. This provides a previously unknown therapeutic mechanism for treating this disease. Additionally, as shown in the work of this application, CD8 is controlled in this situation. + The ability of T cells to proliferate provides a therapeutic mechanism for treating and preventing other inflammatory epidermal diseases or conditions, in which CD8 cells proliferate. + T cell-driven epidermal cell apoptosis, epidermal melanocyte apoptosis, or epidermal appendage cell apoptosis. Attached Figure Description

[0018] To aid in understanding this disclosure and to illustrate how the implementation scheme can be carried out, reference is made to the accompanying drawings by way of example, wherein:

[0019] Figure 1 The amino acid sequences of the heavy and light chains of the antibody named TREGALIZUMAB (BT-061) are shown.

[0020] Figure 2A~ Figure 2F Histological photographs of skin specimens obtained from patients with lichen planus are provided, illustrating the characteristics of the disease. Specifically, Figure 2A reveals banded lymphocytic infiltration with cytotoxic CD8... + T cells make close contact with basal keratinocytes (KC) to induce apoptosis - the boxed portion in Figure 2B is enlarged, and apoptotic cells are marked with a black asterisk. Figures 2C to 2FThe T cell population in *Lysimachia foenum-graecum* was shown. Figure 2C CD3 staining; Figure 2D : CD4 staining; Figure 2E CD8 staining; rectangular markings Figure 2F Details shown; Figure 2F1: Figure 2E (Enlarged image, CD8 staining and Figure 2F2 granzyme B (GrB) staining). Inflammatory infiltration rich in CD8. + T cells, most of which express GrB (Fig. 2F2). Note the presence of GrB at the dermal-epidermal junction. + CD8 + The arrangement of cells, in which they contact the substrate KC (marked with black arrows - Fig. 2F2) (magnification: Fig. 2A and 2F2) Figures 2C to 2E ×200; Figure 2B and Figure 2F ×400).

[0021] Figures 3A to 3D Photographs showing representative immunofluorescence (IF) results from skin specimens obtained from patients with cutaneous lichen planus (LP) are provided. CD4 T cell surface markers were analyzed in the specimens. Figure 3A ) and Foxp3 ( Figure 3B Foxp3 is a transcription factor specific to regulatory T cells (Tregs). DAPI is used as a nuclear control stain. Figure 3C ),and Figure 3D The superposition of triple staining was visualized. One representative study from 10 impact factor (IF) studies is described. These results indicate that CD4 in LP... + FoxP3 + Tregs are abundant in inflammatory skin infiltration ( Figure 3B ).

[0022] Figure 4 CD4 levels were shown in a biopsy specimen of Lichen planus (LP). + FoxP3 + Treg cells were enriched. Compared with healthy controls (HC, n=10) skin, Tregs were abundant in skin LP (LP, n=12). Two blinded investigators determined the mean cell count in three high-power fields (HPF, 200x) for each biopsy. , p < 0.0001.

[0023] Figures 5A-5E A bar graph is provided showing that TREGALIZUMAB (BT-061) controls allogeneic reactivity CD8. + T cell proliferation (Ki-67) enhances regulatory T cell (Treg)-mediated suppression of mixed lymphocyte response (MLR) in LP patients. Figure 5AThe dysfunctional Treg function in suppressing CD8 proliferation during MLR was depicted in LP patients compared with healthy controls (HC, 29.2 ± 2.8% (SEM), n=4) (1.7% ± 3.3% (SEM), n=4). p < 0.001. Conversely, Figure 5B It was revealed that adding TREGALIZUMAB (BT-061) to graded amounts of Tregs in LP patients strongly reconstituted their inhibitory MLR activity. CD8 isolates from MACS from healthy donors... + and CD4 + T cells, as well as Tregs and APCs from LP patients, may or may not be mixed with TREGALIZUMAB (BT-061) (1µg / ml) or TREGALIZUMAB (BT-061) solvent (placebo) to resolve CD8. + Allogeneic Ki-67 response of T cells. Cells were co-cultured for 3 days and then analyzed by flow cytometry using the proliferation marker Ki-67. The dashed line represents the mean MLR without any supplementation. p < 0.05; p < 0.01; one-way ANOVA followed by Fisher's least significant difference (LSD) test, n = 4. Figure 5C Showing Figure 5B The relative inhibition of proliferation is shown. The values ​​represent the percentage inhibition of the average MLR against the absence of any stimulant (culture medium). TREGALIZUMAB (BT-061) significantly enhanced the inhibition of MLR by Treg. p < 0.01; p < 0.001; p < 0.0001; ns, not significant; unpaired t-test (Figure A), one-way ANOVA followed by Fisher's least significant difference (LSD) test, n = 4 (Figures B and C), SEM, standard error of the mean. Figure 5D The study showed that stimulation with either Tregalizumab (BT-061) or placebo (PL) for 3 days in MLR resulted in increased levels of regulatory T cells (Tregs) and CD4+. + CD4 modulation on conventional T cells (Tconv). CD4 modulation is expressed as mean fluorescence intensity (MFI) compared to the culture medium control (=100%). Error bars indicate SEM. p < 0.0001, one-sided ANOVA. Figure 5E The study showed that stimulation with Tregalizumab (BT-061) for 3 days in MLR resulted in increased levels of regulatory T cells (Tregs) and CD4+. +TREGALIZUMAB (BT-061)-induced CD137 expression in conventional T cells (Tconv). CD137 expression is expressed as the difference (Δ) in mean fluorescence intensity (MFI) between MLRs treated with TREGALIZUMAB (BT-061) and those treated with placebo. Error bars indicate SEM. p < 0.05, Mann-Whitney-U test, n = 4. Detailed Implementation

[0024] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context explicitly specifies otherwise. Therefore, a reference to “an antibody” includes a reference to “antibodies.”

[0025] All technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains, unless otherwise expressly stated.

[0026] As described above, the present invention relates to the treatment or prevention of inflammatory epidermal diseases in subjects, wherein the diseases are associated with or characterized by inflammatory infiltrations (e.g., located in the dermis (dermal infiltration)) that contain cytotoxic CD8. + T cells cause damage to epidermal cells, particularly epidermal cell apoptosis, or damage to other cells within the epidermis or epidermal appendages, such as apoptosis of melanocytes in the epidermis or epidermal appendage cells. Epidermal appendages are skin-associated structures located in the dermal region of the skin, including certain glands and hair follicles.

[0027] This epidermal disease is characterized (and can be identified) by histological examination of specimens obtained from diseased areas of the body (e.g., biopsy), which show cytotoxic CD8. + T cells attack and induce apoptosis in epithelial cells (e.g., keratinocytes), melanocytes in the epidermis, or cells of epidermal appendages (such as hair follicle cells). (Apoptotic cells can be identified in samples using DNA-binding staining agents (such as Hoechst 33342), which can be used to display the nuclear aggregation characteristic of apoptotic cells.) Cytotoxic CD8 + T cells can be spontaneously reactive or allogeneic reactive (e.g., in skin or mucosal graft-versus-host disease following allogeneic stem cell transplantation), and in some cases can be triggered by allergic reactions (e.g., in severe skin drug reactions). In some instances, histological examination shows lymphocytic infiltration (e.g., zosteric lymphocytic infiltration) that reaches the dermal-epidermal junction and causes so-called interface dermatitis, in which cytotoxic CD8 cells...+ T cells are in close contact with epidermal cells (such as basal keratinocytes), and numerous apoptotic epidermal cells, particularly apoptotic keratinocytes, are observed. Exemplary histological findings from a lichen planus patient are shown in Figures 2A-20. Figure 2F In particular, in Figure 2B, apoptotic cells are marked with a black asterisk.

[0028] The inflammatory epidermal disease may be an autoimmune epidermal disease, an allogeneic epidermal disease, or an allergic skin disease caused by adverse reactions to drugs or infections, for example.

[0029] Specifically, the inflammatory epidermal diseases may be selected from lichen planus (including skin, mucous membranes, and hair follicles), lichen sclerosus (skin and / or mucous membranes (genitals / or mouth)), cutaneous or mucosal lupus erythematosus (isolated or in the context of systemic lupus erythematosus), dermatomyositis, cutaneous or mucosal graft-versus-host disease, small or large erythema multiforme exudative erythema (including mucocutaneous involvement) and especially caused by infectious (bacterial, viral) antigens, fixed drug eruptions, severe cutaneous drug reactions (including Stevens-Johnson syndrome and toxic epidermal necrolysis (also known as Lyell syndrome)), alopecia areata (including localized alopecia areata (including creeping alopecia), diffuse alopecia areata, partial alopecia areata, total alopecia, and generalized alopecia areata), non-segmental vitiligo (including generalized vitiligo, facial arcuate vitiligo, generalized vitiligo, and generalized vitiligo), and focal vitiligo. The inflammatory epidermal diseases mentioned may be selected from lichen planus, lichen sclerosus, cutaneous lupus erythematosus, dermatomyositis, and skin graft-versus-host disease.

[0030] Preferably, the disease is an inflammatory autoimmune skin disease selected from lichen planus, lichen sclerosus, cutaneous lupus erythematosus, and dermatomyositis. More preferably, the disease is lichen planus, also referred to herein as LP. This encompasses all clinically distinct cutaneous / mucosal lesions of LP, including oral lichen planus, penile or vulvar lichen planus, follicular lichen planus, nail lichen planus, and lichenoid papulosis, the latter being a fulminant variant of lichen planus.

[0031] The subject can be any mammal, including humans, non-human primates, or domesticated mammals such as cats, dogs, or rodents. However, preferably, the subject is a human. The subject can be 6 years of age or older, preferably 16 years of age or older.

[0032] As mentioned above, the disease to be treated uses cytotoxic CD8. + Characterized by T cells, which lead to apoptosis of the patient's keratinocytes. In particular, this still occurs despite the presence of regulatory T cells (see [link to relevant documentation]). Figures 3A-3DSee, for example, Ujiie, H., “Regulatory T cells in autoimmune skin diseases”, Experimental Dermatology, 2019; 28: 642-646.

[0033] Regulatory T cells (Tregs) are CD4+ cells. + A subset of T cells, known to regulate the immune system, and particularly known to be important for maintaining tolerance to self-antigens (Sakaguchi et al., Immunological Reviews (2001); 182: 18-32), and to play a role in regulating immune homeostasis and inflammation.

[0034] The Treg family consists of two key subsets: (i) naturally occurring Tregs (sometimes called nTregs), which develop in the thymus; and (ii) peripherally induced Tregs (sometimes called iTregs), which arise from regular T cells in the peripheral circulation. Tregs are typically characterized by the expression of CD4 and CD25 surface biomarkers, and also by the transcription factor Foxp3. In this article, regulatory T cells (Tregs) are referred to as CD4+ Tregs. + FoxP3 + Regulatory T cells (although they can also be considered CD4) + CD25 + FoxP3 + Regulatory T cells).

[0035] Dysfunction or low levels of Tregs are known to be associated with certain autoimmune or allergic diseases, and also with graft-versus-host disease (Rivas et al., J Allergy Clin Immunol. 2016 September;138(3): 639-652; Ohl and Tenbrock, Eur. J. Immunol. 2015. 45: 344-355; Whangbo et al., Expert Review of Hematology, 2020; 13(2): 141-154). Their inability to control cytotoxic CD8... + The cells were inferred to have functional impairment in lichen planus, despite their significant presence in the diseased skin. For example... Figure 5A As shown, Tregs from LP patients were unable to inhibit CD8 during mixed lymphocyte response (MLR). + Cell proliferation.

[0036] However, as Figure 5B and 5C As shown, despite potential functional impairments, these Tregs can be activated by the activator described herein, and once activated, they can control CD8. + T cell proliferation.

[0037] Therefore, this invention relates to CD4 + FoxP3 + Medical uses of regulatory T-cell activators. In one aspect of the invention, the activator is used to administer to a subject, i.e., for in vivo treatment or prevention of disease. In an alternative aspect of the invention, CD4 can be isolated from a sample (e.g., a blood sample) collected from the subject to be treated. + FoxP3 + Regulatory T cells are extracted, activated in vitro, and then transferred back to the subject to treat or prevent disease.

[0038] CD4 + FoxP3 + Activators of regulatory T cells are known in the art. Specifically, said activators may be antibodies or antibody fragments, such as anti-CD4 antibodies or fragments thereof (e.g., as described in patent application publication number WO2011 / 064407), or proteins, such as gp120 protein or fragments thereof (e.g., as described in patent application publication number WO 2008 / 092905), IL-2, or IL-2 mutants. Some activators known to be activators of both regulatory T cells and effector T cells, or activators known to cause undesirable side effects in vivo, are only suitable for ex vivo use, where their action is effective against isolated regulatory T cells, such as some anti-CD3 antibodies or fragments thereof. Other activators, including the anti-CD4 antibody TREGALIZUMAB (also known in the art and referred to herein as BT-061), discussed below, are capable of selectively activating regulatory T cells.

[0039] The activator "can activate CD4" + FoxP3 + "Regulatory T cells." In one instance, this could be defined as the activator, such as an anti-CD4 antibody or antibody fragment, activating CD4 expression in vitro. + and Foxp3 + The ability of T cells to activate CD4+ is preferred. + FoxP3 +Regulatory T cells are able to suppress the production of pro-inflammatory cytokines (e.g., IL-5, IL-4 and / or IL-13, preferably IL-5) in PBMC cultures after TCR stimulation, particularly in cultures containing T effector cells.

[0040] Assay methods for this activity may include using antibodies or antibody fragments to obtain CD4 from a donor (e.g., a healthy human donor). + FoxP3 + Regulatory T cells are pre-incubated for a time suitable for cell activation (e.g., 48–72 hours), and T effector cell cultures obtained from the same donor are stimulated with TCR. Pre-incubated CD4+ cells are then added. + FoxP3 + Regulatory T cells were transferred into T effector cell cultures, and pro-inflammatory cytokine levels were measured 72 hours later. T effector cell cultures and / or CD4+ were also analyzed. + FoxP3 + Regulatory T cells can be obtained from healthy individuals or individuals suffering from the disease described above according to the invention. In this assay, CD4 cells are capable of activation. + FoxP3 + Activators of regulatory T cells, such as anti-CD4 antibodies or fragments thereof, will lead to a reduction in the amount of pro-inflammatory cytokines compared to CD4 cells not pre-incubated with the activator. + FoxP3 + Compared to a negative control of cells. Preferably, the activator tested results in the same reduction ±25%, more preferably ±10%, as described herein, as with the positive control TREGALZUMAB antibody (BT-061) or an anti-CD4 antibody having the same variable heavy chain and variable light chain regions as TREGALZUMAB antibody (BT-061).

[0041] The activation of CD4 by the activator can also be determined by the methods described in this art. + FoxP3 + The ability of regulatory T cells, such as those described in WO2011 / 064407, to act as an activator by incubating with CD4+. + FoxP3 + Regulatory T cells and CD4 + CD25 - Effector T cell co-culture to detect CD4 + FoxP3 + This ability was measured by the inhibitory activity of regulatory T cells. Activated CD4 + FoxP3 +Regulatory T cells can inhibit the proliferation of these effector T cells in effector T cell proliferation assays. Effector T cells can be labeled with CFSE to identify any proliferation. Alternatively, […]. 3 H] Thymidine incorporation is used to determine the proliferation of effector cells.

[0042] It has also been shown that CD137 + CD154 - Expression is a ubiquitous marker of in vitro Treg activation, which allows for the identification and isolation of antigen-activated Tregs (Nowak et al., Frontiers in Immunology, February 2018, Vol. 9, Article 199). Therefore, as a further option, expression based on CD137 can also be used. + CD154 - Expression to identify activated CD4 produced in the experiment + FoxP3 + Regulatory T cells.

[0043] This activator can also be defined as not causing either antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Methods are known in the art to determine this ability. Specifically, the anti-CD4 antibody TREGALIZUMAB (BT-061) described herein is known not to cause ADCC or CDC (as described in Helling et al., Immunology and Cell Biology (2015) 93: 396-405). Methods for modulating the effector function of antibodies or antibody fragments are known in the art to eliminate effector function (Saunders, KO, June 2019, Frontier in Immunology, Vol. 10, Article No. 1296). The scope of this invention includes antibodies or antibody fragments modified such that they do not cause ADCC or CDC.

[0044] The activator is preferably an antibody, antibody fragment, or bispecific or multispecific antibody that binds to Tregs. Bispecific or multispecific antibodies target at least one other antigen (e.g., another target cell, such as CD8). + Antibodies against antigens of cells can be used to help activate Treg cells come into contact with cells that have an effect on them.

[0045] The antibody may be a human antibody, a humanized antibody, a chimeric antibody, or a fragment thereof. Preferably, the antibody is a humanized antibody or a fragment of a humanized antibody.

[0046] Typically, the antibody also contains a human constant region (Fc). This constant region can be selected from the constant domains of any class of immunoglobulins (including IgM, IgG, IgD, IgA, and IgE) and any subtype (including IgG1, IgG2, IgG3, and IgG4). Preferred constant regions are selected from the constant domains of IgG, particularly the constant domain of IgG1.

[0047] The antibody is preferably an IgG1 antibody, and / or the antibody or antibody fragment preferably contains an Fc portion, enabling the antibody or antibody fragment to bind to an Fc receptor (preferably FcγRI (i.e., CD64)). Most preferably, the antibody or antibody fragment contains the Fc portion of an IgG1 antibody. Alternatively, the antibody or antibody fragment may bind to monocytes via an Fc receptor.

[0048] The invention also includes any fragments of the antibody, including fragments containing its V region. This particularly includes Fab, Fab', F(ab')2, Fv, and scFv fragments.

[0049] The activator may be an anti-CD4 antibody or an antibody fragment thereof, preferably an anti-human CD4 antibody or a fragment thereof.

[0050] Specifically, the anti-CD4 antibody can be the antibody TREGALIZUMAB (also known as BT-061) or an antibody based on or derived from it. These antibodies are non-depleting and can activate regulatory T cells but not effector T cells.

[0051] Therefore, preferably, the antibody is a humanized anti-CD4 antibody or a fragment thereof, which is the Tregalizumab antibody (BT-061), or a variant or fragment thereof. Specifically, the Tregalizumab antibody (BT-061) has a V domain defined by the following polypeptide sequence (further described in the examples below):

[0052]

[0053] Therefore, the anti-CD4 antibody or fragment thereof used in this invention may comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 4. In a preferred embodiment, the anti-CD4 antibody comprises a human IgG1 constant domain.

[0054] Variants of the antibody or antibody fragment are also suitable for use in this invention. Variants include those whose V domain is defined by a polypeptide sequence having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4. More preferably, the antibody or fragment thereof has a heavy chain variable domain having at least 90% sequence identity with SEQ ID NO: 3 and a light chain variable domain having at least 90% sequence identity with SEQ ID NO: 4. Still more preferably, the antibody or fragment thereof has a heavy chain variable domain having at least 95% sequence identity with SEQ ID NO: 3 and a light chain variable domain having at least 95% sequence identity with SEQ ID NO: 4. A preferred embodiment is an antibody comprising these variable domain sequences and an IgG1 constant domain.

[0055] Alternatively, the antibody or antibody fragment may be an antibody or antibody fragment having the CDR sequence (SEQ ID NO: 5-10) of TREGALIZUMAB (BT-061). A particularly preferred embodiment is an anti-CD4 antibody or a fragment thereof comprising a heavy chain variable domain having at least 95% sequence identity with SEQ ID NO: 3 and including the CDR sequences of SEQ ID NO: 5-7, and a light chain variable domain having at least 95% sequence identity with SEQ ID NO: 4 and including the CDR sequences of SEQ ID NO: 8-10.

[0056] The binding mechanism of TREGALIZUMAB (BT-061) to CD4 has been disclosed in Helling et al., Immunology and Cell Biology (2015) 93: 396-405. Further variants of TREGALIZUMAB (BT-061) have also been disclosed in WO 2011 / 064407. Therefore, this information can be used to guide changes in the heavy and light chain sequences. Preferably, the changes do not significantly affect the binding specificity and / or affinity.

[0057] Alternatively, the antibody or antibody fragment may be an antibody or antibody fragment that binds to epitopes on CD4 containing amino acids 148–154, 164–168, 185, 187, 189–190, and 192 (as described in Helling et al., Immunology and Cell Biology (2015) 93: 396-405 and WO 2011 / 064407). Methods for determining the ability to bind specific epitopes are known in the art. The antibody or antibody fragment may also be defined as one that binds substantially the same epitopes as the TREGALIZUMAB antibody (BT-061) described herein. Epitope mapping of antibodies can be performed using methods known in the art (see, for example, Dang et al., “Epitope mapping of monoclonal antibodies: a comprehensive comparison of different technologies”, MARS 2023, vol. 15, no. 1, DOI: 10.1080 / 19420862.2023.2285285).

[0058] In one aspect, the present invention provides an anti-CD4 antibody for treating or preventing lichen planus in a subject, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and the light chain comprising the amino acid sequence of SEQ ID NO: 2.

[0059] In another aspect, the present invention provides an anti-CD4 antibody or a fragment thereof for treating or preventing lichen planus in a subject, wherein the antibody or fragment thereof comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having at least 95% sequence identity with SEQ ID NO: 3 and including the CDR sequences of SEQ ID NO: 5 to 7, and the light chain variable domain having at least 95% sequence identity with SEQ ID NO: 4 and including the CDR sequences of SEQ ID NO: 8 to 10.

[0060] The antibodies or antibody fragments mentioned in this article can be manufactured using encoding polynucleotide sequences. Specifically, recombinant DNA constructs can be obtained and introduced into host cells using known recombinant DNA technologies and genetic engineering techniques. Expression cassettes can be used, in which the polynucleotide sequence is linked to appropriate control sequences that allow for the regulation of transcription and translation in selected host cells. Suitable host cells include mammalian cells such as Sp2 / 0, HeLa, CHO, 3T3, C127, BHK, COS, etc.

[0061] Therefore, antibodies or antibody fragments can be produced in mouse or human cell lines, and particularly in the cell lines mentioned herein.

[0062] The activator may be included in a pharmaceutical composition that also includes pharmaceutically acceptable excipients or carriers. The pharmaceutical composition may also contain stabilizers. The pharmaceutical composition may be suitable for intravenous, subcutaneous, or intramuscular use.

[0063] In some aspects of the invention, the activator is used by administration to a subject. The antibody or antibody fragment can be administered via any suitable route, such as intravenous, subcutaneous, or intramuscular. Preferably, the activator is administered subcutaneously.

[0064] Depending on the route of administration, antibodies or antibody fragments may be contained in vials, pre-filled syringes, or autoinjectors. Vials may be arranged for use in autoinjectors, i.e., arranged so that they can be assembled or inserted into the autoinjector prior to use.

[0065] Based on established methods, the appropriate dosage of the activator can be determined. The activator is an anti-CD4 antibody or a fragment thereof, which can be administered at a dosage between 10 mg and 200 mg, preferably between 75 mg and 200 mg, and more preferably between 100 mg and 200 mg.

[0066] The dosage can also be calculated based on the subject's weight, and can be 1 to 3 mg / kg, preferably 1 to 2.5 mg / kg, and most preferably 1.5 to 2 mg / kg.

[0067] Antibodies or antibody fragments can be administered at regular intervals, such as once a week, once every two weeks, once every three weeks, or once a month.

[0068] As described above, in another aspect of the invention, the activator can be used to in vitro activate CD4 obtained from a subject before the activated cells are reintroduced into the subject to treat or prevent the disease. + FoxP3 + Regulatory T cells.

[0069] Therefore, the present invention also provides a method for treating or preventing a subject suffering from or suffering from the inflammatory autoimmune skin disease described herein, the method comprising removing CD4-containing cells from the subject. + FoxP3 + Samples of regulatory T cells, making CD4 + FoxP3 + Regulatory T cells are exposed to activators as described herein to activate CD4. + FoxP3 +Regulatory T cells and the administration of activated cells to the subject. The method may also include an in vitro step to increase the number of Tregs. This can be accomplished using the amplification strategies described herein (Peters et al., PLoS 2008; 3(9): e3161; Hoffmann et al., Blood 2004; 104(3):895-903).

[0070] Similarly, the present invention provides activated CD4 + FoxP3 + Regulatory T cells, which have been activated in vitro using the activators described herein, were used to treat or prevent the diseases described herein in subjects. CD4 + FoxP3 + Regulatory T cells were obtained from the subject. Optionally, as described above, CD4 was expanded before being reintroduced into the subject. + FoxP3 + Regulatory T cells.

[0071] The following are merely examples and are not intended to limit this disclosure.

[0072]

Example

[0073] [Example 1: Activation of regulatory T cells in patients with lichen planus (LP)]

[0074]

Antibody

[0075] The anti-CD4 antibody Tregalizumab (BT-061) used in the examples is a recombinant humanized IgG1 monoclonal antibody containing two heavy chains and two light chains. The amino acid sequences of both the heavy and light chains were predicted from the nucleotide sequence of the Tregalizumab (BT-061) gene and have been experimentally confirmed. Figure 1 The predicted amino acid sequences of the heavy and light chains, along with the most likely disulfide bond assignments, are shown. The heavy chain has SEQ ID NO: 1, and the light chain has SEQ ID NO: 2.

[0076] The TREGALIZUMAB antibody (BT-061) used in the examples has been produced in the mouse myeloma host cell line Sp2 / 0-Ag14 (Sp2 / 0, corresponding to ATCC CRL-1581). Antibody expression in Sp2 / 0 is also described in WO 2009 / 124815.

[0077] The BT-061 antibody has been given the generic name TREGALIZUMAB.

[0078] Mixed lymphocyte response (MLR)

[0079] Mixed lymphocyte response (MLR) is an experimental procedure used to study the immune system's response to foreign cells in vitro. Because autoreactive CD8 cells in LP recognize a portion of the skin (epidermis) as foreign in the context of an autoimmune process, MLR is suitable as an experimental method for studying the ability of Treg activators to treat this disease.

[0080] To establish the MLR, CD8-positive T cells from donor A were mixed with antigen-presenting cells (APCs) from donor B and cultured in an incubator at 37°C. Over a 3-day period, cytotoxic CD8 cells recognized APCs as foreign and began to proliferate in the neoimmune response. This proliferative response was quantified by flow cytometry using the proliferation marker Ki-67. The addition of CD4-positive T lymphocytes (from donor A) to the cell culture enabled a more physiological approach and ensured measurement of proliferation. The effect of regulatory T cells (Tregs) on the proliferative response of CD8 cells was addressed by incorporating Tregs (from donor B) into the cell culture. Finally, to address the effect of Tregalizumab mAb (BT-061) on the inhibitory effect on Tregs, Tregalizumab (BT-061) was added to the MLR with or without Tregs.

[0081] [Detailed Experiment Setup]

[0082] Anonymous erythrocyte sedimentation rate (ESR) amber layers from different donors at the Institute of Clinical Transfusion Medicine and Hemotherapy, Würzburg University Hospital, were used to isolate the desired cells. In a typical experimental setup, 5 × 10⁶ cells from donor A were used. 4 CD8 cells and 2×10 4 1.5 × 10⁴ CD4 cells and 1.5 × 10⁴ cells from LP patients 5 APC cells (hereafter designated donor B) were mixed in 96-well plates. To address the effect of Tregs on MLR, 5 × 10⁶ cells from donor B were co-cultured from the start of MLR. 4 Three days later, CD8 cell proliferation was determined by flow cytometry using the Ki-67 marker. To analyze the effect of TREGALIZUMAB (BT-061) on MLR when co-cultured with Tregs, mAb was added from the start of the cell culture.

[0083] Specifically, 5×10⁻⁶ of the erythrocyte sedimentation rate (ESR) layer from the healthy donor A was used to measure the erythrocyte sedimentation rate (ESR) layer. 7 10 cells and 2-3 × 10 from donor B 7The cells were thawed from the freezer (-80°C) and cultured overnight in X-Vivo medium (Gibco) at 37°C and 5% CO2.

[0084] On the second day, CD8-positive T cells were first isolated from donor A using the MACS technique (Miltenyi Biotec, #130-045-201). The initial flow medium from the CD8 column was used to purify CD4 cells, from which contamination-free CD4-positive cells with Tregs were obtained in a further step using a Treg isolation kit (Miltenyi Biotec, #130-091-301). In this step, CD25-labeled CD4-positive T cells (Tregs) remained in the MACS column, while Treg-depleted CD4-positive cells were passed through the column as flow medium. To ensure a high level of Treg-depleted CD4 cells, the process was repeated. CD4 cells were added to the MLR at the concentration described above. The Tregs retained in the column were discarded.

[0085] APCs from donor B, such as those from LP patients, were purified by depleting CD3 cells using CD3 microbeads (Miltenyi Biotec, #130-050-101) and used in the MLR at the concentrations described above. Additionally, Tregs from donor B were isolated using a Treg isolation kit (Miltenyi Biotec, #130-091-301) according to the manufacturer's instructions. The range was 1×10⁻⁶. 2 ~1×10 4 Different concentrations of Treg were used in MLR assays for individual cells.

[0086] First, a stock solution of Tregalizumab (BT-061) (120 mg / ml) was diluted 1:120 in X-Vivo medium (Gibco), and then diluted 1:1000 to a final concentration of 1 µg / ml (equivalent to a 1:120,000 dilution of the stock solution). The same dilutions were used for the placebo solution containing Tregalizumab (BT-061) solvent. Three days later, cells were cultured for 4 hours at 37°C, 5% CO2 in the presence of brevidin A (final concentration 20 µg / ml) with PMA (final concentration 100 ng / ml) and iomycin (final concentration 1 µg / ml) for flow cytometry analysis of proliferation markers. Subsequently, cells were analyzed by flow cytometry (with markers CD3, CD4, CD8, CD137, CD154, Ki-67, Foxp3, and live / dead staining).

[0087]

result

[0088] The results showed that Tregalizumab (BT-061) activates Tregs from healthy donors and inhibits CD8 in the MLR. + Following the allogeneic reactive proliferative capacity of T cells (data not shown), we inquired whether the MLR response could also be attenuated by TREGALIZUMAB (BT-061) in the use of Tregs from patients with lichen planus (LP). Therefore, by using Tregs and APCs from PBMCs of LP patients and CD8 from healthy donors… + and CD4 + MLR is established using T cells. This is achieved through allogeneic reactive CD8 cells. + The strength of the MLR response is measured by T cell proliferation. Typically, this is done by adding [a specific ingredient] to the cell culture. 3 Quantification using H-thymidine requires prior treatment of APCs with mitomycin C or proliferation-inhibiting irradiation. Since this pretreatment of damaged cells interferes with MLR, flow cytometry analysis of Ki-67 expression was used to determine CD8 expression. + T cell proliferation response.

[0089] Four independent experiments showed that the average MLR response was converted to 7.5 ± 1.9% (± SEM) of allogeneic reactivity CD8. + T cell proliferation ( Figure 5B The results were not statistically affected by the addition of either Tregalizumab (BT-061) or the Tregalizumab (BT-061) solvent (placebo). Interestingly, the addition of Tregalizumab (BT-061) from LP patients and 10 4 Adding one Treg to the MLR assay showed an MLR response inhibition of 78.7% (1.6 ± 0.4%, ± SEM), while Treg alone or placebo had little effect (7.6 ± 1.9%, ± SEM or 6.2 ± 1.3%, ± SEM, respectively). The range of use is from 10... 3 ~10 2 The enhanced inhibition of MLR by TREGALIZUMAB (BT-061) was also observed in fractionated amounts of Treg. Figure 5B ).

[0090] The relative inhibition of the proliferative CD8 response (where the mean of the MLR without any stimulation was set at 100%) reflects the effect of 10 4 One Treg cell, together with TREGALIZUMAB (BT-061), promoted 75.5 ± 5.0% (± SEM) inhibition of MLR ( Figure 5CIn contrast, adding placebo to MLR-Treg cultures had no statistically significant effect on MLR inhibition, as did TREGALIZUMAB (BT-061) alone.

[0091] Mean CD4 expression on T cells in MLRs was analyzed by flow cytometry, followed by stimulation with either Tregalizumab (BT-061) or placebo (PL) for 3 days. Dead cells were excluded after lymphocyte gating was performed, using live / dead staining. Gating was then performed sequentially for CD3 and CD8. CD4 cells were selected after excluding CD8 cells. Based on Foxp3... + Or Foxp3 - Regulatory T cells (Tregs) and CD4 + Conventional T cells (Tconv) were separated. Results showed... Figure 5D In the study, CD4 regulation was depicted as mean fluorescence intensity (MFI) compared to the culture medium control (=100%). These results indicate that Tregalizumab (BT-061) modulates regulatory T cells (Tregs) and CD4+. + Conventional T cells (Tconv) showed comparable and significant CD4 downregulation induction compared to the other two.

[0092] CD137 expression in Treg and Tconv was analyzed by flow cytometry after 3 days of stimulation in the MLR with either Tregalizumab (BT-061) or placebo (PL). A gating strategy was performed as described in the previous paragraph, followed by Foxp3 assay. + / Foxp3 - Gating is set sequentially with CD137 / CD154. CD137 + The cells are CD154 - (No data displayed). Results are shown below. Figure 5E In the study, CD137 expression was characterized as the difference in MFI (Δ) between BT-061 and placebo-treated MLRs. These results indicate that TREGALIZUMAB (BT-061) specifically induces CD137 expression in Tregs, but not in Tconvs (CD137 is known as a biomarker of Treg activation).

[0093]

in conclusion

[0094] The MLR model is a recognized model for addressing allogeneic reactive immune responses. Using this model, in vitro testing of regulatory T cells from patients with the autoimmune skin disease LP showed that Tregs alone could not suppress MLR. Surprisingly, adding mAb BT-061 to the MLR-Treg co-culture promoted strong Treg-mediated inhibition. In contrast, Tregalizumab (BT-061) alone or placebo (Tregalizumab (BT-061) solvent) showed no significant effect. Further analysis confirmed that Treg cells in the MLR had been activated by Tregalizumab (BT-061) – inducing CD4 downregulation and cell CD137. + and CD154 - This is a known Treg activation phenotype (Nowak et al., Frontiersin Immunology, February 2018, Vol. 9, Article 199). Data show that the TREGALIZUMAB antibody (BT-061), along with general T regulatory cell activators, can enhance Treg effector function in LP patients, thereby leading to treatment of the disease.

[0095] The embodiments described herein should be understood as illustrative examples of implementations of the invention. Further embodiments and embodiments are contemplated. Any feature described in connection with any embodiment or embodiment may be used alone or in combination with other features. Furthermore, any feature described in connection with any embodiment or embodiment may also be used in combination with one or more features of any other embodiment or embodiment, or in any combination of any other embodiment or embodiment. Additionally, equivalents and modifications not described herein may be employed within the scope of the invention, the scope of which is defined in the claims. All articles and patent documents cited herein are incorporated herein by reference in their entirety.

[0096]

sequence

[0097]

Claims

1. CD4 + FoxP3 + A regulatory T-cell activator for the treatment or prevention of an inflammatory epidermal disease in a subject, wherein the disease comprises cytotoxic CD8+ that induces apoptosis of epidermal cells, melanocytes in the epidermis, or epidermal appendage cells. + Characterized by inflammatory infiltration of T cells.

2. The activator according to claim 1, wherein the inflammatory epidermal disease is an autoimmune epidermal disease, an alloimmune reactive epidermal disease, or an allergic dermatitis.

3. The activator according to claim 1 or claim 2, The epidermal cells mentioned above are keratinocytes, or The epidermal appendages mentioned above are hair follicles.

4. An activator for the use according to any one of the preceding claims, wherein the disease is characterized by interfacial dermatitis.

5. An activator for the use according to any one of the preceding claims, wherein the disease is selected from: lichen planus, lichen sclerosus, cutaneous or mucosal lupus erythematosus, dermatomyositis, cutaneous or mucosal graft-versus-host disease, small polymorphic exudative erythema, multimorphic exudative erythema, fixed drug eruption, severe cutaneous drug reactions (including Stevens-Johnson syndrome and toxic epidermal necrolysis (Lyell syndrome)), alopecia areata, non-segmental vitiligo, and focal vitiligo.

6. The activator for the use according to claim 5, wherein the disease is selected from: lichen planus, lichen sclerosus, cutaneous lupus erythematosus, dermatomyositis, and skin graft-versus-host disease.

7. The activator for the use of claim 6, wherein the disease is lichen planus.

8. An activator for the use according to any one of the preceding claims, wherein the activator is a protein.

9. The activator for use according to claim 8, wherein the activator is an antibody, antibody fragment, bispecific antibody, or multispecific antibody.

10. An activator for use according to claim 8 or claim 9, wherein the activator is an anti-CD4 antibody or a fragment thereof, gp120 protein or a fragment thereof, IL2 or an IL2 mutant.

11. The activator for the use of claim 10, wherein the anti-CD4 antibody or a fragment thereof comprises: A variable heavy chain polypeptide sequence having at least 85% sequence identity with SEQ ID NO: 3, and A variable light chain polypeptide sequence having at least 85% sequence identity with SEQ ID NO:

4.

12. The activator for the use of claim 10 or 11, wherein the anti-CD4 antibody or a fragment thereof comprises: A variable heavy chain polypeptide sequence comprising CDR sequences having SEQ ID NO: 5, 6 and 7, and A variable light chain polypeptide sequence containing CDR sequences having SEQ ID NO: 8, 9 and 10.

13. The activator for use according to any one of claims 10 to 12, wherein the anti-CD4 antibody or a fragment thereof comprises: A variable heavy chain polypeptide sequence having SEQ ID NO: 3, and A variable light chain polypeptide sequence having SEQ ID NO:

4.

14. The activator for use according to any one of claims 9 to 13, wherein the antibody is an IgG antibody.

15. The activator for use according to any one of claims 9 to 14, wherein the antibody is an IgG1 antibody.

16. The activator for use according to any one of claims 9 to 15, wherein the antibody is an anti-CD4 antibody, the anti-CD4 antibody having: The heavy chain sequence of SEQ ID NO: 1, and The light chain sequence of SEQ ID NO:

2.

17. An activator for use according to any one of the preceding claims, wherein the activator is contained in a pharmaceutical composition.

18. An activator for use according to any of the preceding claims, wherein the subject is a human subject.

19. An activator for use according to any of the preceding claims, wherein the activator is administered to a subject intravenously, intramuscularly or subcutaneously, and preferably wherein the activator is administered subcutaneously.

20. To prepare cytotoxic CD8 inhibitors suitable for the treatment or prevention of patients with epidermal cells, including those inducing apoptosis of epidermal melanocytes or epidermal appendage cells. + A method of administering a drug to a subject with an inflammatory epidermal disease characterized by inflammatory infiltration of T cells, the method comprising using CD4 + FoxP3 + Regulatory T cell activators activate CD4 cells obtained from subjects in vitro. + FoxP3 + Regulatory T cells.

21. The method of claim 20, wherein the disease is defined as any one of claims 2 to 7.

22. The method of claim 20 or claim 21, wherein the activator is defined as in any one of claims 8 to 17.

23. Activated CD4 + FoxP3 + Regulatory T cells, their use in treating or preventing inflammatory epidermal diseases in subjects. The disease described herein comprises cytotoxic CD8 molecules that induce epidermal cell apoptosis, melanocyte apoptosis in the epidermis, or epidermal appendage cell apoptosis. + Characterized by inflammatory infiltration of T cells. The activated CD4 + FoxP3 + Regulatory T cells utilize CD4 + FoxP3 + Regulatory T cell activators activated CD4 cells obtained from the subjects in vitro. + FoxP3 + It is prepared from regulatory T cells.

24. Activated CD4 for the purpose of claim 22 + FoxP3 + Regulatory T cells, wherein the disease is defined as in any one of claims 2 to 7.

25. Activated CD4 for the purpose of claim 24 + FoxP3 + Regulatory T cells, wherein the activator is defined as in any one of claims 8 to 17.

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