WO2022096727A1 - Anti-cd4 antibody or fragment thereof for medical use - Google Patents
Anti-cd4 antibody or fragment thereof for medical use Download PDFInfo
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- WO2022096727A1 WO2022096727A1 PCT/EP2021/080985 EP2021080985W WO2022096727A1 WO 2022096727 A1 WO2022096727 A1 WO 2022096727A1 EP 2021080985 W EP2021080985 W EP 2021080985W WO 2022096727 A1 WO2022096727 A1 WO 2022096727A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/577—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 tolerising response
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Definitions
- the present disclosure relates to the prevention and treatment of asthma.
- the present disclosure relates to an anti-CD4 antibody or a fragment thereof for use in treating or preventing asthma, including allergic asthma and non-allergic asthma.
- An allergy or allergic disease is a medical condition that involves a damaging immune response by the body to a substance (allergen) in the environment, which is typically common and harmless (e.g. pollen, fur or dust), and to which the body has become hypersensitive.
- allergen e.g. pollen, fur or dust
- the immune system “overreacts” leading to symptoms that can range from mild to severe and life-threatening.
- Asthma is a chronic airway disease characterized by inflammation, recurrent wheezing, bronchial hyperreactivity, and airway obstruction. It is generally divided into two types: 1) allergic asthma, which is triggered by inhaling antigens and is initiated by Th2 cells; and 2) non-allergic asthma, which is caused by triggers such as infections of the respiratory tract, exercise, smoke, air pollution, or cold or dry air (Hurwitz, S.H., 1955. Nonallergic Asthma — Differential Diagnosis and Treatment. Calif Med 83, 61-67).
- allergic asthma is one of the most common chronic diseases and the most common non-communicable chronic disease worldwide. More than 30 million people are estimated to be affected worldwide with the prevalence of allergic asthma on the rise (Backman, H., et al., 2017. Increased prevalence of allergic asthma from 1996 to 2006 and further to 2016-results from three population surveys. Clin. Exp. Allergy 47, 1426-1435). About 5-10% of asthma patients suffer from uncontrollable, severe asthma which is refractory to the current standard of treatment with steroids (Lloyd, C.M., Hessel, E.M., 2010. Functions of T cells in asthma: more than just T(H)2 cells. Nat. Rev. Immunol. 10, 838-848).
- Asthma can result in persistent airway remodelling and permanent structural changes in the airways. These structural changes contribute to the airway obstruction associated with the disease and can result in the progressive loss of lung function over time. In addition to structural changes, as the disease progresses, airflow is further limited by edema, inflammation, cellular infiltration, and overproduction of mucus. Infiltrating immune cells — which are thought to underlie structural changes — include eosinophils, neutrophils, mast cells, and lymphocytes.
- the traditional standard of care for controlling bronchial asthma has focused on the use of potent anti-inflammatory drugs, particularly steroids. Glucocorticoids are effective in most patients but do not provide prolonged relief without repeated administration and do not address the molecular basis of the disease.
- glucocorticoids fail to control disease symptoms in a significant number of asthma patients. These individuals are most at risk of hospitalization and asthma-related death.
- monoclonal antibody treatments are available for the treatment of severe asthma patients, targeting IL-4, IL-5, IL-13 or IgE.
- IL-4, IL-5, IL-13 or IgE monoclonal antibody treatments
- Other monoclonal antibody treatments have been investigated but have been found not to be suitable.
- the chimeric human/macaque IgGl anti-CD4 antibody keliximab
- keliximab was initially thought to benefit severe asthmatics by decreasing the role of CD4 + lymphocytes in the pathogenesis of chronic airway disease.
- studies into the use of this antibody were stopped as a result of the limiting side effect of a reduction in CD4 + cell count in patients (Kon et al., Eur. Respir. J. 2001; 18: 45-52; Tourangeau et al., Therapeutic Advances in Respiratory Disease (2011): 5(3): 183-194).
- the humanized anti-CD25 IgGl antibody, daclizumab was assessed in moderate to severe asthmatic patients and was shown to improve FEV1, reduce daytime asthma symptoms and P2 agonist use, however, there were more serious adverse events in the treatment group as compared to the placebo. (Busse et al,. Am. J. Respir. Crit. Care Med. 2008; 178: 1002-1008).
- the present invention provides an anti-CD4 antibody or a fragment thereof as described herein for use in treating or preventing asthma in a subject.
- the present invention provides an anti-CD4 antibody or a fragment thereof for use in treating or preventing asthma in a subject, wherein the anti-CD4 antibody or fragment thereof is capable of activating CD4 + CD25 + regulatory T cells.
- the present invention provides an anti-CD4 antibody for use in treating or preventing asthma in a subject, wherein the anti-CD4 antibody comprises a variable heavy chain polypeptide sequence and a variable light chain polypeptide sequence, and further comprises a human constant domain of the IgG isotype, wherein: (a) the variable heavy chain polypeptide sequence comprises CDR sequences having SEQ ID NOs: 5, 6 and 7, and the variable light chain polypeptide sequence comprising CDR sequences having SEQ ID NOs: 8, 9 and 10; or (b) the variable heavy chain polypeptide sequence is SEQ ID NO: 3 and the variable light chain polypeptide sequence is SEQ ID NO: 4.
- the variable chain polypeptide sequences may have at least 85% sequence identity to SEQ ID Nos: 3 and
- the present invention provides a method of treating or preventing asthma in a subject, comprising administering an effective amount of an anti-CD4 antibody or fragment thereof to the subject, wherein the anti-CD4 antibody or fragment thereof is capable of activating CD4 + CD25 + regulatory T cells.
- the present invention provides a method of treating or preventing asthma in a subject, comprising administering an effective amount of an anti-CD4 antibody to the subject, wherein the anti-CD4 antibody is as defined above in the third aspect.
- the present invention provides the use of an anti-CD4 antibody or fragment thereof for the manufacture of a medicament for treating or preventing asthma in a subject, wherein the anti-CD4 antibody or fragment thereof is capable of activating CD4 + CD25 + regulatory T cells.
- the present invention provides the use of an anti-CD4 antibody for the manufacture of a medicament for treating or preventing asthma in a subject, wherein the anti-CD4 antibody is as defined above in the third aspect.
- the present invention provides an anti-CD4 antibody or fragment thereof for use in treating or preventing asthma in a subject, wherein the antibody or fragment thereof is capable of activating CD4 + CD25 + regulatory T cells, wherein the use comprises contacting CD4 + CD25 + regulatory T cells obtained from the subject in vitro with the antibody or fragment thereof so as to activate the CD4 + CD25 + regulatory T cells, and wherein the use further comprises administering the activated CD4 + CD25 + regulatory T cells to the subject.
- the present invention also provides, in a further aspect, an anti-CD4 antibody for use in treating or preventing asthma in a subject, wherein the use comprises contacting CD4 + CD25 + regulatory T cells obtained from the subject in vitro with the antibody so as to activate the CD4 + CD25 + regulatory T cells, and wherein the use further comprises administering the activated CD4 + CD25 + regulatory T cells to the subject, wherein the anti- CD4 antibody is as defined above in the third aspect.
- Still further provided is a method of treating or preventing asthma in a subject wherein the method comprises obtaining a sample from the subject comprising CD4 + CD25 + regulatory T cells, contacting the CD4 + CD25 + regulatory T cells in vitro with an anti-CD4 antibody or fragment thereof capable of activating the CD4 + CD25 + regulatory T cells, and administering the activated CD4 + CD25 + regulatory T cells to the subject so as to prevent or treat the asthma.
- the present invention provides a method of treating or preventing asthma in a subject, wherein the method comprises obtaining a sample from the subject comprising CD4 + CD25 + regulatory T cells, contacting the CD4 + CD25 + regulatory T cells in vitro with an anti-CD4 antibody to activate the CD4 + CD25 + regulatory T cells, and administering the activated CD4 + CD25 + regulatory T cells to the subject so as to prevent or treat the asthma, wherein the anti-CD4 antibody is as defined above in the third aspect.
- an anti-CD4 antibody or fragment thereof for the manufacture of a medicament for treating or preventing asthma in a subject, wherein the anti- CD4 antibody or fragment thereof is capable of activating CD4 + CD25 + regulatory T cells, and wherein the use comprises contacting CD4 + CD25 + regulatory T cells obtained from the subject in vitro with the anti-CD4 antibody or fragment so as to activate the CD4 + CD25 + regulatory T cells, and administering the activated CD4 + CD25 + regulatory T cells to the subject.
- an anti-CD4 antibody for the manufacture of a medicament for treating or preventing allergic asthma in a subject, wherein the use comprises contacting CD4 + CD25 + regulatory T cells obtained from the subject in vitro with the anti-CD4 antibody so as to activate the CD4 + CD25 + regulatory T cells, and administering the activated CD4 + CD25 + regulatory T cells to the subject, wherein the anti-CD4 antibody is as defined above in the third aspect.
- the asthma may be allergic asthma.
- Figure 1 shows the amino acid sequences of the heavy chain and light chain of the antibody designated BT061.
- Fi ure 2 provides a graph showing the percentage of Foxp3 -expressing cells 24 hours, 72 hours and 10 days after stimulation of peripheral blood mononuclear cells (PBMCs) with house dust mite HDM allergen (as assessed by intracellular flow cytometry).
- PBMCs peripheral blood mononuclear cells
- HMM allergen as assessed by intracellular flow cytometry.
- PBMCs are derived from patients with house dust mite allergy (RAST Scale 3 and 6) and healthy controls (RAST Scale 0).
- Figure 3a and 3b provide a graph ( Figure 3a) and a bar chart ( Figure 3b) showing the time course of IL-5 production 24, 48 and 72 hours after stimulation of peripheral blood mononuclear cells (PBMCs) derived from patients with house dust mite allergy (“Patients” - RAST Scale 3 and 6) and healthy controls (“Controls” RAST Scale 0) with HDM allergen (“Mite”), cell medium (“Medium”) and “PHA” (phythaemagglutinine) as a +ve control.
- PBMCs peripheral blood mononuclear cells
- Figure 4a and 4b provide a graph ( Figure 4a) and a bar chart ( Figure 4b) showing the time course of IFN-y production 24, 48 and 72 hours after stimulation of peripheral blood mononuclear cells (PBMCs) derived from patients with house dust mite allergy (“Patients” - RAST Scale 3 and 6) and healthy controls (“Controls” RAST Scale 0) with HDM allergen (“Mite”), cell medium (“Medium”) and “PHA” as a +ve control.
- PBMCs peripheral blood mononuclear cells
- Figure 5a and 5b provide bar charts showing the effect of the antibody BT061 used in the examples on IL-5 ( Figure 5a) and IFN-y ( Figure 5b) production of isolated PBMC from patients with dust mite allergy (RAST 6) and controls (RAST 0) after 72 h stimulation with mite extract. Normalization of the bars to set mite stimulation on 1.0 in order to illustrate the effect of the agents as x-fold as compared to the (internal) control (mite stimulation). The effects of different BT061 concentrations (0 pg/ml, 0.1 pg/ml, 1.0 pg/ml, and 10 pg/ml) are shown.
- Figure 6a to f provide bar charts showing RTPCR results of mite-stimulated PBMC from patients with dust mite allergy (RAST 6) and controls (RAST 0) with and without BT061 relative mRNA expression of IL-5 (Figure 6a), IFN-y ( Figure 6b), TGF-P (Figure 6c), GATA- 3 ( Figure 6d), T-bet ( Figure 6e), and Fox-P3 ( Figure 6f).
- Figure 7h shows the fold change of IL-5, IFN-y, IL-10 and TGF-P levels of mite stimulated PBMCSs from patients with dust mite allergy after BT-061 treatment.
- Figure 8a to g provide bar charts showing Treg activation with BT-061.
- Purified Tregs cultured in the cell expansion system for 10 days were harvested and stimulated with anti- CD3 (OKT3)/anti-CD28 antibody (mAb) or BT-061 in the presence of FCS 10% or HS 10%, respectively.
- Activation of cells was measured by BrdU Proliferation test (Fig. 8a, Fig. 8d) and production of (Fig. 8e) TGF-P, (Fig. 8f) IL-5 and (Fig. 8g) IFN-y.
- culture mediums were tested for (Fig. 8b) BrdU background signal and (Fig. 8c) TGF-P level.
- Figure 9a to f provide bar charts showing the effect of BT-061 on Treg activation (Fig. 9a) and the effect of activated Tregs on (Fig. 9b-d) TCR-stimulated and (Fig. 9e,f) mite- stimulated Teffs using isolated and expanded Tregs and Teffs from from patients with dust mite allergy and healthy controls.
- Tregs were pre-cultured with BT-061 or with medium alone for 72 hrs (a) and Teffs were added (1 : 10) to the cell culture, stimulated either (b-d) via the TCR (CD3/CD28) (e,f) or with mite extract and IL-5, IFN-y as well as IL-10 and the T-cell transcription factors Foxp3, T-bet as well as GATA-3 were detected by qRT-PCR.
- Figure 10A to D provide histograms showing CD69 expression on (A) unstimulated Teffs or Teffs cultured in the presence of (B) BT061, (C) Omalizumab or (D) Phorbolester/Ionomycin (PBu) analyzed by flow cytometry.
- (F) Effect of BT061 or Omalizumab on the expression of the activation marker CD69 on unstimulated or Phytohaemagglutinin (PHA)-stimulated Teffs (n 4).
- CD69 expression was analysed using flow cytometry and is shown as fold change (FC).
- G, H Cytokine release of isolated Teffs measured by Cytometric Bead array, n.s.
- FIG. 11 A to E provide a plot (A), graphs (B,C) and bar charts (D,E).
- PBMCs were stimulated via T-cell receptor (TCR) and activation of cells was determined by CD69 expression using flow cytometry.
- A-C Gating strategy.
- PBMCs were differentiated by the surface markers CD4 and CD25, and CD69 expression on Teffs (CD4 + CD25 ) and on Tregs (CD4 + CD25 + ) was analysed.
- CD69 expression is shown as fold change (FC) of TCR activation. ** PO.Ol.
- Figures 12 A to E provide bar charts showing the effect of BT061 on cytokine release. Effect of BT-061 on IL-2, IL-5, IFN-y, IL-10 and TNF-P release of isolated PBMC from controls after 48 h stimulation with anti-CD3 and anti-CD28 mAK. Cytokine concentration is shown as fold change (FC) of TCR activation. *P ⁇ 0.05, ** PO.OL
- asthma is a chronic airway disease characterized by inflammation, bronchial hyperactivity and airway obstruction. Common symptoms are wheezing, breathlessness, a tight chest and coughing.
- Allergic asthma (sometimes also known as allergy-induced asthma) is asthma caused by an allergic reaction to an allergen. Common allergens are pollen, dust mites, and animal fur or feathers.
- IL-4 is important for allergic sensitization and IgE production
- IL-5 is crucial for eosinophil survival
- IL-13 has pleiotropic effects in the lungs, including a central role in the development of airway hyperresponsiveness (AHR) and tissue remodelling.
- AHR airway hyperresponsiveness
- inflammatory mediators released from mast cells act on airway smooth muscle, resulting in bronchoconstriction and the clinical features of asthma.
- the release of inflammatory mediators also results in mucus secretion, which further contributes to airway obstruction.
- Th2 lymphocytes may have a role in non-allergic asthma patients (Gauthier, M., et al., 2015. Evolving Concepts of Asthma. Am. J. Respir. Crit. Care Med. 192, 660-668).
- Regulatory T cells are a subpopulation of CD4+ T cells that are known to modulate the immune system, and in particular are known to be important for maintaining tolerance to self-antigens (Sakaguchi et al., Immunological Reviews (2001); 182: 18-32).
- Treg cells The family of Treg cells consists of two key subsets: (i) naturally arising Tregs (sometimes known as nTregs), which develop in the thymus; and (ii) peripherally induced Tregs (sometimes known as iTregs) which arise in peripheral circulation from conventional T cells.
- Treg cells are generally characterised by the expression of CD4 and CD25 surface biomarkers, and the transcription factor FoxP3.
- Treg dysfunction is known to be associated with certain autoimmune disease.
- Tregs are also known to play a role in controlling allergic asthma by ensuring the maintenance of immunological tolerance and the prevention of Th2 -induced inflammatory response (Robinson, D.S., 2009. Regulatory T cells and asthma. Clin. Exp. Allergy 39, 1314-1323; Martin-Orozco, E., et al., May 2017. Regulatory T Cells in Allergy and Asthma. Front Pediatr 5. Article 117).
- Th2 cell responses to allergens can also be formed in individuals without a genetic propensity for developing allergic disease (non-atopic), these responses are normally silenced by Tregs.
- Tregs The modulatory role of Tregs is extensive and affects effector cells either directly or indirectly to ultimately block inappropriate immune response. Tregs exert their actions on effector cells through several pathways: 1) cell to cell contact, 2) inhibitory cytokines such as IL-10 and TGF-P, 3) cytotoxicity mediated by perforins and granzymes, and 4) competition for T cell growth factors such as IL-2.
- Treg-mediated suppression of effector cells is deficient, leading to inflammation of the airways via a Th2-cell response to an allergen (Robinson, D.S., 2009. Regulatory T cells and asthma. Clin. Exp. Allergy 39, 1314-1323; Martin-Orozco, E., et al., May 2017. Regulatory T Cells in Allergy and Asthma. Front Pediatr 5. Article 117).
- Treg number and allergy development have been shown (Tulic, M.K., et al., 2012. Changes in thymic regulatory T-cell maturation from birth to puberty: differences in atopic children. J. Allergy Clin. Immunol. 129, 199-206. el-4).
- studies investigating the role of Tregs in the peripheral blood of asthmatic children have demonstrated that activated pulmonary Tregs are decreased in children with atopic allergy and fail to suppress pulmonary Th2 responses (Hartl, D., et al., 2007. Quantitative and functional impairment of pulmonary CD4+CD25hi regulatory T cells in pediatric asthma. J. Allergy Clin. Immunol.
- Tregs In allergic asthma, and potentially also in non-allergic asthma, there is no approved therapeutic approach to date which capitalizes on the activation of naturally-occurring Tregs and currently approved biologies for this indication target either IgE or pro-inflammatory cytokines (IL-4, IL-5 and IL-13).
- an anti-CD4 antibody or fragment thereof for use in treating or preventing asthma in a subject.
- the anti- CD4 antibody or fragment is described herein and may be defined as an anti-CD4 antibody or fragment thereof is capable of activating CD4 + CD25 + regulatory T cells and/or by reference to the sequence features described herein.
- the medical use relates to the treatment of asthma, which may be allergic asthma or non-allergic asthma.
- Allergic asthma is caused by an allergic reaction triggered by inhalation of an allergen. It may also be defined as allergy-induced asthma.
- the allergen may be one or more of dust mites, pollen, smoke (including cigarette smoke), mould, and pet dander (including fur, hair and features).
- the subject may be suffering from any form of asthma (e.g. allergic asthma), i.e. mild intermittent asthma, mild persistent asthma, moderate persistent asthma or severe persistent asthma.
- asthma e.g. allergic asthma
- mild intermittent asthma mild persistent asthma
- moderate persistent asthma e.g. mild persistent asthma
- severe persistent asthma e.g. mild intermittent asthma
- the subject is suffering from moderate persistent asthma or severe persistent asthma.
- the subject may be one who has previously been treated with one or more agents suitable for treating asthma which are not the anti-CD4 antibody or fragment thereof described herein.
- the subject may be one who has previously been treated with one or more agents suitable for treating allergic asthma.
- the one or more agents may be a corticosteroid, a biologic, an anti -leukotriene, or a bronchodilator.
- the subject may have been treated with the one or more agents for more than 1 year.
- the subject may be one who is resistant or intolerant to the one or more agents.
- the medical use of the present invention is particularly suitable for a subject who has been previously treated with a corticosteroid, in particular a glucocorticosteroid.
- the subject may have been treated with the corticosteroid for more than 1 year and/or the subject may be one who is resistant or intolerant to corticosteroid treatment.
- the subject may be one who has been treated with inhaled corticosteroids (ICS), optionally also with short- and/or long-acting beta agonists.
- ICS inhaled corticosteroids
- the subject may be one with poor symptom control and/or exacerbations (acute or sub-acute worsening in symptoms and lung function from the subject’s usual status) despite such treatments.
- the subject can be any mammal, including a human, non-human primate, or a domesticated mammal such as a cat or a dog. However, preferably the subject is a human.
- the subject can be six years or older, preferably sixteen years or older.
- the use may reduce inflammation (e.g. allergic inflammation), and in particular may reduce levels of IL-5, IFNy, TNF-a, and/or IL-2 in the plasma or sputum of the subject (as compared to the levels pre-treatment).
- the use may reduce IL-5 levels in the plasma or sputum of the subject.
- the use may increase the level of anti-inflammatory cytokines, such as TGF-P, in the plasma or sputum of the subject (as compared to the level pre-treatment). These changes may be detected by obtaining samples of plasma or sputum from the subject and assaying the samples, for example using ELISA.
- the reduction / increase may be seen around 1 hour post administration, around 72 hours post administration, after 2 weeks, after 4 weeks or after 12 weeks of weekly administration of the antibody or antibody fragment, or 1 week after the end of treatment (in particular 1 week after the end of a 12 week treatment of weekly administration of the antibody or antibody fragment).
- bronchoconstriction can be determined based on the forced expiry volume in 1 second (FEVi) in the period following challenge with an asthma trigger, e.g. with FEVi measured at baseline at 0 hours and then at hourly intervals in the 8 hour period following allergen challenge (see e.g., Gaownau et al., NEJM 2014; 370: 2102-10).
- FEVi forced expiry volume in 1 second
- Reduced bronchoconstriction may be shown after a 2 week, a 4 week or a 12 week treatment period of weekly administration of the antibody or antibody fragment, or 1 week after the end of treatment (in particular 1 week after the end of a 12 week treatment of weekly administration of the antibody or antibody fragment.
- the anti-CD4 antibody or antibody fragment for the medical use described herein can be defined as being an agonistic antibody.
- the antibody or antibody fragment is “capable of activating CD4 + CD25 + regulatory T cells”.
- this can be defined as the ability of the anti-CD4 antibody or antibody fragment to activate T cells expressing CD4 + and CD25 + in vitro, preferably such that the activated CD4 + CD25 + regulatory T cells are able to inhibit the production of pro-inflammatory cytokines (e.g. IL-5, IL-4 and/or IL-13, preferably IL-5) in a PBMC culture after TCR stimulation, in particular in a culture containing T effector cells.
- pro-inflammatory cytokines e.g. IL-5, IL-4 and/or IL-13, preferably IL-5
- the testing method may comprise pre-incubating CD4 + CD25 + regulatory T cells obtained from a donor with the antibody or antibody fragment for a period suitable to activate the cells (e.g. 48 - 72 hours), TCR stimulating a T effector cell culture obtained from the same donor, transferring the pre-incubated CD4 + CD25 + regulatory T cells to the T effector cell culture, and measuring the level of the pro-inflammatory cytokine after 72 hours.
- the T effector cell culture and/or the CD4 + CD25 + regulatory T cells may be obtained from a healthy individual, or an individual suffering from allergic asthma.
- an anti-CD4 antibody or fragment thereof that is capable of activating CD4 + CD25 + regulatory T cells will lead to reduction in the amount of the pro-inflammatory cytokine detected as compared to a negative control in which the CD4 + CD25 + cells have not been pre-incubated with the anti- CD4 antibody or fragment thereof.
- the anti-CD4 antibody or fragment thereof leads to the same reduction +/- 25%, more preferably +/- 10%, as a positive control with the BT061 antibody described herein.
- the ability of the antibody or antibody fragment to activate CD4 + CD25 + regulatory T cells can also be determined by methods described in the art, such as those described in WO201 1/064407. For example, the ability can be assayed by examining the suppressive activity of the CD4 + CD25 + regulatory T cells after incubation with the antibody or antibody fragment by co-culturing the CD4 + CD25 + regulatory T cells with CD4 + CD25- effector T cells. Activated CD4 + CD25 + regulatory T cells are able to inhibit proliferation of such effector T cells in effector T cell proliferation assays.
- the effector T cells can be labelled with CFSE such that any proliferation can be determined.
- proliferation of effector cells can be determined by [3H] thymidine incorporation.
- the antibody or antibody fragment may also be defined as one that does not cause antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). This ability can be determined by methods known in the art.
- ADCC antibody-dependent cell-mediated cytotoxicity
- CDC complement-dependent cytotoxicity
- the anti-CD4 antibody BT061 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 of modulating the effector functions of an antibody or antibody fragment, so as to ablate effector functions are known in the art (Saunders, K.O., June 2019, Frontier in Immunology, Vol. 10, Article No. 1296).
- Antibodies or antibody fragments that are modified such that they do not cause ADCC or CDC are within the scope of the invention.
- CD4 + CD25 + regulatory T cells enable the antibody or antibody fragment to inhibit activation of CD4 + effector T cells in vitro when in the presence of CD4 + CD25 + regulatory T cells.
- the anti-CD4 antibody activates CD4 + CD25 + regulatory T cells in vitro leading to suppression of the release of pro-inflammatory cytokines IL-5 and IFN-y.
- activation of T effector cells can be determined in vivo by monitoring levels of inflammatory cytokines released by activated T effector cells in samples of the subject’s plasma or sputum.
- the use of the antibody or antibody fragment of the present invention is advantageous over the prior art use of the anti-CD4 antibody keliximab antibody in asthma patients, since, as noted above, keliximab was found to cause a reduction in CD4 cell count, and further did not improve FEVi (Kon et al., Eur. Respir. J. 2001; 18: 45-52; Tourangeau et al., Therapeutic Advances in Respiratory Disease (2011): 5(3): 183-194).
- the present inventors consider that the use of the present invention can treat asthma patients without inducing ADCC.
- the present inventors consider that surprisingly in the use of the present invention pro-inflammatory cytokines are downregulated, which was not shown with keliximab treatment.
- the ability to activate CD4 + CD25 + T regulatory cells from asthma patients as shown in the present examples is unexpected as these cells may be decreased and functionally impaired in such patients.
- the anti-CD4 antibody or antibody fragment thereof is preferably an anti-human CD4 antibody or fragment thereof.
- the antibody may be a human antibody, or may be a humanized or chimeric antibody. Preferably the antibody is a humanized antibody.
- the antibody further comprises a human constant region (Fc).
- This constant region can be selected among constant domains from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4.
- Preferred constant regions are selected among constant domains of IgG, in particular IgGl.
- the antibodies are preferably IgGl antibodies, and/or the antibody or antibody fragment preferably comprises an Fc portion such that the antibody or antibody fragment is capable of binding to an Fc receptor, preferably FcyRI (i.e. CD64). Most preferably the antibody or antibody fragment comprises the Fc portion of an IgGl antibody. In addition, or alternatively, the antibody or antibody fragment is capable of binding to monocytes via an Fc receptor.
- FcyRI i.e. CD64
- the antibody or antibody fragment comprises the Fc portion of an IgGl antibody.
- the antibody or antibody fragment is capable of binding to monocytes via an Fc receptor.
- the present invention also includes any fragment of the antibody including fragments comprising the V regions thereof. This comprises in particular Fab, Fab', F(ab)'2, Fv and scFv fragments.
- the antibody is a humanized anti-CD4 antibody or fragment thereof which is a variant of the BT061 antibody, which has V domains defined by the following polypeptide sequences (and which is described further in the examples below):
- the anti-CD4 antibody or fragment thereof for use in the present 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 SEQ ID NO: 4.
- the anti-CD4 antibody comprises a human IgGl constant domain.
- Variants of this antibody or antibody fragment are also suitable for use in the present invention.
- Variants include those with V domains defined by polypeptide sequences having at least 85%, preferably at least 90 %, most 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 with at least 90% sequence identity with SEQ ID NO: 3 and a light chain variable domain with at least 90% sequence identity with SEQ ID NO: 4. Still more preferably the antibody or fragment thereof has a heavy chain variable domain with at least 95% sequence identity with SEQ ID NO: 3 and a light chain variable domain with at least 95% sequence identity with SEQ ID NO: 4.
- a preferred embodiment is an antibody comprising these variable domain sequences and an IgGl constant domain.
- the antibody or antibody fragment is one which has the CDR sequences of BT061 (SEQ ID Nos: 5 to 10).
- a particularly preferred embodiment is an anti- CD4 antibody or fragment thereof comprising a heavy chain variable domain with at least 95% sequence identity with SEQ ID NO: 3 and which includes the CDR sequences of SEQ ID Nos: 5 to 7, a light chain variable domain with at least 95% sequence identity with SEQ ID NO: 4 and which includes the CDR sequences of SEQ ID Nos: 8 to 10.
- BT-061 binds to CD4 has been disclosed in Helling et al., Immunology and Cell Biology (2015) 93: 396-405. Further variants of BT-061 have also been disclosed in WO 2011/064407. Accordingly, this information can be used to guide the variation of the sequences of the heavy and light chains. Preferably the variations do not substantially affect the specificity and/or affinity of binding.
- the antibody or antibody fragment is one that binds to an epitope on CD4 comprising 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 in WO201 1/064407). Methods for determining the ability to bind to a particular epitope are known in the art.
- the antibody or antibody fragment can also be defined as one that binds to substantially the same epitope as the BT061 antibody described herein. This property can be determined by alanine scanning.
- the invention provides an anti-CD4 antibody for use in treating or preventing allergic asthma in a subject, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2.
- the antibody or antibody fragment may be manufactured using encoding polynucleotide sequence.
- recombinant DNA constructs can be obtained and introduced in host cells by the well-known techniques of recombinant DNA and genetic engineering.
- Expression cassettes can be used in which the polynucleotide sequence(s) are linked to appropriate control sequences that allow for regulation of transcription and translation in a chosen host cell.
- Suitable host cells include mammalian cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc.
- the antibody or antibody fragment may be one which has been produced in a mouse or a human cell line, and in particular in a cell line mentioned herein.
- the antibody or antibody fragment may be comprised in a pharmaceutical composition which further comprises a pharmaceutically acceptable excipient or carrier.
- the pharmaceutical composition may further comprise a stabilizing agent.
- the pharmaceutical composition may be suitable for intravenous, subcutaneous, or intramuscular use, or use as an inhalative.
- the antibody or antibody fragment is for use by administration to the subject.
- the antibody or antibody fragment may be administered by any appropriate route, e.g. intravenous, subcutaneous, intramuscular or via inhalation.
- the antibody or antibody fragment is administered subcutaneously or via inhalation.
- Most preferably the antibody or antibody fragment is administered subcutaneously.
- the antibody or antibody fragment may be comprised in a vial, a pre-filled syringe, an autoinjector or an inhaler (or puffer).
- the vial may be arranged for use in an autoinjector or an inhaler, i.e. arranged such that it can be fitted or slotted into an autoinjector or an inhaler just prior to use.
- the present invention provides an inhaler, or a vial for use with an inhaler, comprising the anti-CD4 antibody or fragment thereof described above.
- This inhaler can be used to deliver the antibody or fragment into the body via the lungs.
- the medical use of the present invention will not suffer from the drawback described in the art for pulmonary-delivered antibodies of short residence time of the antibody in the lungs. (In particular it has been suggested that some pulmonary-delivered monoclonal antibodies are mostly cleared from the lungs within 1-2 days (Desoubeaux G., et al., 2016, MABS, 8(6): 999-1009)).
- the inhaler may be a metered dose inhaler which comprises components that generate a metered dose of the medication in aerosol form, or a soft mist inhaler (e.g. a mesh inhaler), which comprises components that generate a mist of medication for inhalation by the user.
- a metered dose inhaler which comprises components that generate a metered dose of the medication in aerosol form
- a soft mist inhaler e.g. a mesh inhaler
- the anti-CD4 antibody or fragment thereof is comprised within the inhaler, or vial for use with an inhaler, in a pharmaceutical composition.
- the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient suitable for use in the lungs.
- the pharmaceutical composition may further comprise a surfactant suitable for use in the lungs.
- the antibody or antibody fragment may be administered in a dose of between 25 mg and 200 mg, preferably between 75 mg and 200 mg, more preferably between 100 mg and 200 mg. Where the route of administration is via inhalation the dose of the antibody or antibody fragment is preferably from 25 mg to 150 mg, more preferably from 75 mg to 125 mg.
- Doses may also be calculated based on the body weight of the subject, and may be from 1 to 3mg/kg, preferably 1 to 2.5 mg/kg, and most preferably 1.5 to 2 mg/kg.
- the antibody or antibody fragment may be administered at regular time intervals, e.g. once per week, once every 2 weeks, once every 3 weeks or once every four weeks.
- the antibody or antibody fragment may be used in combination with one or more further agents known to treat asthma (e.g. allergic asthma), provided that the one or more further agents are non-immunosuppressive.
- one or more further agents known to treat asthma (e.g. allergic asthma)
- the one or more further agents are non-immunosuppressive.
- the anti-CD4 antibody or fragment thereof may be used to activate CD4 + CD25 + regulatory T cells obtained from the subject in vitro, before the activated cells are reintroduced back into the subject to treat or prevent allergic asthma.
- the present invention further provides a method of treating a subject suffering from or preventing a subject suffering from asthma (e.g. allergic asthma), the method comprising removing a sample comprising CD4 + CD25 + regulatory T cells from the subject, contacting the CD4 + CD25 + regulatory T cells with an antibody or antibody fragment as described herein to activate CD4 + CD25 + regulatory T cells and administering the activated cells to the subj ect.
- a method may additionally include an in vitro step of increasing the number of Treg cells. This can be done using the expansion strategies described herein (Peters et al., PLoS 2008; 3(9): e3161; Hoffmann et al., Blood 2004; 104(3):895-903).
- the present invention provides activated CD4 + CD25 + regulatory T cells, which have been activated in vitro using the antibody or antibody fragment described herein, for use in treating or preventing asthma (e.g. allergic asthma) in a subject.
- the CD4 + CD25 + regulatory T cells are obtained from the subject.
- the CD4 + CD25 + regulatory T cells are expanded prior to reintroduction into the subject as described above.
- the anti-CD4 antibody (BT061) that is used in the examples is a recombinant, humanized IgGl monoclonal antibody comprised of two heavy chains and two light chains.
- the amino acid sequence for both heavy and light chains has been predicted from the translation of the nucleotide sequence for the gene of BT061 and have been confirmed experimentally.
- Figure 1 displays the predicted amino acid sequences for the heavy and light chains, as well as the most likely disulphide bond assignment.
- the heavy chain has SEQ ID NO: 1 and the light chain has SEQ ID NO: 2.
- the BT061 antibody used in the examples has been produced in a mouse myeloma host cell line Sp2/0-Agl4 (Sp2/0, corresponding to ATCC CRL-1581). Expression of the antibody in Sp2/0 is also described in WO 2009/124815.
- the BT061 antibody has been given the generic name tregalizumab.
- RAST radioallergosorbent test
- Figure 2 shows the time kinetic of Foxp3 -expression 24 hours, 72 hours and 10 days after HDM stimulation, as detected by intracellular flow cytometry. It can be seen that cells derived from patients with different RAST scales (RAST Scale 3 and 6) exhibited a different Treg proliferation kinetic compared to that of cells derived from healthy control subjects (RAST Scale 0).
- PBMCs from patients with mite dust allergy also showed a different proliferating response compared to controls as detected by CFSE-flow cytometry (data not shown). Stimulation with HDM extract led to a time dependent raise in cytokine production. After 72 hours PBMCs from patients showed a strong IL-5 response to mite stimulation, whereas no effect was detectable for controls ( Figures 3a and 3b - the IL-5 amounts with “Control Medium”, “Controls Mite” (i.e. cells from healthy donors incubated with either medium or allergen) and “Patients Medium” (i.e. cells from patient donors incubated with medium) all track on or just above the x-axis in Figure 3a). In contrast, IFN-y levels (as shown in Figure 4a and 4b) were much higher in control cell culture as in patients.
- BT-061 decreased IL-5 as well as IFN-y cytokine production after 72 hours of stimulation (Fig. 5a, 5b). However, no concentration-dependent effect of BT-061 was found when 0.1, 1.0 and 10.0 pg/ml of the BT-061 -antibody were used suggesting that at a concentration of 0.1 pg/mL saturation may already have been achieved.
- RTPCR underlined the results of the cytokine production (Fig. 6a, 6b). IL- 5 mRNA was found higher in patients PBMCs after mite extract stimulation and was decreases in the presence of BT-061. Vice versa, IFN-y mRNA was higher in controls and down-regulated with BT-061.
- TGF-P and Fox-P3 mRNA were higher in patients, especially TGF-P mRNA was increased after BT-061 stimulation (Fig.6c, 6f). No effect of the stimulation system could be detected for the transcription factors GATA-3 and T-bet (Fig.6d, 6e).
- Example 3 Influence of BT-061 on activation and proliferation of T-regulatory cells from patients with house dust mite allergy in a Treg cell expansion system.
- MCS Magnetic Activated Cell Sorting-System
- STEMCELL Technologies EasySep® Human CD4 + CD25 + T Cell Isolation Kit
- Tregs were cultured in the presence of irradiated (60 Gray) L-cells (L929-derived murine Ltk- cell line (a mouse fibroblast cell line) stably transfected with human FcyRII (CD32)), anti- CD3 (OKT3), anti-CD28 antibody and IL-2.
- irradiated (60 Gray) L-cells L929-derived murine Ltk- cell line (a mouse fibroblast cell line) stably transfected with human FcyRII (CD32)), anti- CD3 (OKT3), anti-CD28 antibody and IL-2.
- Treg activation with BT-061 was tested.
- Purified Tregs cultured in the cell expansion system for 10 days were harvested and stimulated with anti- CD3 (OKT3)/anti- CD28 antibody or BT-061 in the presence of FCS 10% or HS 10%, respectively (Fig. 8).
- Proliferation testing by BrdU incorporation showed increased optical density (OD) after stimulation with anti CD3/CD28, but did not increase after BT-061 stimulation. However, we found very high OD levels (>1.5) for unstimulated probes in the cell culture with HS 10% (Fig. 8a).
- Example 4 Effect of (A) BT-061 on Treg activation and the effect of activated Tregs on, (B) TCR-stimulated and, (C) mite-stimulated Teffs using isolated and expanded Tregs and Teffs
- Tregs were pre-incubated with BT-061 or with medium alone for 72 hours and T-cell transcription factors Foxp3, T-bet and GATA-3 were measured by qRT- PCR. Tregs stimulated with BT-061 showed a significant increase in Foxp3 -expression, whereas no effects could be detected in T-bet and GATA-3 expression ( Figure 9a).
- BT061 has the potential to combat inflammation triggered by allergic asthma.
- BT061 exerts a positive effect on Tregs (isolated from allergic patients) and a modulatory effect on TCR-stimulated Teffs.
- the cytokine patterns observed in in vitro studies with patient PBMCs correspond to the in vivo situation observed in allergic mice subjected to methacholine challenge (Xu et al 2012 as above).
- PBMCs Peripheral mononuclear blood lymphocytes
- Tregs were expanded using a Treg expansion system as described [Hoffmann et al. Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells. Blood. 2004 Aug 1; 104(3):895-903],
- cells were cultured in the presence of irradiated L-cells (L929-derived murine Ltk- cell line stably transfected with human FcyRII (CD32)), anti-CD3 (OKT3), anti-CD28 (CD28.2) antibody and IL-2 in the presence of human serum (HS 10%) for 5 days.
- Enrichment of CD4 + CD25‘ cells and CD4 + CD25 + as well as Treg expansion was monitored by flow cytometry.
- Cells were cultured in 96-well flat-bottom plates in RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 1% HEPES, 2% glutamine and 2% gentamicin at a concentration of l x 10 6 cells/ml.
- TCR anti-T cell receptor
- CD3 OKT3, 1 pg/ml
- CD28.2, 1 pg/ml costimulated with anti-CD28 mAb
- lymphocytes were incubated 72h in the presence of phythaemagglutinine (PHA) (10 pg/ml), or phorbol- 12, 13 -dibutyrate (PBu, 1 ng/ml) in conjunction with ionomycin (0.5 pg/ml).
- PHA phythaemagglutinine
- PBu phorbol- 12, 13 -dibutyrate
- Cells were stained with the following monoclonal antibodies (MoAb): CD25 APC Clone 2 A3, CD4 FITC Clone RPA-T4, CD 127 PE Clone hIL-7R-M21, HLA-DR PE Clone TU36, CD3 BV450 Clone UCHT1, CD45 V500 Clone HI30, CD69 PE Clone FN50, CD 44 PE Clone G44-26, CD 64 PE Clone lO.lfrom Becton Dickinson (San Jose, CA, USA). After staining, cells were incubated for 15 min at room temperature (RT).
- RT room temperature
- cytokines/chemokines were determined in culture supernatants using the BDTM CBA Flex Set System for the measurement of IL-2, IL-5, IL-10, INF-y, TNF-a and TGF-P (BD Bioscience-PharMingen, San Diego, CA, USA).
- Each BDTM CBA Flex Set contained one bead population with distinct fluorescence intensity, as well as the appropriate phycoerythrin (PE) detection reagent and standard. The tests were performed according to the manufacturer’s advice as described before [Eickmeier et al. Sputum biomarker profiles in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) and association between pulmonary function. Cytokine. 2010 May;50(2): 152-7], Results
- Tregs were expanded using a Treg expansion system noted above.
- cells were cultured in the presence of irradiated L-cells (L929-derived murine Ltk- cell line stably transfected with human FcyRII (CD32)), anti-CD3 (OKT3), anti-CD28 antibody, and IL-2 in the presence of human serum (HS 10%) for 10 days.
- irradiated L-cells L929-derived murine Ltk- cell line stably transfected with human FcyRII (CD32)
- OKT3 anti-CD3
- anti-CD28 antibody anti-CD28 antibody
- IL-2 IL-2
- Teffs were stimulated via the T-cell receptor (TCR) using anti-CD3 and anti-CD28 monoclonal antibodies to establish a system for demonstrating the effect of BT061 on stimulated T cells.
- Stimulation of PBMCs via the TCR is a well-established system in the lab and has been shown to induce a strong proliferation and cytokine response on Teffs.
- stimulation with Phytohemagglutinin (PHA) led to an appropriate CD69 expression and therefore could be used as positive control for further experiments on Teffs.
- PHA Phytohemagglutinin
- TCR-stimulated PBMCs showed a significant release of IL-5 and IFN-y and of IL-2, IL-10 and TNF-p.
- BT061 The effect of BT061 on the modulation of activation markers on isolated Teffs was measured by the expression of CD69, CD25, CD44, CD127 and HLA-DR.
- Omalizumab anti- IgE, a recombinant humanised IgGlk monoclonal antibody
- Teffs were cultured in RPMI medium alone or in the presence of BT061, Omalizumab, or Phorbolester/Ionomycin (PBu) (Fig. 10 A-F).
- Figure 10 A-F shows an example flow cytometric experiment for the induction of CD69. No changes in the expression of any used activation markers on Teffs could be detected after treatment with BT061 or Omalizumab (Fig.
- BT061 did not induce cytokine release such as IL-5, INF-g (Fig. 10 G, H), IL-2, IL-10 or TNF-P (data not shown) of isolated Teffs, whereas PHA treatment did.
- PBMCs were used and CD69 expression was analysed on CD4 + /CD25‘ and CD4 + /CD25 + T cells by flow cytometry. PBMCs were differentiated by the surface markers CD4 and CD25, and CD69 expression on Teffs (CD4 + CD25 ) and on Tregs (CD4 + CD25 + ) in unstimulated and TCR-stimulated cells was analysed (Fig. 11 A-C).
- BT061 had no effect on unstimulated Teffs or Tregs (Fig. 11 D, E). Stimulation via the TCR resulted in an activation of both cell populations. The addition of BT061 led to a significantly reduced CD69 expression on Teffs but not on Tregs. In contrast, Omalizumab did not have any effect on T cell activation.
- BT061 did not have such an effect on the TCR-induced cytokines.
- BT061 has no direct effect on Teff activation or Teff cytokine release. BT061 is also shown to significantly reduced activation as well as release of the cytokines IL-2, IL-5, IFN-y and TNF-P of TCR-stimulated Teffs in the presence of Tregs.
- Example 6 Clinical Trial in Asthmatic Patients
- a randomised double blind placebo-controlled human phase II clinical trial was performed in 42 patients to demonstrate the effects of tregalizumab on allergen-induced airway responses and airway inflammation in asthmatic patients under the EudraCT number 2020 000585 41 (sponsor T-Balance Therapeutics GmbH). There were two treatment arms; those patients receiving tregalizumab and those receiving the placebo.
- Tregalizumab (Product code: BT061) was used in the form of a 100 mg/mL solution for injection (for subcutaneous administration), and was administered once weekly for a period of 12 weeks at a dose of 100 mg per week (i.e. 1 mL subcutaneously per week).
- Placebo Solution for injection also administered subcutaneously.
- BMI Body mass index
- BHR i.e., a decrease in FEV1 of at least 20% measured by methacholine challenge.
- EAR is defined as a decrease in FEV1 of > 20% within 0 to 3 hours after allergen challenge
- LAR is defined as a decrease in FEV1 of > 15% within 4 to 7 hours after BAP.
- methylxanthines e.g., theophyllin
- anticholinergics e.g., ipratropium bromide
- leukotriene modifiers e.g., montelukast
- tiotropium bromide cromolyn or nedocromil
- LAT alanine transaminase
- AS AT aspartate transaminase
- Kidney insufficiency as defined by creatinine level > 133 pmol/L at screening.
- Presence or history of clinically significant major disease e.g., severe heart/lung disease New York Heart Association [NYHA] Class > 3, autoimmune disease [apart from rheumatoid arthritis], acute uncontrolled hyper- or hypo-thyroidism, severe uncontrolled hypo or hypertension).
- major disease e.g., severe heart/lung disease New York Heart Association [NYHA] Class > 3, autoimmune disease [apart from rheumatoid arthritis], acute uncontrolled hyper- or hypo-thyroidism, severe uncontrolled hypo or hypertension).
- EBV Epstein-Barr Virus
- CMV Cytomegalovirus
- lymphoproliferative disease including lymphoma and lymphadenopathy.
- Non-vasectomized man who, during the study or in the 3 months after the last administration of study drug, is not using two (2) independent effective contraceptive methods (as specified above) or is planning a sperm donation.
- Imprisonment or placement in an institution (AMG ⁇ 40 (1), sentence 4).
- the primary endpoint will be the baseline-corrected LAR measured by the area under the curve (AUC) for FEV 1 at 4 to 7 hours after BAP (AUC4-7FEV1) on Day 84.
- the AUC4 7FEV1 will be calculated as the area under the curve of the normalized FEV1 values (post-BAP FEV1 values divided by the pre-BAP FEV1 value in %) over time Timepoint(s) of evaluation of this end point: Visit 2, Visit 4 and visit 17
- DIVMTQSPDS LAVSLGERAT INCRASKSVS TSGYSYIYWY QQKPGQPPKL LIYLAS ILES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQHSRELPW TFGQGTKVEI KRTVAAPSVF I FPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC
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Abstract
The present invention provides an anti-CD4 antibody or fragment thereof for use in treating or preventing asthma in a subject.
Description
ANTI-CD4 ANTIBODY OR FRAGMENT THEREOF FOR MEDICAL USE
Technical Field
The present disclosure relates to the prevention and treatment of asthma. In particular, the present disclosure relates to an anti-CD4 antibody or a fragment thereof for use in treating or preventing asthma, including allergic asthma and non-allergic asthma.
Background
An allergy or allergic disease is a medical condition that involves a damaging immune response by the body to a substance (allergen) in the environment, which is typically common and harmless (e.g. pollen, fur or dust), and to which the body has become hypersensitive. In particular, the immune system “overreacts” leading to symptoms that can range from mild to severe and life-threatening.
Asthma is a chronic airway disease characterized by inflammation, recurrent wheezing, bronchial hyperreactivity, and airway obstruction. It is generally divided into two types: 1) allergic asthma, which is triggered by inhaling antigens and is initiated by Th2 cells; and 2) non-allergic asthma, which is caused by triggers such as infections of the respiratory tract, exercise, smoke, air pollution, or cold or dry air (Hurwitz, S.H., 1955. Nonallergic Asthma — Differential Diagnosis and Treatment. Calif Med 83, 61-67).
In particular, allergic asthma is one of the most common chronic diseases and the most common non-communicable chronic disease worldwide. More than 30 million people are estimated to be affected worldwide with the prevalence of allergic asthma on the rise (Backman, H., et al., 2017. Increased prevalence of allergic asthma from 1996 to 2006 and further to 2016-results from three population surveys. Clin. Exp. Allergy 47, 1426-1435). About 5-10% of asthma patients suffer from uncontrollable, severe asthma which is refractory to the current standard of treatment with steroids (Lloyd, C.M., Hessel, E.M., 2010. Functions of T cells in asthma: more than just T(H)2 cells. Nat. Rev. Immunol. 10, 838-848).
Asthma can result in persistent airway remodelling and permanent structural changes in the airways. These structural changes contribute to the airway obstruction associated with the disease and can result in the progressive loss of lung function over time. In addition to structural changes, as the disease progresses, airflow is further limited by edema, inflammation, cellular infiltration, and overproduction of mucus. Infiltrating immune cells — which are thought to underlie structural changes — include eosinophils, neutrophils, mast cells, and lymphocytes.
The traditional standard of care for controlling bronchial asthma has focused on the use of potent anti-inflammatory drugs, particularly steroids. Glucocorticoids are effective in most patients but do not provide prolonged relief without repeated administration and do not address the molecular basis of the disease. In addition, long-term, high-dose usage in moderate to severe asthma patients is generally linked to considerable side-effects. Furthermore, glucocorticoids fail to control disease symptoms in a significant number of asthma patients. These individuals are most at risk of hospitalization and asthma-related death. In addition to glucocorticoids, monoclonal antibody treatments are available for the treatment of severe asthma patients, targeting IL-4, IL-5, IL-13 or IgE. However, due to their mode of action in the downstream part of the inflammatory cascade, they are associated with side effects, including infections, and a significant portion of patients become refractory over time. Other monoclonal antibody treatments have been investigated but have been found not to be suitable. For example, the chimeric human/macaque IgGl anti-CD4 antibody, keliximab, was initially thought to benefit severe asthmatics by decreasing the role of CD4+ lymphocytes in the pathogenesis of chronic airway disease. However, studies into the use of this antibody were stopped as a result of the limiting side effect of a reduction in CD4+ cell count in patients (Kon et al., Eur. Respir. J. 2001; 18: 45-52; Tourangeau et al., Therapeutic Advances in Respiratory Disease (2011): 5(3): 183-194). Also, the humanized anti-CD25 IgGl antibody, daclizumab, was assessed in moderate to severe asthmatic patients and was shown to improve FEV1, reduce daytime asthma symptoms and P2 agonist use, however, there were more serious adverse events in the treatment group as compared to the placebo. (Busse et al,. Am. J. Respir. Crit. Care Med. 2008; 178: 1002-1008).
These facts highlight the need for more specific therapies with the potential to provide long-term relief and fewer side effects to asthma-sufferers.
Summary
Accordingly, in a first aspect the present invention provides an anti-CD4 antibody or a fragment thereof as described herein for use in treating or preventing asthma in a subject.
In a second aspect the present invention provides an anti-CD4 antibody or a fragment thereof for use in treating or preventing asthma in a subject, wherein the anti-CD4 antibody or fragment thereof is capable of activating CD4+CD25+ regulatory T cells.
In a third aspect, the present invention provides an anti-CD4 antibody for use in treating or preventing asthma in a subject, wherein the anti-CD4 antibody comprises a variable heavy chain polypeptide sequence and a variable light chain polypeptide sequence, and further comprises a human constant domain of the IgG isotype, wherein: (a) the variable heavy chain polypeptide sequence comprises CDR sequences having SEQ ID NOs: 5, 6 and 7, and the variable light chain polypeptide sequence comprising CDR sequences having SEQ ID NOs: 8, 9 and 10; or (b) the variable heavy chain polypeptide sequence is SEQ ID NO: 3 and the variable light chain polypeptide sequence is SEQ ID NO: 4. In (a) the variable chain polypeptide sequences may have at least 85% sequence identity to SEQ ID Nos: 3 and 4.
In a fourth aspect the present invention provides a method of treating or preventing asthma in a subject, comprising administering an effective amount of an anti-CD4 antibody or fragment thereof to the subject, wherein the anti-CD4 antibody or fragment thereof is capable of activating CD4+CD25+ regulatory T cells.
In a further aspect the present invention provides a method of treating or preventing asthma in a subject, comprising administering an effective amount of an anti-CD4 antibody to the subject, wherein the anti-CD4 antibody is as defined above in the third aspect.
In a further aspect the present invention provides the use of an anti-CD4 antibody or fragment thereof for the manufacture of a medicament for treating or preventing asthma in a subject, wherein the anti-CD4 antibody or fragment thereof is capable of activating CD4+CD25+ regulatory T cells.
In a still further aspect the present invention provides the use of an anti-CD4 antibody for the manufacture of a medicament for treating or preventing asthma in a subject, wherein the anti-CD4 antibody is as defined above in the third aspect.
In a further aspect the present invention provides an anti-CD4 antibody or fragment thereof for use in treating or preventing asthma in a subject, wherein the antibody or fragment thereof is capable of activating CD4+CD25+ regulatory T cells, wherein the use comprises contacting CD4+CD25+ regulatory T cells obtained from the subject in vitro with the antibody or fragment thereof so as to activate the CD4+CD25+ regulatory T cells, and wherein the use further comprises administering the activated CD4+CD25+ regulatory T cells to the subject.
The present invention also provides, in a further aspect, an anti-CD4 antibody for use in treating or preventing asthma in a subject, wherein the use comprises contacting CD4+CD25+ regulatory T cells obtained from the subject in vitro with the antibody so as to activate the CD4+CD25+ regulatory T cells, and wherein the use further comprises
administering the activated CD4+CD25+ regulatory T cells to the subject, wherein the anti- CD4 antibody is as defined above in the third aspect.
Still further provided is a method of treating or preventing asthma in a subject, wherein the method comprises obtaining a sample from the subject comprising CD4+CD25+ regulatory T cells, contacting the CD4+CD25+ regulatory T cells in vitro with an anti-CD4 antibody or fragment thereof capable of activating the CD4+CD25+ regulatory T cells, and administering the activated CD4+CD25+ regulatory T cells to the subject so as to prevent or treat the asthma.
In a still further aspect, the present invention provides a method of treating or preventing asthma in a subject, wherein the method comprises obtaining a sample from the subject comprising CD4+CD25+ regulatory T cells, contacting the CD4+CD25+ regulatory T cells in vitro with an anti-CD4 antibody to activate the CD4+CD25+ regulatory T cells, and administering the activated CD4+CD25+ regulatory T cells to the subject so as to prevent or treat the asthma, wherein the anti-CD4 antibody is as defined above in the third aspect.
Still further provided is use of an anti-CD4 antibody or fragment thereof for the manufacture of a medicament for treating or preventing asthma in a subject, wherein the anti- CD4 antibody or fragment thereof is capable of activating CD4+CD25+ regulatory T cells, and wherein the use comprises contacting CD4+CD25+ regulatory T cells obtained from the subject in vitro with the anti-CD4 antibody or fragment so as to activate the CD4+CD25+ regulatory T cells, and administering the activated CD4+CD25+ regulatory T cells to the subject.
In addition, still further provided is an aspect which is use of an anti-CD4 antibody for the manufacture of a medicament for treating or preventing allergic asthma in a subject, wherein the use comprises contacting CD4+CD25+ regulatory T cells obtained from the subject in vitro with the anti-CD4 antibody so as to activate the CD4+CD25+ regulatory T cells, and administering the activated CD4+CD25+ regulatory T cells to the subject, wherein the anti-CD4 antibody is as defined above in the third aspect.
In all of the above aspects the asthma may be allergic asthma.
Preferred features of the invention are set out in the dependent claims.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be
used to limit the scope of the claimed subject matter. Nor is the claimed subject matter limited to implementations that solve any or all of the disadvantages noted herein.
Brief Description of the Drawings
To assist understanding of the present disclosure and to show how embodiments may be put into effect, reference is made by way of example to the accompanying drawings in which: Figure 1 shows the amino acid sequences of the heavy chain and light chain of the antibody designated BT061.
Fi ure 2 provides a graph showing the percentage of Foxp3 -expressing cells 24 hours, 72 hours and 10 days after stimulation of peripheral blood mononuclear cells (PBMCs) with house dust mite HDM allergen (as assessed by intracellular flow cytometry). PBMCs are derived from patients with house dust mite allergy (RAST Scale 3 and 6) and healthy controls (RAST Scale 0).
Figure 3a and 3b provide a graph (Figure 3a) and a bar chart (Figure 3b) showing the time course of IL-5 production 24, 48 and 72 hours after stimulation of peripheral blood mononuclear cells (PBMCs) derived from patients with house dust mite allergy (“Patients” - RAST Scale 3 and 6) and healthy controls (“Controls” RAST Scale 0) with HDM allergen (“Mite”), cell medium (“Medium”) and “PHA” (phythaemagglutinine) as a +ve control. The results after stimulation with HDM allergen and cell medium are shown in Figure 3a, while the results after stimulation with PHA are shown in Figure 3b (in which “Controls PHA” is the left-hand bar at each time point, with “Patients PHA” as the right-hand bar at each time point).
Figure 4a and 4b provide a graph (Figure 4a) and a bar chart (Figure 4b) showing the time course of IFN-y production 24, 48 and 72 hours after stimulation of peripheral blood mononuclear cells (PBMCs) derived from patients with house dust mite allergy (“Patients” - RAST Scale 3 and 6) and healthy controls (“Controls” RAST Scale 0) with HDM allergen (“Mite”), cell medium (“Medium”) and “PHA” as a +ve control. The results after stimulation with HDM allergy and cell medium are shown in Figure 4a, while the results after stimulation with PHA are shown in Figure 4b (in which “Controls PHA” is the left-hand bar at each time point, with “Patient PHA” as the right-hand bar at each time point).
Figure 5a and 5b provide bar charts showing the effect of the antibody BT061 used in the examples on IL-5 (Figure 5a) and IFN-y (Figure 5b) production of isolated PBMC from patients with dust mite allergy (RAST 6) and controls (RAST 0) after 72 h stimulation with
mite extract. Normalization of the bars to set mite stimulation on 1.0 in order to illustrate the effect of the agents as x-fold as compared to the (internal) control (mite stimulation). The effects of different BT061 concentrations (0 pg/ml, 0.1 pg/ml, 1.0 pg/ml, and 10 pg/ml) are shown.
Figure 6a to f provide bar charts showing RTPCR results of mite-stimulated PBMC from patients with dust mite allergy (RAST 6) and controls (RAST 0) with and without BT061 relative mRNA expression of IL-5 (Figure 6a), IFN-y (Figure 6b), TGF-P (Figure 6c), GATA- 3 (Figure 6d), T-bet (Figure 6e), and Fox-P3 (Figure 6f).
Figure 7a to h provide bar charts showing the effect of BT-061 on mite-stimulated PBMCs derived from patients (n=19) with dust mite allergy and healthy controls (n=7) on (Fig. 7a) IL-5, (Fig. 7b) IFN-y, (Fig. 7c) IL-2, (Fig. 7d) IL-10, (Fig. 7e) TGF-P, (Fig. 7f) TNF-a production of isolated PBL after 72 h stimulation with mite extract. Cytokines were measured by cytometric bead assay CBA-technique. Figure 7h shows the fold change of IL-5, IFN-y, IL-10 and TGF-P levels of mite stimulated PBMCSs from patients with dust mite allergy after BT-061 treatment.
Figure 8a to g provide bar charts showing Treg activation with BT-061. Purified Tregs cultured in the cell expansion system for 10 days were harvested and stimulated with anti- CD3 (OKT3)/anti-CD28 antibody (mAb) or BT-061 in the presence of FCS 10% or HS 10%, respectively. Activation of cells was measured by BrdU Proliferation test (Fig. 8a, Fig. 8d) and production of (Fig. 8e) TGF-P, (Fig. 8f) IL-5 and (Fig. 8g) IFN-y. In addition, culture mediums were tested for (Fig. 8b) BrdU background signal and (Fig. 8c) TGF-P level.
Figure 9a to f provide bar charts showing the effect of BT-061 on Treg activation (Fig. 9a) and the effect of activated Tregs on (Fig. 9b-d) TCR-stimulated and (Fig. 9e,f) mite- stimulated Teffs using isolated and expanded Tregs and Teffs from from patients with dust mite allergy and healthy controls. Tregs were pre-cultured with BT-061 or with medium alone for 72 hrs (a) and Teffs were added (1 : 10) to the cell culture, stimulated either (b-d) via the TCR (CD3/CD28) (e,f) or with mite extract and IL-5, IFN-y as well as IL-10 and the T-cell transcription factors Foxp3, T-bet as well as GATA-3 were detected by qRT-PCR.
Figure 10A to D provide histograms showing CD69 expression on (A) unstimulated Teffs or Teffs cultured in the presence of (B) BT061, (C) Omalizumab or (D) Phorbolester/Ionomycin (PBu) analyzed by flow cytometry.
Figure 10 E to H provide bar charts showing (E) Effect of BT061 compared to Omalizumab on the expression of the activation markers CD69, CD25, CD44, CD127 and HLA-DR on isolated
Teffs (n=3). (F) Effect of BT061 or Omalizumab on the expression of the activation marker CD69 on unstimulated or Phytohaemagglutinin (PHA)-stimulated Teffs (n=4). CD69 expression was analysed using flow cytometry and is shown as fold change (FC). (G, H) Cytokine release of isolated Teffs measured by Cytometric Bead array, n.s. = not significant Figures 11 A to E provide a plot (A), graphs (B,C) and bar charts (D,E). PBMCs were stimulated via T-cell receptor (TCR) and activation of cells was determined by CD69 expression using flow cytometry. (A-C) Gating strategy. PBMCs were differentiated by the surface markers CD4 and CD25, and CD69 expression on Teffs (CD4+CD25 ) and on Tregs (CD4+CD25+) was analysed. BT061 (green) or Omalizumab (red) were added to unstimulated or TCR-stimulated PBMCs and CD69 expression was detected on (D) Teffs and (E) Tregs (n=6). CD69 expression is shown as fold change (FC) of TCR activation. ** PO.Ol.
Figures 12 A to E provide bar charts showing the effect of BT061 on cytokine release. Effect of BT-061 on IL-2, IL-5, IFN-y, IL-10 and TNF-P release of isolated PBMC from controls after 48 h stimulation with anti-CD3 and anti-CD28 mAK. Cytokine concentration is shown as fold change (FC) of TCR activation. *P<0.05, ** PO.OL
Detailed Description
As used herein, singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Accordingly, reference to “an antibody” includes reference to “antibodies”.
All technical and scientific terms used herein have the meaning that would be understood by a person of skill in the art to which the invention belongs unless clearly indicated otherwise.
As noted above, asthma is a chronic airway disease characterized by inflammation, bronchial hyperactivity and airway obstruction. Common symptoms are wheezing, breathlessness, a tight chest and coughing.
Allergic asthma (sometimes also known as allergy-induced asthma) is asthma caused by an allergic reaction to an allergen. Common allergens are pollen, dust mites, and animal fur or feathers.
The current scientific consensus is that the basis of allergic asthma is an inappropriate Th2 response to common environmental allergens such as house dust mite (HDM) (Gauthier, M., et al., 2015. Evolving Concepts of Asthma. Am. J. Respir. Crit. Care Med. 192, 660-668). In patients with asthma, inhaled allergens activate mast cells following cross-linking of
allergen-specific IgE and binding to high affinity IgE receptors. This, in turn, promotes the release of histamine and leukotrienes, which then triggers acute symptoms (early allergic response). Further infiltration of the airway by eosinophils and Th2 lymphocytes coupled to the release of pro-inflammatory cytokines such as IL-4, IL-5, and IL-13, occurs hours later and leads to the late allergic response. IL-4 is important for allergic sensitization and IgE production; IL-5 is crucial for eosinophil survival; and IL-13 has pleiotropic effects in the lungs, including a central role in the development of airway hyperresponsiveness (AHR) and tissue remodelling. Thus, inflammatory mediators released from mast cells act on airway smooth muscle, resulting in bronchoconstriction and the clinical features of asthma. The release of inflammatory mediators also results in mucus secretion, which further contributes to airway obstruction.
Studies also suggest that Th2 lymphocytes may have a role in non-allergic asthma patients (Gauthier, M., et al., 2015. Evolving Concepts of Asthma. Am. J. Respir. Crit. Care Med. 192, 660-668).
Regulatory T cells (Tregs) are a subpopulation of CD4+ T cells that are known to modulate the immune system, and in particular are known to be important for maintaining tolerance to self-antigens (Sakaguchi et al., Immunological Reviews (2001); 182: 18-32).
The family of Treg cells consists of two key subsets: (i) naturally arising Tregs (sometimes known as nTregs), which develop in the thymus; and (ii) peripherally induced Tregs (sometimes known as iTregs) which arise in peripheral circulation from conventional T cells. Treg cells are generally characterised by the expression of CD4 and CD25 surface biomarkers, and the transcription factor FoxP3.
Treg dysfunction is known to be associated with certain autoimmune disease. Tregs are also known to play a role in controlling allergic asthma by ensuring the maintenance of immunological tolerance and the prevention of Th2 -induced inflammatory response (Robinson, D.S., 2009. Regulatory T cells and asthma. Clin. Exp. Allergy 39, 1314-1323; Martin-Orozco, E., et al., May 2017. Regulatory T Cells in Allergy and Asthma. Front Pediatr 5. Article 117). Although Th2 cell responses to allergens can also be formed in individuals without a genetic propensity for developing allergic disease (non-atopic), these responses are normally silenced by Tregs. The modulatory role of Tregs is extensive and affects effector cells either directly or indirectly to ultimately block inappropriate immune response. Tregs exert their actions on effector cells through several pathways: 1) cell to cell contact, 2) inhibitory cytokines such as IL-10 and TGF-P, 3) cytotoxicity mediated by perforins and
granzymes, and 4) competition for T cell growth factors such as IL-2. In allergic asthma, it is likely that Treg-mediated suppression of effector cells is deficient, leading to inflammation of the airways via a Th2-cell response to an allergen (Robinson, D.S., 2009. Regulatory T cells and asthma. Clin. Exp. Allergy 39, 1314-1323; Martin-Orozco, E., et al., May 2017. Regulatory T Cells in Allergy and Asthma. Front Pediatr 5. Article 117).
Generally, an inverse correlation of Treg number and allergy development has been shown (Tulic, M.K., et al., 2012. Changes in thymic regulatory T-cell maturation from birth to puberty: differences in atopic children. J. Allergy Clin. Immunol. 129, 199-206. el-4). In addition, studies investigating the role of Tregs in the peripheral blood of asthmatic children have demonstrated that activated pulmonary Tregs are decreased in children with atopic allergy and fail to suppress pulmonary Th2 responses (Hartl, D., et al., 2007. Quantitative and functional impairment of pulmonary CD4+CD25hi regulatory T cells in pediatric asthma. J. Allergy Clin. Immunol. 119, 1258-1266; Stelmaszczyk-Emmel, A., et al., 2013. Frequency and Activation of CD4+CD25high FoxP3+ Regulatory T Cells in Peripheral Blood from Children with Atopic Allergy. IAA 162, 16-24). Additional studies in asthmatics have shown that pulmonary Tregs are decreased, functionally impaired, and cannot suppress pulmonary inflammation and the late asthmatic response (Aggarwal, N.R., et al., 2014. Immunological Priming Requires Regulatory T Cells and IL-10-Producing Macrophages To Accelerate Resolution from Severe Lung Inflammation. The Journal of Immunology 192, 4453-4464; Matsumoto, K., et al., 2002. IL-10 production in circulating T cells differs between allergen- induced isolated early and dual asthmatic responders. Journal of Allergy and Clinical Immunology 109, 281-286, Kinoshita, T., et al., 2014. Natural regulatory T cells in isolated early responders compared with dual responders with allergic asthma. Journal of Allergy and Clinical Immunology 133, 696-703). Moreover, Kinoshita et al (2014) demonstrated that the percentage of Tregs in the sputum of patients with mild allergic asthma significantly decreased 24 hours after allergen challenge. Despite the pivotal role of Tregs in allergic asthma, and potentially also in non-allergic asthma, there is no approved therapeutic approach to date which capitalizes on the activation of naturally-occurring Tregs and currently approved biologies for this indication target either IgE or pro-inflammatory cytokines (IL-4, IL-5 and IL-13).
As indicated above, the present disclosure relates to the medical use of an anti-CD4 antibody or fragment thereof for use in treating or preventing asthma in a subject. The anti- CD4 antibody or fragment is described herein and may be defined as an anti-CD4 antibody or
fragment thereof is capable of activating CD4+CD25+ regulatory T cells and/or by reference to the sequence features described herein.
In particular, the medical use relates to the treatment of asthma, which may be allergic asthma or non-allergic asthma.
Allergic asthma is caused by an allergic reaction triggered by inhalation of an allergen. It may also be defined as allergy-induced asthma. The allergen may be one or more of dust mites, pollen, smoke (including cigarette smoke), mould, and pet dander (including fur, hair and features).
The subject may be suffering from any form of asthma (e.g. allergic asthma), i.e. mild intermittent asthma, mild persistent asthma, moderate persistent asthma or severe persistent asthma. Preferably the subject is suffering from moderate persistent asthma or severe persistent asthma.
The subject may be one who has previously been treated with one or more agents suitable for treating asthma which are not the anti-CD4 antibody or fragment thereof described herein. In particular, the subject may be one who has previously been treated with one or more agents suitable for treating allergic asthma. The one or more agents may be a corticosteroid, a biologic, an anti -leukotriene, or a bronchodilator. The subject may have been treated with the one or more agents for more than 1 year. The subject may be one who is resistant or intolerant to the one or more agents. In one embodiment the medical use of the present invention is particularly suitable for a subject who has been previously treated with a corticosteroid, in particular a glucocorticosteroid. The subject may have been treated with the corticosteroid for more than 1 year and/or the subject may be one who is resistant or intolerant to corticosteroid treatment. The subject may be one who has been treated with inhaled corticosteroids (ICS), optionally also with short- and/or long-acting beta agonists. In addition, the subject may be one with poor symptom control and/or exacerbations (acute or sub-acute worsening in symptoms and lung function from the subject’s usual status) despite such treatments.
The subject can be any mammal, including a human, non-human primate, or a domesticated mammal such as a cat or a dog. However, preferably the subject is a human. The subject can be six years or older, preferably sixteen years or older.
The use may reduce inflammation (e.g. allergic inflammation), and in particular may reduce levels of IL-5, IFNy, TNF-a, and/or IL-2 in the plasma or sputum of the subject (as compared to the levels pre-treatment). Preferably the use may reduce IL-5 levels in the plasma
or sputum of the subject. Alternatively, the use may increase the level of anti-inflammatory cytokines, such as TGF-P, in the plasma or sputum of the subject (as compared to the level pre-treatment). These changes may be detected by obtaining samples of plasma or sputum from the subject and assaying the samples, for example using ELISA. The reduction / increase may be seen around 1 hour post administration, around 72 hours post administration, after 2 weeks, after 4 weeks or after 12 weeks of weekly administration of the antibody or antibody fragment, or 1 week after the end of treatment (in particular 1 week after the end of a 12 week treatment of weekly administration of the antibody or antibody fragment).
The use described herein may also reduce bronchoconstriction in the subject, in particular as compared to pre-treatment levels. As is known in the art, bronchoconstriction can be determined based on the forced expiry volume in 1 second (FEVi) in the period following challenge with an asthma trigger, e.g. with FEVi measured at baseline at 0 hours and then at hourly intervals in the 8 hour period following allergen challenge (see e.g., Gauvreau et al., NEJM 2014; 370: 2102-10). Reduced bronchoconstriction may be shown after a 2 week, a 4 week or a 12 week treatment period of weekly administration of the antibody or antibody fragment, or 1 week after the end of treatment (in particular 1 week after the end of a 12 week treatment of weekly administration of the antibody or antibody fragment.
The anti-CD4 antibody or antibody fragment for the medical use described herein can be defined as being an agonistic antibody. In particular the antibody or antibody fragment is “capable of activating CD4+CD25+ regulatory T cells”. In one example this can be defined as the ability of the anti-CD4 antibody or antibody fragment to activate T cells expressing CD4+ and CD25+ in vitro, preferably such that the activated CD4+CD25+ regulatory T cells are able to inhibit the production of pro-inflammatory cytokines (e.g. IL-5, IL-4 and/or IL-13, preferably IL-5) in a PBMC culture after TCR stimulation, in particular in a culture containing T effector cells.
In particular, the testing method may comprise pre-incubating CD4+CD25+ regulatory T cells obtained from a donor with the antibody or antibody fragment for a period suitable to activate the cells (e.g. 48 - 72 hours), TCR stimulating a T effector cell culture obtained from the same donor, transferring the pre-incubated CD4+CD25+ regulatory T cells to the T effector cell culture, and measuring the level of the pro-inflammatory cytokine after 72 hours. The T effector cell culture and/or the CD4+CD25+ regulatory T cells may be obtained from a healthy individual, or an individual suffering from allergic asthma. In this assay an anti-CD4
antibody or fragment thereof that is capable of activating CD4+CD25+ regulatory T cells will lead to reduction in the amount of the pro-inflammatory cytokine detected as compared to a negative control in which the CD4+CD25+ cells have not been pre-incubated with the anti- CD4 antibody or fragment thereof. Preferably the anti-CD4 antibody or fragment thereof leads to the same reduction +/- 25%, more preferably +/- 10%, as a positive control with the BT061 antibody described herein.
The ability of the antibody or antibody fragment to activate CD4+CD25+ regulatory T cells can also be determined by methods described in the art, such as those described in WO201 1/064407. For example, the ability can be assayed by examining the suppressive activity of the CD4+CD25+ regulatory T cells after incubation with the antibody or antibody fragment by co-culturing the CD4+CD25+ regulatory T cells with CD4+CD25- effector T cells. Activated CD4+CD25+ regulatory T cells are able to inhibit proliferation of such effector T cells in effector T cell proliferation assays. The effector T cells can be labelled with CFSE such that any proliferation can be determined. Alternatively, proliferation of effector cells can be determined by [3H] thymidine incorporation.
The antibody or antibody fragment may also be defined as one that does not cause antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). This ability can be determined by methods known in the art. In particular, the anti-CD4 antibody BT061 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 of modulating the effector functions of an antibody or antibody fragment, so as to ablate effector functions are known in the art (Saunders, K.O., June 2019, Frontier in Immunology, Vol. 10, Article No. 1296). Antibodies or antibody fragments that are modified such that they do not cause ADCC or CDC are within the scope of the invention.
The ability to activate CD4+CD25+ regulatory T cells enables the antibody or antibody fragment to inhibit activation of CD4+ effector T cells in vitro when in the presence of CD4+CD25+ regulatory T cells. As shown in Figure l id and in Figures 9a to 9c, the anti-CD4 antibody activates CD4+CD25+ regulatory T cells in vitro leading to suppression of the release of pro-inflammatory cytokines IL-5 and IFN-y. Similarly, activation of T effector cells can be determined in vivo by monitoring levels of inflammatory cytokines released by activated T effector cells in samples of the subject’s plasma or sputum.
The use of the antibody or antibody fragment of the present invention is advantageous over the prior art use of the anti-CD4 antibody keliximab antibody in asthma patients, since,
as noted above, keliximab was found to cause a reduction in CD4 cell count, and further did not improve FEVi (Kon et al., Eur. Respir. J. 2001; 18: 45-52; Tourangeau et al., Therapeutic Advances in Respiratory Disease (2011): 5(3): 183-194). In contrast, the present inventors consider that the use of the present invention can treat asthma patients without inducing ADCC. Moreover, the present inventors consider that surprisingly in the use of the present invention pro-inflammatory cytokines are downregulated, which was not shown with keliximab treatment. In particular, the ability to activate CD4+CD25+ T regulatory cells from asthma patients as shown in the present examples is unexpected as these cells may be decreased and functionally impaired in such patients.
The anti-CD4 antibody or antibody fragment thereof is preferably an anti-human CD4 antibody or fragment thereof.
The antibody may be a human antibody, or may be a humanized or chimeric antibody. Preferably the antibody is a humanized antibody.
Generally, the antibody further comprises a human constant region (Fc). This constant region can be selected among constant domains from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4. Preferred constant regions are selected among constant domains of IgG, in particular IgGl.
The antibodies are preferably IgGl antibodies, and/or the antibody or antibody fragment preferably comprises an Fc portion such that the antibody or antibody fragment is capable of binding to an Fc receptor, preferably FcyRI (i.e. CD64). Most preferably the antibody or antibody fragment comprises the Fc portion of an IgGl antibody. In addition, or alternatively, the antibody or antibody fragment is capable of binding to monocytes via an Fc receptor.
The present invention also includes any fragment of the antibody including fragments comprising the V regions thereof. This comprises in particular Fab, Fab', F(ab)'2, Fv and scFv fragments.
Preferably the antibody is a humanized anti-CD4 antibody or fragment thereof which is a variant of the BT061 antibody, which has V domains defined by the following polypeptide sequences (and which is described further in the examples below):
- H chain V domain: EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWLRQA PGKGLEWIGVISVKSENYGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVY YCSAS YYRYDVGAWFAYWGQGTLVTVSS (SEQ ID NO: 3)
- L chain V domain:
DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQ
KPGQPPKLLIYLASILESGVPDRF SGSGSGTDFTLTIS SLQAED VAVYYCQHSRELPWT FG QGTKVEIK (SEQ ID NO: 4).
Accordingly, the anti-CD4 antibody or fragment thereof for use in the present 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 SEQ ID NO: 4. In a preferred embodiment the anti-CD4 antibody comprises a human IgGl constant domain.
Variants of this antibody or antibody fragment are also suitable for use in the present invention. Variants include those with V domains defined by polypeptide sequences having at least 85%, preferably at least 90 %, most 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 with at least 90% sequence identity with SEQ ID NO: 3 and a light chain variable domain with at least 90% sequence identity with SEQ ID NO: 4. Still more preferably the antibody or fragment thereof has a heavy chain variable domain with at least 95% sequence identity with SEQ ID NO: 3 and a light chain variable domain with at least 95% sequence identity with SEQ ID NO: 4. A preferred embodiment is an antibody comprising these variable domain sequences and an IgGl constant domain.
In addition or alternatively the antibody or antibody fragment is one which has the CDR sequences of BT061 (SEQ ID Nos: 5 to 10). A particularly preferred embodiment is an anti- CD4 antibody or fragment thereof comprising a heavy chain variable domain with at least 95% sequence identity with SEQ ID NO: 3 and which includes the CDR sequences of SEQ ID Nos: 5 to 7, a light chain variable domain with at least 95% sequence identity with SEQ ID NO: 4 and which includes the CDR sequences of SEQ ID Nos: 8 to 10.
The manner in which BT-061 binds to CD4 has been disclosed in Helling et al., Immunology and Cell Biology (2015) 93: 396-405. Further variants of BT-061 have also been disclosed in WO 2011/064407. Accordingly, this information can be used to guide the variation of the sequences of the heavy and light chains. Preferably the variations do not substantially affect the specificity and/or affinity of binding.
Alternatively or in addition the antibody or antibody fragment is one that binds to an epitope on CD4 comprising 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 in WO201 1/064407). Methods for determining the ability to bind to a particular epitope are known in the art. The antibody or antibody fragment can also be defined as one that binds to
substantially the same epitope as the BT061 antibody described herein. This property can be determined by alanine scanning.
In one aspect the invention provides an anti-CD4 antibody for use in treating or preventing allergic asthma in a subject, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2.
The antibody or antibody fragment may be manufactured using encoding polynucleotide sequence. In particular, recombinant DNA constructs can be obtained and introduced in host cells by the well-known techniques of recombinant DNA and genetic engineering. Expression cassettes can be used in which the polynucleotide sequence(s) are linked to appropriate control sequences that allow for regulation of transcription and translation in a chosen host cell. Suitable host cells include mammalian cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc.
Accordingly, the antibody or antibody fragment may be one which has been produced in a mouse or a human cell line, and in particular in a cell line mentioned herein.
The antibody or antibody fragment may be comprised in a pharmaceutical composition which further comprises a pharmaceutically acceptable excipient or carrier. The pharmaceutical composition may further comprise a stabilizing agent. The pharmaceutical composition may be suitable for intravenous, subcutaneous, or intramuscular use, or use as an inhalative.
In some aspects of the invention the antibody or antibody fragment is for use by administration to the subject. The antibody or antibody fragment may be administered by any appropriate route, e.g. intravenous, subcutaneous, intramuscular or via inhalation. Preferably the antibody or antibody fragment is administered subcutaneously or via inhalation. Most preferably the antibody or antibody fragment is administered subcutaneously.
Depending on the route of administration, the antibody or antibody fragment may be comprised in a vial, a pre-filled syringe, an autoinjector or an inhaler (or puffer). The vial may be arranged for use in an autoinjector or an inhaler, i.e. arranged such that it can be fitted or slotted into an autoinjector or an inhaler just prior to use.
In a further aspect the present invention provides an inhaler, or a vial for use with an inhaler, comprising the anti-CD4 antibody or fragment thereof described above. This inhaler can be used to deliver the antibody or fragment into the body via the lungs. In particular, without wishing to be bound by theory the inventors consider that due to internalization of the
anti-CD4 antibody or fragment thereof after binding to the CD4 epitope described above, the medical use of the present invention will not suffer from the drawback described in the art for pulmonary-delivered antibodies of short residence time of the antibody in the lungs. (In particular it has been suggested that some pulmonary-delivered monoclonal antibodies are mostly cleared from the lungs within 1-2 days (Desoubeaux G., et al., 2016, MABS, 8(6): 999-1009)).
The inhaler may be a metered dose inhaler which comprises components that generate a metered dose of the medication in aerosol form, or a soft mist inhaler (e.g. a mesh inhaler), which comprises components that generate a mist of medication for inhalation by the user.
The anti-CD4 antibody or fragment thereof is comprised within the inhaler, or vial for use with an inhaler, in a pharmaceutical composition. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient suitable for use in the lungs. In particular, the pharmaceutical composition may further comprise a surfactant suitable for use in the lungs.
The antibody or antibody fragment may be administered in a dose of between 25 mg and 200 mg, preferably between 75 mg and 200 mg, more preferably between 100 mg and 200 mg. Where the route of administration is via inhalation the dose of the antibody or antibody fragment is preferably from 25 mg to 150 mg, more preferably from 75 mg to 125 mg.
Doses may also be calculated based on the body weight of the subject, and may be from 1 to 3mg/kg, preferably 1 to 2.5 mg/kg, and most preferably 1.5 to 2 mg/kg.
The antibody or antibody fragment may be administered at regular time intervals, e.g. once per week, once every 2 weeks, once every 3 weeks or once every four weeks.
The antibody or antibody fragment may be used in combination with one or more further agents known to treat asthma (e.g. allergic asthma), provided that the one or more further agents are non-immunosuppressive.
In a further aspect of the invention the anti-CD4 antibody or fragment thereof may be used to activate CD4+CD25+ regulatory T cells obtained from the subject in vitro, before the activated cells are reintroduced back into the subject to treat or prevent allergic asthma.
Accordingly, the present invention further provides a method of treating a subject suffering from or preventing a subject suffering from asthma (e.g. allergic asthma), the method comprising removing a sample comprising CD4+CD25+ regulatory T cells from the subject, contacting the CD4+CD25+ regulatory T cells with an antibody or antibody fragment as
described herein to activate CD4+CD25+ regulatory T cells and administering the activated cells to the subj ect. Such a method may additionally include an in vitro step of increasing the number of Treg cells. This can be done using the expansion strategies described herein (Peters et al., PLoS 2008; 3(9): e3161; Hoffmann et al., Blood 2004; 104(3):895-903).
Similarly, the present invention provides activated CD4+CD25+ regulatory T cells, which have been activated in vitro using the antibody or antibody fragment described herein, for use in treating or preventing asthma (e.g. allergic asthma) in a subject. The CD4+CD25+ regulatory T cells are obtained from the subject. Optionally the CD4+CD25+ regulatory T cells are expanded prior to reintroduction into the subject as described above.
The following are intended as examples only and do not limit the present disclosure.
Examples
The anti-CD4 antibody (BT061) that is used in the examples is a recombinant, humanized IgGl monoclonal antibody comprised of two heavy chains and two light chains. The amino acid sequence for both heavy and light chains has been predicted from the translation of the nucleotide sequence for the gene of BT061 and have been confirmed experimentally. Figure 1 displays the predicted amino acid sequences for the heavy and light chains, as well as the most likely disulphide bond assignment. The heavy chain has SEQ ID NO: 1 and the light chain has SEQ ID NO: 2.
The BT061 antibody used in the examples has been produced in a mouse myeloma host cell line Sp2/0-Agl4 (Sp2/0, corresponding to ATCC CRL-1581). Expression of the antibody in Sp2/0 is also described in WO 2009/124815.
The BT061 antibody has been given the generic name tregalizumab.
Example \ - In Vitro System Set-Up
To test the immunological response of subjects with mite dust allergy in vitro a mite stimulation system was set up using peripheral blood mononuclear cells (PBMCs) isolated from subjects with house dust mite (HDM) allergy (n=19) and from healthy control subjects (n=7). The disease status of the subjects was determined using the radioallergosorbent test (RAST), which is used to detect specific IgE antibodies to house dust mites. Patients were segregated into either treatment or control groups based on RAST scores (0 = control; 3 = high level of allergen-specific IgE; 6 = extremely high level of allergen-specific IgE).
(Schulze J,Leberkuehne L, Salzmann-Manrique E, Schubert R, Zielen S, Rosewich M. Comparison of two different assays and the predictive value of allergen components in house dust mite allergy. Immunotherapy. 2017 Nov;9(15): 1253-1262.)
Samples were analyzed for specific antibodies against Der p and Der f, IgE using a two-sided chemiluminescent assay (Immulite DPC). Analysis was performed according to the manufacturer’s instructions.
Figure 2 shows the time kinetic of Foxp3 -expression 24 hours, 72 hours and 10 days after HDM stimulation, as detected by intracellular flow cytometry. It can be seen that cells derived from patients with different RAST scales (RAST Scale 3 and 6) exhibited a different Treg proliferation kinetic compared to that of cells derived from healthy control subjects (RAST Scale 0).
PBMCs from patients with mite dust allergy also showed a different proliferating response compared to controls as detected by CFSE-flow cytometry (data not shown). Stimulation with HDM extract led to a time dependent raise in cytokine production. After 72 hours PBMCs from patients showed a strong IL-5 response to mite stimulation, whereas no effect was detectable for controls (Figures 3a and 3b - the IL-5 amounts with “Control Medium”, “Controls Mite” (i.e. cells from healthy donors incubated with either medium or allergen) and “Patients Medium” (i.e. cells from patient donors incubated with medium) all track on or just above the x-axis in Figure 3a). In contrast, IFN-y levels (as shown in Figure 4a and 4b) were much higher in control cell culture as in patients.
These results demonstrate that a suitable system has been set up to measure the effects of the anti-CD4 antibody BT-061 on activation and proliferation of T-regs and on TH2-driven immunity in patients with house dust mite allergy. In particular, the inventors consider that the system is capable of simulating the cytokine/chemokine pattern observed in in vivo asthma models and humans. (The cytokine/chemokine pattern observed in vitro is comparable to patterns observed in in vivo asthma models (Xu et al 2012. Adoptive transfer of induced- Treg cells effectively attenuates murine airway allergic inflammation. PLoS ONE 7, e40314).) For all further experiments we used the cytometric bead array (CBA) for analysis of cytokine production and qRT-PCR for detection of mRNA-levels.
Example 2 - Effect of BT-061 on mite-stimulated PMBC derived from patients with dust mite allergy
We investigated the time course of IL-5 and IFN-y production after stimulating PBMCs from subjects with dust mite allergy (RAST 6) and controls (RAST 0) with mite extract with different BT-061 concentrations (0 pg/ml, 0.1 pg/ml, 1.0 pg/ml, and 10 pg/ml).
BT-061 decreased IL-5 as well as IFN-y cytokine production after 72 hours of stimulation (Fig. 5a, 5b). However, no concentration-dependent effect of BT-061 was found when 0.1, 1.0 and 10.0 pg/ml of the BT-061 -antibody were used suggesting that at a concentration of 0.1 pg/mL saturation may already have been achieved. RTPCR underlined the results of the cytokine production (Fig. 6a, 6b). IL- 5 mRNA was found higher in patients PBMCs after mite extract stimulation and was decreases in the presence of BT-061. Vice versa, IFN-y mRNA was higher in controls and down-regulated with BT-061. Interestingly, in this preliminary experiments, TGF-P and Fox-P3 mRNA were higher in patients, especially TGF-P mRNA was increased after BT-061 stimulation (Fig.6c, 6f). No effect of the stimulation system could be detected for the transcription factors GATA-3 and T-bet (Fig.6d, 6e).
We implemented PBMCs from 19 Patients with dust mite allergy and 7 healthy controls in the in vitro system. We found a significant down-regulation of BT- 061 on IL-5 and IFN-y production of mite stimulated PBMCs of patients with dust mite allergy and controls, respectively, pointing to the down-regulatory effect of BT-061 on inflammation but also on “allergic inflammation” (Figure 7a to h).
Example 3 - Influence of BT-061 on activation and proliferation of T-regulatory cells from patients with house dust mite allergy in a Treg cell expansion system.
Tregs were isolated using either the Magnetic Activated Cell Sorting-System (MACS, Miltenyi Biotec) or the EasySep® Human CD4+CD25+T Cell Isolation Kit (STEMCELL Technologies). Derived from heparinized whole blood, cells were separated in several steps from PBMCs, CD4+ cells to CD4+CD25‘ cells (Teffector cells = Teffs) and CD4+CD25+ cells (Tregs), respectively. Enrichment of CD4+CD25+ was monitored by flow cytometry showing an approximate purity of 75% after the last isolation step. Foxp3 expression of this population was approximately 50 %. Recovery of Tregs was higher with the EasySep method (0.33 xl05 cells/ml) compared to the MACS system (0.16 xl05 cells/ml). No differences were seen in the purity of separation between the methods.
For expansion of purified Tregs, cells were cultured in the presence of irradiated (60 Gray) L-cells (L929-derived murine Ltk- cell line (a mouse fibroblast cell line) stably transfected with human FcyRII (CD32)), anti- CD3 (OKT3), anti-CD28 antibody and IL-2. (Antibodies against CD3 and CD28 as well as BT061 bind to FcyRII, enabling improved cross-linking of the receptors of the target cell and thus signal amplification.) Because cell numbers kept very low in cell culture with fetal calf serum (FCS 10%), we added experiments culturing cells in the presence of human serum (HS 10%) for 10 days. Tregs proliferated very well in HS 10% starting at culture day 2 and resulted in a 30-fold expansion of the cells until day 10. Treg proliferation was accompanied by a high Foxp3 expression, which was stable over the stimulation period. In contrast, Tregs did not proliferate well in FCS 10%.
Next, Treg activation with BT-061 was tested. Purified Tregs cultured in the cell expansion system for 10 days were harvested and stimulated with anti- CD3 (OKT3)/anti- CD28 antibody or BT-061 in the presence of FCS 10% or HS 10%, respectively (Fig. 8). Proliferation testing by BrdU incorporation showed increased optical density (OD) after stimulation with anti CD3/CD28, but did not increase after BT-061 stimulation. However, we found very high OD levels (>1.5) for unstimulated probes in the cell culture with HS 10% (Fig. 8a). In addition, blank values in the presence of HS 10% were more than two-fold as high as in the presence of FCS 10% and more important TGF-P levels of more than 250 pg/ml was found in medium containing HS 10% (Fig. 8b, c).
For that reason, we also used FCS 10% to reduce background in 72 hrs Treg activation. As seen before, stimulation with BT-061 did not influence proliferation of Tregs (Fig. 8d). But BT-061 stimulated Tregs to produce TGF-b showing the potential of BT-061 to activate Tregs. Fig. 8e) BT-061 did not stimulate IL-5 and IFN-y production (Fig. 8f, g).
Example 4 - Effect of (A) BT-061 on Treg activation and the effect of activated Tregs on, (B) TCR-stimulated and, (C) mite-stimulated Teffs using isolated and expanded Tregs and Teffs
In (A) isolated Tregs were pre-incubated with BT-061 or with medium alone for 72 hours and T-cell transcription factors Foxp3, T-bet and GATA-3 were measured by qRT- PCR. Tregs stimulated with BT-061 showed a significant increase in Foxp3 -expression, whereas no effects could be detected in T-bet and GATA-3 expression (Figure 9a).
In (B) the effect of BT-061 -activated Tregs on Teffs after TCR-stimulation was investigated. Due to its unspecific character of the TCR-stimulation, we first investigated the
effect of BT-061 on CD3/CD28 stimulated Teffs from patients with dust mite allergy and healthy controls. Tregs were added to the Teff cell culture in a 1 : 10 ratio. Cytokines and T- cell transcription factors were detected by CBA or qRT-PCR.
We found a significant increase in IL-5, IFN-y and IL-10 expression (Figure 9b-d) compared to the unstimulated cells. Pre-incubation with BT-061 significantly down-regulated the expression of IL-5 and IFN-y pointing to the inhibitory effect of the activated Tregs on the TCR stimulated Teffs.
In (C) the effect of BT-061 -activated Tregs on mite-stimulated Teffs from patients with dust mite allergy was studied. Mite-stimulated Teffs from patients with dust mite allergy were cultured in the presence of Tregs pre-incubated with BT-061. BT-061 decreased IL-5 expression (>25%) and to a lesser extend IFN-y expression but strongly increased IL- 10 expression in our Treg/Teff-culture system (Figure 9e). In addition, BT-061 up-regulated Foxp3 expression and down-regulated the TH2-transcrition factor GATA-3 (Figure 9f).
The data from the above in vitro studies demonstrate that BT061 has the potential to combat inflammation triggered by allergic asthma. In particular it has been shown that BT061 exerts a positive effect on Tregs (isolated from allergic patients) and a modulatory effect on TCR-stimulated Teffs. In addition, the cytokine patterns observed in in vitro studies with patient PBMCs correspond to the in vivo situation observed in allergic mice subjected to methacholine challenge (Xu et al 2012 as above).
Further examples were conducted on regulatory T cells and effector T cells obtained from healthy volunteers.
Example 5 - Effect of BT061 on T-effector cells (Teffs)
Further examples were conducted on regulatory T cells and effector T cells obtained from healthy volunteers.
Cell separation
Peripheral mononuclear blood lymphocytes (PBMCs) from buffy coat or healthy volunteers were obtained by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Teffs and Tregs were isolated using the EasySep® Human CD4+CD25+ T Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer’s instruction.
Tregs were expanded using a Treg expansion system as described [Hoffmann et al. Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells. Blood. 2004 Aug 1; 104(3):895-903], For expansion of purified Tregs, cells were cultured in
the presence of irradiated L-cells (L929-derived murine Ltk- cell line stably transfected with human FcyRII (CD32)), anti-CD3 (OKT3), anti-CD28 (CD28.2) antibody and IL-2 in the presence of human serum (HS 10%) for 5 days. Enrichment of CD4+CD25‘ cells and CD4+CD25+ as well as Treg expansion was monitored by flow cytometry.
Cell culture
Cells were cultured in 96-well flat-bottom plates in RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 1% HEPES, 2% glutamine and 2% gentamicin at a concentration of l x 106 cells/ml. For induction of cell proliferation and cytokine production, cells were stimulated with anti-T cell receptor (TCR) mAb, anti CD3 (OKT3, 1 pg/ml) and costimulated with anti-CD28 mAb (CD28.2, 1 pg/ml). Alternatively, lymphocytes were incubated 72h in the presence of phythaemagglutinine (PHA) (10 pg/ml), or phorbol- 12, 13 -dibutyrate (PBu, 1 ng/ml) in conjunction with ionomycin (0.5 pg/ml).
Cell proliferation (BrdU-Test)
For measurement of DNA synthesis cells, were incubated for 72h under conditions described above. BrdU (5-bromo-2'-deoxyuridine) incorporation was detected by a colorimetic cell proliferation ELISA (BrdU-ELISA, Boehringer Mannheim) after 18h incubation according to the manufacturer’s instruction.
Flow cytometry
Cells were stained with the following monoclonal antibodies (MoAb): CD25 APC Clone 2 A3, CD4 FITC Clone RPA-T4, CD 127 PE Clone hIL-7R-M21, HLA-DR PE Clone TU36, CD3 BV450 Clone UCHT1, CD45 V500 Clone HI30, CD69 PE Clone FN50, CD 44 PE Clone G44-26, CD 64 PE Clone lO.lfrom Becton Dickinson (San Jose, CA, USA). After staining, cells were incubated for 15 min at room temperature (RT). For intracellular staining, cells were surface-stained with a cocktail of anti-CD4-fluorescein isothiocyanate (FITC) (OKT- 4) and anti-CD25-phycoerythrin (PE) (BC96) monoclonal antibodies. After fixation and permeabilization, the cells were blocked with 2% normal rat serum and intracellular staining was performed using anti-human FoxP3 allophycocyanin (APC) (PCH101) antibody (Natutec, Frankfurt am Main, Germany). On each sample, 10,000 events were analyzed by a BD FACSVerse™ flow cytometer (Becton Dickinson, San Jose, CA, USA). Data analysis was performed using BD Facssuite software (Becton Dickinson, San Jose, CA, USA).
Cytometric Bead Array
Concentrations of cytokines/chemokines were determined in culture supernatants using the BD™ CBA Flex Set System for the measurement of IL-2, IL-5, IL-10, INF-y, TNF-a and
TGF-P (BD Bioscience-PharMingen, San Diego, CA, USA). Each BD™ CBA Flex Set contained one bead population with distinct fluorescence intensity, as well as the appropriate phycoerythrin (PE) detection reagent and standard. The tests were performed according to the manufacturer’s advice as described before [Eickmeier et al. Sputum biomarker profiles in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) and association between pulmonary function. Cytokine. 2010 May;50(2): 152-7], Results
Teffs and Tregs were isolated as described above. Cells were separated in several steps from PBMCs, CD4+ cells to CD4+CD25‘ cells (Teffector cells = Teffs) and CD4+CD25+ cells (Tregs), respectively. Enrichment of CD4+CD25‘ cells and CD4+CD25+ was monitored by flow cytometry. The separation of Teffs and Tregs resulted in a high purity of isolated cells and a sufficient number of Teffs (13.9 xlO6 cells, 5.2-47.0 xlO6). However, number of Tregs was not sufficient for the subsequent experiments (0.31 xlO6 cells, 0.15-0.70 xlO6). Therefore, Tregs were expanded using a Treg expansion system noted above. For expansion of purified Tregs, cells were cultured in the presence of irradiated L-cells (L929-derived murine Ltk- cell line stably transfected with human FcyRII (CD32)), anti-CD3 (OKT3), anti-CD28 antibody, and IL-2 in the presence of human serum (HS 10%) for 10 days. Expansion resulted in a 3.2-fold (1.3 - 9.8) increase in Treg numbers.
Characterization and Stimulation of T-effector cells
Teffs were stimulated via the T-cell receptor (TCR) using anti-CD3 and anti-CD28 monoclonal antibodies to establish a system for demonstrating the effect of BT061 on stimulated T cells. Stimulation of PBMCs via the TCR is a well-established system in the lab and has been shown to induce a strong proliferation and cytokine response on Teffs. Unexpectedly, no effect on proliferation of Teffs (data not shown), CD69 expression, and on cytokine release was seen after TCR stimulation. In contrast, stimulation with Phytohemagglutinin (PHA) led to an appropriate CD69 expression and therefore could be used as positive control for further experiments on Teffs. In contrast, stimulation of PBMCs via TCR resulted in a strong upregulation of CD69 expression on CD4+/CD25‘ cells (Teffs) as indicated by flow cytometry. In addition, TCR-stimulated PBMCs showed a significant release of IL-5 and IFN-y and of IL-2, IL-10 and TNF-p.
Effect of BT061 and Omalizumab on activation marker expression on T-effector cells
The effect of BT061 on the modulation of activation markers on isolated Teffs was measured by the expression of CD69, CD25, CD44, CD127 and HLA-DR. Omalizumab (anti-
IgE, a recombinant humanised IgGlk monoclonal antibody) was used as a negative control. Teffs were cultured in RPMI medium alone or in the presence of BT061, Omalizumab, or Phorbolester/Ionomycin (PBu) (Fig. 10 A-F). Figure 10 A-F shows an example flow cytometric experiment for the induction of CD69. No changes in the expression of any used activation markers on Teffs could be detected after treatment with BT061 or Omalizumab (Fig. 10 E). In addition, there was also no effect of BT061 on CD69 expression, even when cells were stimulated with PHA (Fig. 10 F). BT061 did not induce cytokine release such as IL-5, INF-g (Fig. 10 G, H), IL-2, IL-10 or TNF-P (data not shown) of isolated Teffs, whereas PHA treatment did.
Effect of BT061 on activation of Teffs in the presence of Tregs
To investigate the effect of BT061 on activated Teffs in the presence of Tregs, T cell activation by CD69 expression and cytokine release after TCR-stimulation was analysed (Fig. 11, 12). Due to the poor activation of isolated Teffs, PBMCs were used and CD69 expression was analysed on CD4+/CD25‘ and CD4+/CD25+ T cells by flow cytometry. PBMCs were differentiated by the surface markers CD4 and CD25, and CD69 expression on Teffs (CD4+CD25 ) and on Tregs (CD4+CD25+) in unstimulated and TCR-stimulated cells was analysed (Fig. 11 A-C). BT061 had no effect on unstimulated Teffs or Tregs (Fig. 11 D, E). Stimulation via the TCR resulted in an activation of both cell populations. The addition of BT061 led to a significantly reduced CD69 expression on Teffs but not on Tregs. In contrast, Omalizumab did not have any effect on T cell activation.
Effect of BT061 on cytokine release
The effect of BT061 on cytokine release of TCR-stimulated Teffs in the presence of Tregs was analysed by Cytometric Bead Array. This multiplex assay detection allows the measurement of several cytokines simultaneously (Fig. 12). BT061 alone did not stimulate cytokine production. Stimulation via the TCR resulted in an extensive production and release of all measured cytokines. The addition of BT061 led to a significantly reduced release of IL- 2 (Fig. 12 A), IL-5 (Fig. 12B), IFN-y (Fig. 12C) and TNF-P (Fig. 12E), but not IL- 10 (Fig. 12D). In contrast, Omalizumab did not have such an effect on the TCR-induced cytokines.
The results in this example show that BT061 has no direct effect on Teff activation or Teff cytokine release. BT061 is also shown to significantly reduced activation as well as release of the cytokines IL-2, IL-5, IFN-y and TNF-P of TCR-stimulated Teffs in the presence of Tregs.
Example 6 - Clinical Trial in Asthmatic Patients
A randomised double blind placebo-controlled human phase II clinical trial was performed in 42 patients to demonstrate the effects of tregalizumab on allergen-induced airway responses and airway inflammation in asthmatic patients under the EudraCT number 2020 000585 41 (sponsor T-Balance Therapeutics GmbH). There were two treatment arms; those patients receiving tregalizumab and those receiving the placebo.
Active: Tregalizumab (Product code: BT061) was used in the form of a 100 mg/mL solution for injection (for subcutaneous administration), and was administered once weekly for a period of 12 weeks at a dose of 100 mg per week (i.e. 1 mL subcutaneously per week).
Methacholine inhalation challenge referred to below was performed with Provokit® 0,33% (33 mg) (Aristo Pharma GmbH)
Pharmaceutical form: Powder and solvent for nebuliser solution
Routes of administratiomlnhalation use
Placebo: Solution for injection also administered subcutaneously.
Medical condition(s) being investigated: mild controlled allergic asthma and house-dust mite (HDM) allergy
Main objective of the trial: to demonstrate the effect of 100 mg tregalizumab treatment administered weekly (via subcutaneous injection) over 12 weeks to patients with mild controlled allergic asthma and HDM allergy, who have been exposed to allergen induced bronchoconstriction under controlled conditions in this challenge study. The primary endpoint supporting this objective will be the change from baseline in the Late Asthmatic Response (LAR) after bronchial allergen provocation (BAP)
Secondary objectives of the trial:
•Evaluate the Early Asthmatic Response (EAR) after BAP in adult patients with mild controlled allergic asthma and HDM allergy over 12-week treatment with tregalizumab. •Determine the anti-inflammatory profile of tregalizumab on the airways in induced sputum, by exhaled nitric oxide (eNO) and in blood after 12-week treatment with tregalizumab;
•Examine bronchial hyperresponsiveness (BHR) after a methacholine inhalation challenge after 12-week treatment with tregalizumab;
•Determine the pharmacodynamics by CD4 modulation and activation of regulatory T cells (Tregs), the pharmacokinetics and immunogenicity of tregalizumab treatment;
•Assess the safety and tolerability of tregalizumab;
•Assess the effect of tregalizumab treatment on the asthma-related quality of life.
Principal inclusion criteria:
1.Willing and able to give written informed consent
2. Male or female subject aged 18 to 65 years (both inclusive).
3. Established diagnosis of mild controlled allergic asthma (GINA 2019) and history of allergic bronchial asthma for at least 1 year.
4.Body mass index (BMI) of 18.0 to 30.0 (both inclusive).
5.Non-smoker (all substances).
6. Specific IgE to HDM > class 2 in radioallergosorbent test (RAST).
7. BHR (i.e., a decrease in FEV1 of at least 20%) measured by methacholine challenge.
8.FEV1 > 75% of predicted value (according to height, weight and sex).
9. Subject must demonstrate a significant EAR and LAR without rescue medication use within the first 7 hours after BAP. EAR is defined as a decrease in FEV1 of > 20% within 0 to 3 hours after allergen challenge; LAR is defined as a decrease in FEV1 of > 15% within 4 to 7 hours after BAP.
10.No clinically relevant abnormalities in 12-lead ECG at screening.
Principal exclusion criteria:
1. Severe, unstable bronchial asthma.
2. Exacerbation of asthma < 4 weeks prior to screening.
3. Treatment with parenteral and oral corticosteroids 6 weeks prior to screening and during the study.
4. Treatment with inhaled corticosteroids, methylxanthines (e.g., theophyllin), anticholinergics (e.g., ipratropium bromide), leukotriene modifiers (e.g., montelukast), tiotropium bromide, cromolyn or nedocromil within 2 weeks prior to screening and during the study.
5. Current treatment with any immunosuppressants (e.g., monoclonal antibodies, methotrexate, cyclosporin).
6. Specific immunotherapy (SCIT) to mite within 3 years prior to screening.
7. Serious adverse drug reaction to previous biological treatment.
8. Previous therapy with a monoclonal antibody (mAb) targeting CD4, including tregalizumab.
9. Known hypersensitivity to any constituents of tregalizumab, and/or other mAbs, that, in the opinion of the investigator or Medical Monitor, contraindicates participation.
10. Previous inclusion in this study.
11. Serum transaminases, alanine transaminase (ALAT) and/or aspartate transaminase (AS AT) > 2.5-fold upper level of normal (ULN) at screening.
12. Bilirubin > 34.2 pmol/L at screening.
13. Alkaline phosphatase (AP) > 2-fold ULN at screening.
14. Urea nitrogen > 1.5-fold ULN at screening.
15. Kidney insufficiency as defined by creatinine level > 133 pmol/L at screening.
16. History of severe allergic or anaphylactic reaction to proteins of human origin (e.g. vaccination reaction, biological therapy).
17. Presence or history of malignancy within the previous 5 years (except completely resected squamous or basal cell carcinoma of the skin).
18. Presence or history of clinically significant major disease (e.g., severe heart/lung disease New York Heart Association [NYHA] Class > 3, autoimmune disease [apart from rheumatoid arthritis], acute uncontrolled hyper- or hypo-thyroidism, severe uncontrolled hypo or hypertension).
19. Serious local (e.g., abscess) or systemic (e.g., pneumonia, septicemia) infection or recurrent chronic infections within 6 weeks prior to screening visit or during the screening period.
20. Any infection requiring antibiotic therapy by any route of administration within 4 weeks prior to screening.
21. Vaccination with live, live attenuated, and/or killed vaccines in the 12 weeks prior to the first administration of the study drug and during the study.
22. Positive diagnosis for acute or chronic infections (e.g. Hepatitis C Virus [HCV], Hepatitis B Virus [HBV], Human Immunodeficiency Virus [HIV]) at screening or history of previous chronic infection.
23. Acute or clinically symptomatic Epstein-Barr Virus (EBV) (infectious mononucleosis) or Cytomegalovirus (CMV) infection.
24. Presence or history of latent or active tuberculosis.
25. Known immune deficiency.
26. Presence or history of lymphoproliferative disease, including lymphoma and lymphadenopathy.
27. Presence or history of clinically significant drug or alcohol abuse.
28. Employee at study site or any institution involved in this study (including the sponsor), or spouse/partner or relative of an investigator.
29. Pregnant or nursing woman or woman considering to become pregnant during the study or in the 3 months after the last administration of study drug.
30.Woman of childbearing potential (unless surgically sterile or post-menopausal > 52 weeks) who is not using two (2) independent effective contraceptive methods (e.g., oral or injectable contraceptives, intra-uterine devices, double barrier method, contraceptive patch or female sterilization) during the study and for at least 3 months after the last administration of study drug
OR
Non-vasectomized man who, during the study or in the 3 months after the last administration of study drug, is not using two (2) independent effective contraceptive methods (as specified above) or is planning a sperm donation.
31. Donation of blood within 30 days prior to screening until end of study.
32. Participation in another clinical trial within 90 days before screening or during the study.
33. Inability or lacking motivation to adhere to the study requirements and to comply with the study schedule.
34. Imprisonment or placement in an institution (AMG § 40 (1), sentence 4).
Primary end point(s): The primary endpoint will be the baseline-corrected LAR measured by the area under the curve (AUC) for FEV 1 at 4 to 7 hours after BAP (AUC4-7FEV1) on Day 84. The AUC4 7FEV1 will be calculated as the area under the curve of the normalized FEV1 values (post-BAP FEV1 values divided by the pre-BAP FEV1 value in %) over time Timepoint(s) of evaluation of this end point: Visit 2, Visit 4 and visit 17
Secondary end point(s):
•Change in LAR on Day 84, as measured from 4 to 7 hours after BAP by the maximum decrease in the normalized FEV1 values;
•Change in EAR on Day 84, as measured from 0 to 3 hours after BAP by the maximum decrease in the normalized FEV1 values;
•Change in EAR on Day 84, as measured from 0 to 3 hours after BAP by the AUC of the normalized FEV1 values;
•Fraction of eNO (FeNO) absolute levels and delta increase after BAP;
•Dose of methacholine causing a decrease in FEV1 of at least 20% (methacholine PD20);
•Impact of tregalizumab on TH1, TH2 and TH17 cytokines in serum and sputum and its correlation to LAR in tregalizumab-treated subjects in comparison to placebo;
•Impact of tregalizumab on known markers of TH2 -induced inflammation (total IgE, mitespecific IgE, eosinophils) and eNO in tregalizumab-treated subjects in comparison to placebo after 12 weeks;
•Mite-specific IgG4 and total IgG after 12 weeks;
•Induced sputum markers: oEosinophils, neutrophils, macrophages and lymphocytes in cytospin samples oTHl, TH2 and TH17 cytokines oPCR for TH1, TH2 and TH17 cytokines and T-cell transcription factors oEosinophil cationic protein (ECP)
•Impact of tregalizumab on the expression of markers of PBMC lineage;
•Safety evaluations include the incidence and severity of adverse events (AEs), changes in clinical laboratory profiles, and the serum levels of specific anti-tregalizumab antibodies;
•Number of rescue 132-agonist puffs ;
•VAS Nasal Symptom Score at screening and 12 weeks;
•Asthma-related quality of life questionnaires [Asthma Control Test (ACT) and Asthma Quality of Life Questionnaire for 12 years and older (AQLQ+12)] at 0 and 12 weeks.
Additional/Exploratory Endpoints
•Changes in miRNA expression profiles in blood in tregalizumab-treated subjects in comparison to placebo as determined by next generation sequencing (optional);
•Tregalizumab trough serum levels.
The examples described herein are to be understood as illustrative examples of embodiments of the invention. Further embodiments and examples are envisaged. Any feature described in relation to any one example or embodiment may be used alone or in
combination with other features. In addition, any feature described in relation to any one example or embodiment may also be used in combination with one or more features of any other of the examples or embodiments, or any combination of any other of the examples or embodiments. Furthermore, equivalents and modifications not described herein may also be employed within the scope of the invention, which is defined in the claims. All articles and patent publications referred to herein are incorporated by reference in their entirety.
Sequences:
SEQ ID No: 1 - BT061 Heavy chain sequence
EEQLVESGGG LVKPGGSLRL SCAASGFSFS DCRMYWLRQA PGKGLEWIGV ISVKSENYGA NYAESVRGRF TISRDDSKNT VYLQMNSLKT EDTAVYYCSA SYYRYDVGAW FAYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SWTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK
SEQ ID No: 2 - BT-061 Light chain sequence
DIVMTQSPDS LAVSLGERAT INCRASKSVS TSGYSYIYWY QQKPGQPPKL LIYLAS ILES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQHSRELPW TFGQGTKVEI KRTVAAPSVF I FPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC
SEQ ID No: 3 - BT-061 Heavy chain variable sequence:
EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWLRQAPGKGLEWIGVISVKSEN YGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAWFA YWGQGTLVTVSS
SEQ ID No: 4 - BT-061 Light chain variable sequence:
DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILE
SGVPDRF SGSGSGTDFTLTIS SLQ AED VAVYYCQHSRELPWTFG QGTKVEIK
SEQ ID No: 5 - BT-061 Heavy chain CDR1 sequence:
DCRMY
SEQ ID No: 6 - BT-061 Heavy chain CDR2 sequence: VISVKSENYGANYAESVRG
SEQ ID No: 7 - BT-061 Heavy chain CDR3 sequence: SYYRYDVGAWFAY
SEQ ID No: 8 - BT-061 Light chain CDR1 sequence: RASKSVSTSGYSYIY
SEQ ID No: 9 - BT-061 Light chain CDR2 sequence: LASILES
SEQ ID No: 10 - BT-061 Light chain CDR3 sequence: QHSRELPWT
Claims
1. An anti-CD4 antibody or fragment thereof for use in treating or preventing asthma in a subject, wherein the anti-CD4 antibody or fragment thereof is capable of activating CD4+CD25+ regulatory T cells.
2. The anti-CD4 antibody or fragment thereof for use according to claim 1, wherein the asthma is allergic asthma.
3. The anti-CD4 antibody or fragment thereof for use according to claim 1 or claim 2, wherein the subject is suffering from one or more symptoms of inflammation.
4. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the use reduces inflammation.
5. The anti-CD4 antibody or fragment thereof for use according to claim 3 or claim 4, wherein the inflammation is allergic inflammation.
6. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the use reduces IL-5, IFNy, TNFa, and/or IL-2 levels in the plasma or sputum of the subject.
7. The anti-CD4 antibody or fragment thereof for use according to claim 6, wherein the use reduces IL-5 levels in the plasma or sputum of the subject.
8. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the use increases levels of TGF-P in the plasma or sputum of the subject.
9. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the use reduces bronchoconstriction in the subject.
10. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the subject is a human subject.
33
11. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the antibody or fragment thereof inhibits the activation of CD4+ effector T cells in vitro in the presence of CD4+CD25+ regulatory T cells.
12. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the anti-CD4 antibody or fragment thereof binds to an epitope on CD4 comprising amino acids 148-154, 164-168, 185, 187, 189-190 and 192.
13. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the anti-CD4 antibody or 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.
14. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the anti-CD4 antibody or fragment thereof comprises a variable heavy chain polypeptide sequence comprising CDR sequences having SEQ ID NOs: 5, 6 and 7, and a variable light chain polypeptide sequence comprising CDR sequences having SEQ ID NOs: 8, 9 and 10.
15. The anti-CD4 antibody or fragment thereof for use according to claim 13 or claim 14, wherein the anti-CD4 antibody or 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.
16. The anti-CD4 antibody for use according to any preceding claim, wherein the anti-CD4 antibody is an IgG antibody.
17. The anti-CD4 antibody for use according to any preceding claim, wherein the anti-CD4 antibody is an IgGl antibody.
18. The anti-CD4 antibody for use according to claim 17, wherein the anti-CD4 antibody has a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2.
19. The anti-CD4 antibody or fragment thereof for use according to any preceding claim wherein the anti-CD4 antibody or fragment thereof is comprised in a pharmaceutical composition.
20. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the antibody or the fragment thereof is to be administered to the subject intravenously, intramuscularly, subcutaneously or via inhalation.
21. The anti-CD4 antibody or fragment thereof for use according to claim 20, wherein the antibody or fragment thereof is to be administered subcutaneously.
22. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the antibody or the fragment thereof is to be administered at a dose of between 25 mg and 200 mg.
23. The anti-CD4 antibody or fragment thereof for use according to claim 22, wherein the antibody or the fragment thereof is to be administered at a dose of between 75 mg and 200 mg.
24. The anti-CD4 antibody or fragment thereof for use according to claim 23, wherein the antibody or the fragment thereof is to be administered at a dose of between 100 mg and 200 mg.
25. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the antibody or fragment thereof is to be administered once every week, once every two weeks, once every three weeks, or once every four weeks.
26. The anti-CD4 antibody or fragment thereof for use according to claim 25, wherein the antibody or fragment thereof is to be administered once every week.
27. The anti-CD4 antibody or fragment thereof for use according to any preceding claim, wherein the anti-CD4 antibody or fragment thereof is contained in a vial, a pre-filled syringe, an autoinjector, or an inhaler.
28. The anti-CD4- antibody or fragment thereof for use according to any preceding claim, wherein the anti-CD4 antibody or fragment thereof is contained in a vial that is arranged for use in an autoinjector or an inhaler.
29. The anti-CD4 antibody or fragment thereof for use according to claim 1, wherein the use comprises contacting CD4+CD25+ regulatory T cells obtained from the subject in vitro with the antibody or fragment thereof so as to activate the CD4+CD25+ regulatory T cells, wherein the use further comprises administering the activated CD4+CD25+ regulatory T cells to the subject.
30. The anti-CD4 antibody or fragment thereof for use according to claim 29, wherein the anti-CD4 antibody or fragment thereof is as defined in any of claims 11 to 19, and/or wherein the subject is a human subject.
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WO2010034590A1 (en) * | 2008-09-29 | 2010-04-01 | Biotest Ag | Composition for treating disease |
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WO2009124815A1 (en) | 2008-03-13 | 2009-10-15 | Biotest Ag | Agent for treating disease |
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