Products for use in the treatment and prevention of allergy and lung conditions / diseases associated with an allergy or with a pathogen-driven immune response

Abstract

The present invention provides an activator of CD4+Foxp3+ regulatory T cells for use in treating or preventing an allergy to Aspergillus or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response in a subject, wherein the activator of CD4+Foxp3+ regulatory T cells binds to domain 2 of CD4.

Description

[0001] PRODUCTS FOR USE IN THE TREATMENT AND PREVENTION OF ALLERGY AND LUNG CONDITIONS / DISEASES ASSOCIATED WITH AN ALLERGY OR WITH A PATHOGEN-DRIVEN IMMUNE RESPONSE

[0002] Technical Field

[0003] The present disclosure relates to medicinal products for use in the prevention or treatment of an allergy to moulds belonging to the Aspergillus genus, a lung condition or lung disease triggered or aggravated by moulds belonging to the Aspergillus genus, (wherein the Aspergillus genus is most prominently represented by the species Aspergillus fumigatus), or a lung condition or lung disease triggered or aggravated by another pathogen, such as a respiratory virus or bacteria. In particular, the lung condition may be allergic bronchopulmonary aspergillosis, Aspergillus- driven aggravation of chronic obstructive pulmonary disease (COPD), or pathogen-driven aggravation of COPD.

[0004] Background

[0005] Aspergillus species are moulds that are abundant in the environment, with notable species being Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus. In particular, the mould Aspergillus fumigatus is widespread in nature and is an opportunistic human pathogen that can cause life-threatening invasive aspergillosis in immunocompromised patients. However, in immunocompetent individuals, inhaling A. fumigatus spores and A. fumigatus infections of the respiratory tract can trigger conditions caused by an over-reaction of the immune system to allergens from the mould and its spores like e.g., allergic bronchopulmonary aspergillosis (ABPA). ABPA has five stages: acute, remission, exacerbation, corticosteroid-dependent asthma, and fibrotic lung disease. Apart from ABPA, allergic overreaction to Aspergillus antigens may also manifest itself in so-called Farmer’s lung or may aggravate chronic obstructive pulmonary disease (COPD).

[0006] Major contributors to the success of A. fumigatus as a pathogen are so-called immune evasion proteins secreted by the fungus, which help to establish an infection in patients whose immune system is not fully functioning. However, the pathogeneses of ABPA, Farmer’s lung and the aggravation of COPD by Aspergillus are not completely understood. Overreactivity towards Aspergillus antigens is commonly diagnosed by skin prick testing and / or determination of Aspergillus- specific IgE and IgG antibodies in the blood. These diseases / conditions constitute major health burdens. For ABPA it is thought that there are more than 4 million sufferers worldwide, with the disease particularly affecting those with the predisposing conditions of asthma and cystic fibrosis. There are very few therapeutic options. Current treatments mainly involve the use of the steroid prednisolone. Anti-fungals (specifically azoles) may also be used, with omalizumab (an anti-IgE antibody) being a further option.

[0007] Farmer’s lung (also known as Farmer’s lung disease or FLD) is a type of hypersensitivity pneumonitis and is one of the most reported causes of occupational health hypersensitivity pneumonitis in Europe. It has been estimated to have a prevalence of around 5 per 1000 in British agricultural workers, in keeping with that reported from other countries (Barber et al., European Respiratory Journal (2017); 50 (suppl. 61): PA1216). Steroids can be used as treatment in the acute stages. However, avoidance of work is thought to be the best measure since patients with five or more symptom recurrences have been found usually to have progressive and significant lung damage (Braun et al., Am. Rev. Respir. Dis. (Feb 1979); 119(2): 185-91).

[0008] COPD is a chronic airway non-inflammatory disease characterised by airway obstruction and alveolar destruction (Guo et al., The Clinical Respiratory Journal (2023); 17(3): 129-138). Aspergillus sensitisation has been found to occur in patients with COPD and in some studies was considered to highly associate with greater symptoms, more frequent exacerbations, and poorer lung function (Hammond et al., BMC Pulm. Med. (2020); 10: 241). Respiratory infections, in particular bacterial and viral infections, are also important triggers of acute exacerbations of COPD (Love et al., Cells (2022); 11(9): 1416).

[0009] As such, there is an on-going need to develop new treatments for these conditions.

[0010] Summary of the Invention

[0011] Accordingly, the present invention provides an activator of CD4+Foxp3+regulatory T cells for use in treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response, in a subject, wherein the activator of CD4+Foxp3+regulatory T cells binds to domain In a second aspect the present invention provides an anti-CD4 antibody or fragment thereof for use in treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response, in a subject, wherein the antibody or fragment thereof comprises a variable heavy chain polypeptide sequence having SEQ ID NO: 3 and a variable light chain polypeptide sequence having at SEQ ID NO: 4 .

[0012] In a third aspect, the present invention provides an anti-CD4 antibody or fragment thereof for use in treating or preventing AB PA, Farmer’s lung, Aspergillus- v\\'cn aggravation of COPD, or a pathogen-driven aggravation of COPD, in a subject, wherein the 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.

[0013] In a fourth aspect, the present invention provides a method of treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response, in a subject, the method comprising administering an effective amount of an activator of CD4+Foxp3+regulatory T cells to the subject, wherein the activator of CD4+Foxp3+regulatory T cells binds to domain 2 of CD4.

[0014] Preferred features of the invention are set out in the dependent claims.

[0015] In particular, the present inventors have surprisingly shown that Enolase 1 protein from A. fumigatus (AfEnol) binds to human CD4 at domain 2, like the CD4+Foxp3+ regulatory T cell activator Tregalizumab. They have further shown that AfEnol binding to CD4+ T cells induces the same distinctive pattern of lymphocyte-specific protein tyrosine kinase (LCK) phosphorylation in CD4+Foxp3+ regulatory T cells (Treg cells) and non-regulatory T cells (Tconv cells), i.e., CD4+ T cells that are Tregs depleted, that is induced by binding of Tregalizumab to these cells. This distinctive pattern of LCK phosphorylation is associated with Tregalizumab ’s ability specifically to activate Treg cells and not Tconv cells. These findings provide basis for a rationale that the activation of Tregs by AfEnol in most individuals leads to control of the body’s immune reaction to allergens from Aspergillus species, such as A. fumigatus, such that a hypersensitivity reaction to the allergen, and subsequent disease / condition such as AB PA, does not develop. It also reveals a new therapeutic mechanism for sufferers of such allergic reactions, and associated lung disease or conditions, involving enhancing the signal provided by the binding of AfEnol to domain 2 of CD4 in CD4+Foxp3+ regulatory T cell using other activators of CD4+Foxp3+ regulatory T cells, including Tregalizumab.

[0016] The present inventors also show with Tregalizumab that activators of Treg cells are able to reduce effector cytokine release from PBMCs after antigenic recall, indicating that they are able to inhibit effector cytokine release from memory T cells. This provides the opportunity to use the activators to address pathogen-driven aggravation that occurs in other lung diseases or conditions such as COPD.

[0017] Brief Description of the Drawings

[0018] 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:

[0019] Figure 1 shows the amino acid sequences of the heavy chain and light chain of the antibody designated BT-061.

[0020] Figures 2A and 2B show results of flow cytometry analysis of Treg cells (Figure 2B) isolated from peripheral blood mononuclear cells (PBMCs) (Figure 2A) of healthy blood donors using Miltenyi Biotec’s ‘CD4+CD25+ Regulatory T Cell Isolation Kit, human’ (catalogue number: 130-091-301), as described further below. Cells were stained for CD4, CD25 and Foxp3 and were CD4 gated.

[0021] Figures 3A and 3B show that AfEnol binds human CD4. AfEnol (1 pg / ml) was incubated with human CD4-coated Dynabeads M-270 Epoxy for 20 minutes at room temperature. The binding of AfEnol to human CD4 was detected by indirect immunofluorescence. Data were pooled from n=4 independent experiments. The bar graphs show normalized data (MFI positive control = 1). 0 AfEnol: No addition of AfEnol (Neg cntrl). The ability of different anti-CD4 antibodies to inhibit binding of AfEnol to human CD4 was assessed - anti-CD4 mAbs : RPA- T4, Tregalizumab, and OKT-4, at Figure 3A) 1 pg / ml and Figure 3B) 10 pg / ml. Values were tested with Dunnef s multiple comparison test (* p < 0.05, ** p < 0.01, **** p < 0.0001). Figures 4A to D shows the results of LCK phosphorylation experiments. Figure 4A shows LCK phosphorylation at Tyr394 in Treg and Tconv cells after stimulation with medium alone (-ve control), RPA-T4 anti-CD4 antibody, HIT3a anti-CD3 antibody, H2O2, Tregalizumab, and AfEnol (Kruskal Wallis test; ** p < 0.005). For each condition both a specific staining and an FMO control was carried out. Median fluorescence intensities (MFI) obtained with the specific stainings were divided by those of FMO controls. This MFI ratio was then normalised to the MFI ratio of cells cultured in medium alone. Figures 4B to 4D show that phosphorylation of LCK at Tyr394 induced by H2O2 (Figure 4B; coefficient r: 0.6029; p-value: 0.0325), Tregalizumab (Figure 4C; coefficient r: 0.7093; p-value: 0.0108), and AfEnol (Figure 4D; coefficient r: 0.8613; p-value: 0.0007) were positively correlated for Treg and Tconv. Each symbol represents the result of one individual donor.

[0022] Figures 5A to D shows the results of LCK phosphorylation experiments. Figure 5A shows LCK phosphorylation at Tyr505 in Treg and Tconv cells after stimulation with medium alone (-ve control), RPA-T4 anti-CD4 antibody, HIT3a anti-CD3 antibody, H2O2, Tregalizumab, and AfEnol (Kruskal Wallis test; *** p < 0.0005). For each condition both a specific staining and an FMO control was carried out. Median fluorescence intensities (MFI) obtained with the specific stainings were divided by those of FMO controls. This MFI ratio was then normalised to the MFI ratio of cells cultured in medium alone. Figures 5B to 5D show that phosphorylation of LCK at Tyr505 induced by H2O2 (Figure 5B; coefficient r: 0.1163; p-value: 0.3919), Tregalizumab (Figure 5C; coefficient r: -0.2658; p-value: 0.2623), and AfEnol (Figure 5D; coefficient r: -0.1338; p-value: 0.3761) were not correlated for Treg and Tconv. Each symbol represents the result of one individual donor.

[0023] Figure 6 shows the results of cell culture experiments with 4- IBB expression as the read-out. Expression of 4- IBB by Tconv and Tregs was determined by flow cytometry after stimulation with medium alone, RPA-T4 anti-CD4 antibody, HIT3a anti-CD3 antibody, Tregalizumab, AfEnol, and the combination of Tregalizumab and AfEnol. Each dot represents the result of a single experiment with Tconv and Tregs from one donor used in parallel in each experiment. Kruskal Wallis test; * p < 0.05, ** p < 0.005, *** p < 0.0005.

[0024] Figures 7 A to 71 show the results of cell culture experiments to determine the ability of a preculture step with Tregalizumab to impact the cytokine secretion profile of a PBMC culture subsequently stimulated with: medium only (as a -ve control); anti-CD3 monoclonal antibody (as a +ve control); the mycobacterial antigen (PPD); and an A.fumigatus-deriNed peptide pool. All figures show the change from medium control (in pg / ml) on the y-axis for: Figure 7A - IFNy; Figure 7B - IL-5; Figure 7C - IL-13; Figure 7D - IL-17A; Figure 7E - IL-22; Figure 7F - IL-2; Figure 7G - IL- 10; Figure 7H - IL-6; Figure 71 - TNFa. Significance was assessed using a paired one-tailed Mann- Whitney U-test; * p < 0.05; ** p < 0.01; ns: not significant.

[0025] Detailed Description

[0026] 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”.

[0027] 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.

[0028] As described above, the present inventors have identified a new therapeutic mechanism for treating an allergy or overreaction of the immune system to Aspergillus or to another pathogen, such as viruses or bacteria responsible for respiratory tract infections. Accordingly, the present invention is directed to an activator of CD4+Foxp3+regulatory T cells for use in treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response, in a subject, wherein the activator of CD4+Foxp3+regulatory T cells binds to domain 2 of CD4. The present invention is also directed to a method of treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response, in a subject, the method comprising administering an effective amount of an activator of CD4+Foxp3+regulatory T cells to the subject, wherein the activator of CD4+Foxp3+regulatory T cells binds to domain 2 of CD4.

[0029] The present invention is further directed to an anti-CD4 antibody or fragment thereof for use in treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response, in a subject, wherein the 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. This aspect can also be referred to as a method of treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response, in a subject, the method comprising administering an effective amount of an anti-CD4 antibody or fragment thereof to the subject, wherein the 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.

[0030] The present invention is still further directed to an anti-CD4 antibody or fragment thereof for use in treating or preventing AB PA, Farmer’s lung, Aspergillus- v\\'cn aggravation of COPD, or a pathogen-driven aggravation of COPD, in a subject wherein the 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. This aspect can also be referred to as a method of treating or preventing AB PA, Farmer’s lung, Aspergillus- v\\'cn aggravation of COPD, or a pathogen-driven aggravation of COPD, in a subject, the method comprising administering an effective amount of an anti-CD4 antibody or fragment thereof to the subject, wherein the 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.

[0031] Regulatory T cells are a subpopulation of CD4+T cells that are known to modulate the immune system. In particular, activated regulatory T cells are known to be important in maintaining tolerance to self-antigens (Sakaguchi et al., Immunological Reviews (2001); 182: 18-32), and in regulating immune homeostasis and inflammation.

[0032] The family of Tregs 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. Tregs are generally characterised by the expression of CD4 and CD25 surface biomarkers, and also by the transcription factor Foxp3. Herein regulatory T cells (Tregs) are referred to as CD4+Foxp3+regulatory T cells (although they can also be considered as CD4+CD25+Foxp3+regulatory T cells). It is known that major contributors to the success of fungi of the genus Aspergillus, particularly A. fumigatus, as a pathogen (causing infections) are so-called immune evasion proteins secreted by the fungus. Among these, enolase 1 (Enol) sticks out as it is one of the most abundantly expressed fungal proteins capable of interfering with complement activation. However, Enol of A. fumigatus (AfEnol) has also been shown to constitute an antigen which preferentially activates human Tregs over conventional non-Treg CD4+T cells (Tconv) (Bacher et al., Cell (Nov. 3, 2016); 167(4): 1067- 1078) suggesting that Enol contributes to preventing overshooting immune responses o Aspergillus antigens. Treg cell activation by Enol might, thus, constitute a mechanism that prevents allergic diseases developing in the vast majority of individuals. A. fumigatus enolase 1 (Enol) protein has 73% identity with enolase 1 from Candida albicans, which is a related fungal pathogen. Mapping of CaEnol binding to CD4 has previously revealed that domain 3 of CD4 is critically involved in the CaEnol-CD4 interaction (Daud et al., Eur. J. Immunol., 2023; 53: 2250284).

[0033] However, as noted above, the present inventors have now surprisingly shown that A. fumigatus enolase 1 (AfEnol) interacts with domain 2 of CD4 and its binding is blocked by the agonistic anti-CD4 antibody Tregalizumab (as shown in Figure 3). Tregalizumab is known to be a selective activator of Treg cells (Kbnig et al., Front. Immunol., 2016; 7:11); binding of Tregalizumab to CD4 of Treg cells in vitro causes the activation of intracellular signalling pathways that lead to the activation of Treg cells. This specific functionality of Tregalizumab has been associated with the recognition of a unique epitope on domain 2 of the CD4 molecule that is not recognised by other anti-CD4 antibodies (Helling et al., Immunology and Cell Biology, 2015; 93: 396-405).

[0034] The present inventors have further shown that AfEnol and Tregalizumab both induced distinctive patterns of LCK phosphorylation in Tregs and Tconv (as shown in Figures 4A to D and 5A to D). In Tregs preferential phosphorylation of Tyr394 in LCK occurs. This phosphorylation is associated with LCK activation. Surprisingly, in Tconv Tyr394 phosphorylation is seen but also Tyr505 phosphorylation, the latter being associated with LCK inhibition. This phosphorylation pattern provides a biochemical rationale for the preferential activation of Tregs over Tconv by Tregalizumab and suggests that AfEnol acts in a similar fashion. Still further, the inventors have shown that activation of Tregs by Tregalizumab, but not AfEnol, is associated with expression of the co- stimulatory marker 4- IBB (see Figure 6) which, in contrast to CD40L, is otherwise known to be upregulated on Tregs after antigenic stimulation including fungal antigens (Schoenbrunn et al., J. Immunol. (2012, Dec. 15); 189(12):5985-94; Bacher et al., Mucosal Immunol. (July 2014); 7(4):916-28). Without wishing to be bound by theory, the present inventors consider that due to prolonged / more intensive signalling induced by Tregalizumab versus AfEnol, only Tregalizumab caused upregulation of the costimulatory molecule 4- IBB on Tconv and Treg. The results therefore support the rationale that the immunoregulatory effects of AfEnol which do not suffice to protect patients from ABPA might be enhanced by treatment with Tregalizumab and other Treg activators binding domain 2 of CD4.

[0035] Moreover, noting the high sequence similarity between the Enol protein of different medicallyrelevant Aspergillus species (as highlighted in the Examples section below), the rationale applies to treating or preventing allergic reaction to Aspergillus in a subject, and to treating or preventing a lung condition or a lung disease associated with an allergy to Aspergillus .

[0036] This is additionally supported by the demonstration herein that Tregalizumab is able to inhibit effector cytokine release from memory T cells after restimulation with pathogen antigens, showing that activators of Tregs can in addition be used to treat pathogen-driven aggravation of lung diseases or conditions such as COPD.

[0037] Accordingly, the present invention relates to the above-mentioned medical use of an activator of CD4+Foxp3+regulatory T cells, and the above-mentioned methods of treatment and prevention. The activator (or antibody / antibody fragment described below) is for administration to the subject, i.e., for treatment or prevention of the disease in vivo. In particular, the activator (or antibody / antibody fragment described below) may be for intravenous, subcutaneous, or intramuscular use, or for use as an inhalative.

[0038] Activators of CD4+Foxp3+regulatory T cells are known in the art. In particular, the activator may be an anti-CD4 antibody or a fragment thereof (e.g. as described in patent application publication no. WO 2011 / 064407). The activator is “capable of activating CD4+Foxp3+regulatory T cells”. In one example this can be defined as the ability of the activator, e.g. the anti-CD4 antibody or antibody fragment thereof, to activate T cells expressing CD4+and Foxp3+in vitro, preferably such that the activated CD4+Foxp3+regulatory T cells are able to inhibit the production of one or more pro- inflammatory cytokine in a PBMC culture after TCR stimulation, in particular in a culture containing T effector cells. The pro-inflammatory cytokine whose production is inhibited may be one or more of IL-5, IL-4 and / or IL-13, and preferable is IL-5.

[0039] A testing method for such activity may comprise pre-incubating CD4+Foxp3+regulatory T cells obtained from a donor (e.g. a healthy human 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+Foxp3+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+Foxp3+regulatory T cells may be obtained from a healthy individual, or an individual suffering from a disease according to the present invention as described above. In this assay an activator, e.g., an anti-CD4 antibody or fragment thereof, that is capable of activating CD4+Foxp3+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+Foxp3+cells have not been pre-incubated with the activator. Preferably the activator being tested leads to the same reduction + / - 25%, more preferably + / - 10%, as a positive control with the BT-061 (Tregalizumab) antibody, or an anti-CD4 antibody having the same variable heavy and variable light chain regions as the BT-061 (Tregalizumab) antibody, which is described herein.

[0040] The ability of the activator to activate CD4+Foxp3+regulatory T cells can also be determined by methods described in the art, such as those described in WO2011 / 064407. For example, the ability can be assayed by examining the suppressive activity of the CD4+Foxp3+regulatory T cells after incubation with the activator by co-culturing the CD4+Foxp3+regulatory T cells with CD4+CD25‘ effector T cells. Activated CD4+Foxp3+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. As noted above, the activator is one that specifically binds to domain 2 of CD4. In particular, the CD4 is human CD4. Human CD4 is encoded on chromosome 12 and belongs to the immunoglobulin (Ig) superfamily. The 458 amino acid sequence of the protein is shown in SEQ ID No: 16. The protein has 4 immunoglobulin domains (domain 1 to domain 4, or DI to D4), a transmembrane domain and a cytoplasmic tail domain. The UniProt entry P01730 provides the domain structure of CD4 as shown below in Table 1. The first 25 amino acids are a signal peptide, which is cleaved off in the biologically active form. Positions 26 through 396 constitute the extracellular domain, which is followed by the transmembrane region, positions 397 through 418. The last part, positions 419 to 458, is the cytoplasmic domain.

[0041] Table 1 Table showing the domain structure of CD4 (according to UniProt P01730)

[0042] The ability of an activator to bind to domain 2 of CD4 may be determined using a competitive binding assay, e.g., using anti-CD4 antibodies that bind to one of each of domains 1, 2, and 3. In particular, such a method is described in Example 1 below, as this was used to demonstrate the binding of Enol from A. fumigatus to domain 2 of CD4. Further determination of binding location be completed with X-ray crystallography or other epitope mapping technologies that are known in the art (as mentioned below).

[0043] The activator 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 BT-061 (Tregalizumab) 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.

[0044] The activator is preferably an antibody, an antibody fragment, or a multispecific or bispecific antibody that binds to the Tregs. A multispecific or bispecific antibody is one which targets at least one other antigen and can be used to assist in bringing the activated Treg cell into contact with the cells on which it has an effect.

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

[0046] 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.

[0047] 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.

[0048] However, as shown by the Examples below, purified Tregs were successfully activated by AfEnol and Tregalizumab without any form of crosslinking, suggesting that an ,FcR-dead‘ version of an antibody like a LALAPG-mutated version can also be considered as an alternative to the unmutated IgGl molecule. Accordingly, in the present invention the antibody may comprise a modified Fc region with a reduced or ablated effector function. Other suitable modifications to reduce or remove effector functions are known in the art (such as those described in Wang et al. (Protein Cell, 2018; 91(1): 63-73. By “modified Fc region” it is meant that a native Fc region has been mutated or engineered in order to reduce or remove the effector function. 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.

[0049] The activator can be an anti-CD4 antibody or antibody fragment thereof, preferably an antihuman CD4 antibody or fragment thereof. As will be appreciated the anti-CD4 antibody or antibody fragment is non-depleting CD4+ antibody / antibody fragment i.e., does not deplete CD4+ cells.

[0050] Preferably the antibody is a humanized anti-CD4 antibody or fragment thereof which is the BT- 061 antibody, or a variant or fragment thereof. In particular, the BT-061 antibody (Tregalizumab) has V domains defined by the following polypeptide sequences (and which are described further in the “Sequences” section below):

[0051] - H chain V domain: EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWLRQA PGKGLEWIGVISVKSENYGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVY YCSAS YYRYDVGAWFAYWGQGTLVTVSS (SEQ ID NO: 3)

[0052] - L chain V domain:

[0053] DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQ

[0054] KPGQPPKLLIYLASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPWT FG QGTKVEIK (SEQ ID NO: 4).

[0055] 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.

[0056] 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.

[0057] In addition or alternatively the antibody or antibody fragment is one which has the CDR sequences of BT-061 (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.

[0058] The manner in which BT-061 (Tregalizumab) 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.

[0059] 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 WO2011 / 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 BT-061 antibody described herein. Epitope mapping of antibodies can be done by methods that are known in the art (See e.g., Dang et al., “Epitope mapping of monoclonal antibodies: a comprehensive comparison of different technologies”, MARS 2023, vol. 15, no. 1, 2285285).

[0060] The description above regarding the anti-CD4 antibodies and fragments thereof also apply to the anti-CD4 antibodies and fragments thereof based on the sequences of BT-061 (Tregalizumab).

[0061] In one specific aspect the invention provides an anti-CD4 antibody for use in treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus (e.g., AB PA, Farmer’s lung or Aspergillus-'mduccd aggravation of COPD) or associated with a pathogen-driven immune response (e.g., a pathogen-driven aggravation of COPD), 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.

[0062] In a further specific aspect, the invention provides an anti-CD4 antibody or fragment thereof for use in treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus (e.g., AB PA, Farmer’s lung or Aspergillus-induccd aggravation of COPD) or associated with a pathogen-driven immune response (e.g., a pathogen- driven aggravation of COPD), in a subject, wherein the antibody or fragment thereof comprises a heavy chain variable domain with at least 85% (preferably at least 95%) sequence identity with SEQ ID NO: 3 and which includes the CDR sequences of SEQ ID Nos: 5 to 7, and a light chain variable domain with at least 85% (preferably at least 95%) sequence identity with SEQ ID NO: 4 and which includes the CDR sequences of SEQ ID Nos: 8 to 10. In particular, the antibody or antibody fragment is one that is capable of activating CD4+Foxp3+regulatory T cells, as described above.

[0063] The antibody or antibody fragment referred to herein may be manufactured using an 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 Sp2 / 0, HeLa, CHO, 3T3, C127, BHK, COS, etc.

[0064] 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.

[0065] The activator / antibody / 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.

[0066] As described above, the activator, (or antibody or fragment thereof) is for use in treating or preventing an allergy to Aspergillus, or a lung disease or lung condition associated with an allergy to Aspergillus. An allergy (or overreactivity) towards Aspergillus antigens can be diagnosed by skin prick testing and / or determination of Aspergillus-specific IgE and IgG antibodies in the blood.

[0067] A lung disease or lung condition associated with an allergy to Aspergillus is one in which the overreaction of the subject’s immune system o Aspergillus antigens leads to Aspergillus- v'wcn aggravation of the disease or condition. In particular, allergic bronchopulmonary aspergillosis (AB PA), Farmer’s lung and Aspergillus Adduced aggravation of COPD are conditions in which fungi of the genus Aspergillus, particularly A. fumigatus, cause an allergic or inflammatory reaction in the lungs. Overreactivity towards Aspergillus antigens is commonly diagnosed by skin prick testing and / or determination of Aspergillus-s citic IgE and IgG antibodies in the blood. In addition, total IgE will be very high and blood eosinophilia might be present, particularly during flares. The pathological changes to the lung together with a proven overreactivity towards Aspergillus antigens leads to the diagnosis of the conditions / diseases mentioned above.

[0068] Further a lung disease or lung condition associated with a pathogen-driven immune response is one in which the overreaction of a subject’s immune system in response to the pathogen leads to an inflammatory reaction in the lungs. This reaction may aggravate or exacerbate an existing lung disease or condition. The pathogen may be a bacteria and / or a virus, specifically a bacteria and / or virus that causes a respiratory infection, preferably a chest infection. As noted above, the lung disease or lung condition associated with a pathogen-driven immune response is preferably a pathogen-driven aggravation of COPD (and can more preferably be a viral and / or bacterial-driven aggravation of COPD). In particular, it is known that a significant proportion of acute exacerbations / aggravations of COPD are associated with viral and / or bacterial infections (Love et al., Cells (2022); 11(9): 1416). Subjects typically present with symptoms of the chest infection, including laboured breathing and increase sputum volume and purulence. In the medical uses described herein, the subject can be any mammal, including a human, nonhuman primate, or a domesticated mammal such as a cat, dog, or rodent. However, preferably the subject is a human. The subject can be six years or older, or sixteen years or older.

[0069] According to the invention the activator (or antibody or fragment thereof) 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 activator (or antibody or fragment thereof) is administered subcutaneously or via inhalation. Most preferably the activator is administered subcutaneously.

[0070] Depending on the route of administration, the activator (or antibody or antibody fragment) may be comprised in a vial, a pre-filled syringe, an autoinjector, or an inhaler (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.

[0071] 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.

[0072] The activator (or 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.

[0073] Suitable doses for the activator (or antibody or fragment thereof) can be determined based on established methods. Where the activator is an anti-CD4 antibody or fragment thereof, the antibody or antibody fragment may be administered in a dose of between 10 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.

[0074] 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.

[0075] The following are intended as examples only and do not limit the present disclosure.

[0076] EXAMPLES

[0077] Antibody

[0078] The anti-CD4 antibody (BT-061) 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 BT-061 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.

[0079] The BT-061 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.

[0080] The BT-061 antibody has been given the generic name Tregalizumab.

[0081] Expression and purification of AfEnol

[0082] Recombinant AfEnol, i.e., Enol from A. fumigatus, was expressed and purified as previously described (Dasari et al., Frontiers in Immunology, November 2019, volume 10, article 2573). Results obtained with recombinant AfEnol most likely apply to other medically relevant Aspergillus species as the amino acid sequences of Enol expressed by A. fumigatus, A. flavus, A. niger, A. terreus and A. nidulans are > 91.8% identical. (The amino acid sequences of these Enol proteins are SEQ ID Nos: 11 to 15, respectively, as shown below.)

[0083] Isolation of CD4+ Foxp3+ regulatory T cells (Treg cells) and non-regulatory CD4+ T cells (Tconv cells)

[0084] Treg and Tconv cells were isolated from peripheral blood mononuclear cells of healthy blood donors (n=l 1) using Miltenyi Biotec’s ‘CD4+CD25+ Regulatory T Cell Isolation Kit, human’ (catalogue number: 130-091-301) according to the manufacturer’s instructions. In brief, an antibody cocktail recognizing CD8, CD14, CD15, CD16, CD19, CD36, CD56, CD123, TCRy / 5 and CD235a (glycophorin A) was used to obtain ‘untouched’ CD4+T cells. In the second step Treg cells were labelled via CD25 and positively selected, while the CD25-negative fraction constituted Tconv cells. Representative data of purified Treg and Tconv are shown in Figure 2B.

[0085] Example 1 -AfEnol recognizes domain 2 of the extracellular part of CD4

[0086] We have previously shown in a FACS-based detection system that Enol from A. fumigatus (AfEnol) binds to CD4-coated beads confirming the molecular interaction between this protein and human CD4 (Daud et al., Eur. J. Immunol., 2023; 53: 2022284). Human CD4 has four different extracellular domains. In order to map the location of binding of AfEnol to CD4 we performed a competitive binding assay using anti-CD4 antibodies that bind to one of each of domains 1, 2 and 3.

[0087] Experimental method

[0088] AfEnol (1 pg / ml) was incubated with human CD4-coated Dynabeads M-270 Epoxy for 20 minutes at room temperature. The binding of AfEnol to human CD4 was detected by indirect immunofluorescence. Data were pooled from n=4 independent experiments. The bar graphs in Figure 3 show normalized data (MFI positive control = 1). 0 AfEnol: No addition of AfEnol (Neg cntrl). The binding of AfEnol to human CD4 was inhibited by different anti-CD4 mAbs A) 1 pg / ml and B) 10 pg / ml. Values were tested with Dunnet's multiple comparison test (* p < 0.05, ** p < 0.01, **** p < 0.0001).

[0089] Results Human CD4 contains four extracellular Ig-like domains, known as domains 1 to 4. As shown in Figure 3, the results indicate that AfEnol also directly bound to the extracellular part of recombinantly expressed and purified human CD4 with the AfEnol -CD4 interface comprising parts of domains 1 and 2 of CD4. Binding of AfEnol to CD4 was partially blocked by Tregalizumab which itself binds domain 2 of CD4 (Helling et al., Immunology and Cell Biology (2015); 93: 396-405). (This is in contrast to Candida albicans enolase 1 protein (CaEnol). Previous work has shown that CaEnol binds extracellular domain 3, but not domains 1 and 2 of human CD4 (Daud et al., Eur. J. Immunol., 2023; 53: 2022284).) Anti-CD4 antibody OKT- 4 was found not to block binding of AfEnol to CD4. Anti-CD4 antibody RPA-T4 and Tregalizumab blocked binding of AfEno to CD4 with the results showing that Tregalizumab had a more pronounced tendency to block binding than RPA-T4. OKT-4 binds to domain 3 of CD4, while RPA-T4 is indicated in the published literature to bind to domain 1 (as noted by Helling et al., Immunology and Cell Biology (2015); 93: 396-405). Based on these results we conclude that AfEnol binding to CD4 at a similar location to Tregalizumab.

[0090] Example 2 -Effect of CD4 binding on LCK phosphorylation in purified human CD4+T cell subsets

[0091] Physiologically, CD4 stabilises peptide / HLA - T cell receptor (TCR) interactions during T cell activation and recruits the lymphocyte- specific protein tyrosine kinase (LCK) to the TCR complex. LCK activation is associated with phosphorylation of Tyrosine 394 which dominates over the inhibitory phosphorylation of Tyrosine 505. To study the biochemical consequences of fungal Enol binding to CD4 we incubated purified human CD4+Treg and Tconv cells with AfEnol in solution. For comparison as positive controls we used the anti-CD3 mAb HIT3a, H2O2, and the anti-CD4 mAbs RPA-T4 and Tregalizumab.

[0092] Experimental method

[0093] Purified Treg or Tconv cells (l x 105) were incubated with AfEnol (1 pg / ml), H2O2 (5 mM), anti-CD4 mAb RPA-T4 (BD Biosciences, 1 pg / ml), or anti-CD3s mAb HIT3a (BioLegend, 1 pg / ml) in pre- warmed RPMI+ medium in a 96- well round-bottom plate (30 min, 37 °C). After the incubation, cells were transferred to a 96- well V-bottom plate, fixed with 2% paraformaldehyde (20 min, room temperature), washed with PBS, and then, permeabilized with 96% methanol (-20°C overnight). The next day, cells were split into two wells, and intracellular staining was performed with an anti-phospho-LCK(Tyr505) (clone A17013A) or an anti- phospho-LCK(Tyr394) (clone A18002D) mAb (45 min, room temperature, in the dark).

[0094] Results

[0095] The results are shown in Figures 4A to 4D (phosphorylation of LCK at Tyr394) and in Figures 5A to 5D (phosphorylation of LCK at Tyr505). Comparing purified human Treg and Tconv showed that both AfEno 1 and Tregalizumab induced phosphorylation of LCK at Tyr394 in both CD4+T cell subsets of some donors (see Figure 4A to 4D), while phosphorylation of Tyr505 of LCK induced by AfEno 1 and Tregalizumab was only seen in Tconv of some donors but not in Treg (see Figure 5A to 5D). This provides detail of the mechanism behind AfEno l’s activation of Tregs.

[0096] Example 3 -Effect of binding on expression of activation marker in CD4+ T cell subsets

[0097] To investigate the further downstream effects of AfEno 1 on T cell activation, we performed experiments to determine expression of 4- IBB (as a marker of T cell activation) by Treg and Tconv after CD4 binding by AfEno 1 and Tregalizumab.

[0098] Experimental Method.

[0099] Purified human Treg and Tconv cells (5 x 104cells / well) were cultured in the absence or presence of AfEno 1, Tregalizumab, the combination of AfEno 1 and Tregalizumab or anti-CD4 mAb RPA-T4 (each reagent at 1 pg / ml) for 48 h in the presence of recombinant human Interleukin-2 (Proleukin®, 30 U / ml). At the end of the culturing period the cells were stained with a viability dye as well as for Foxp3, CD25, and 4- IBB expression and analyzed by flow cytometry.

[0100] Results

[0101] The results for 4- IBB expression are shown in Figure 6. In contrast to AfEno 1, Tregalizumab induced expression of this costimulatory in Tconv and Treg, which was still seen when AfEno 1 and Tregalizumab were added in parallel (four- fold molar excess of AfEno 1 over Tregalizumab), i.e., AfEnol did not inhibit 4-1BB induction by Tregalizumab. From these results, and without wishing to be bound by theory, we conclude that Tregalizumab is able to activate Tregs more strongly than AfEnol. Example 4 -Effect of Tregalizumab on PBMC antigen recall response

[0102] Assays were performed with PBMC cell cultures to investigate the ability of Tregalizumab to dampen “allergic” T helper 2(Th2)-like responses to A. fumigatus T cell recall antigens.

[0103] Experimental Method.

[0104] Conditions for the assays were first tested to optimise the amount of Tregalizumab required for Treg activation in the context of the PBMC cultures. After incubation (day 1 or day 2) FACS analysis of the re-cultured cells was done (CD4, CD3, 4- IBB, CD25 extracellularly and Foxp3, CD40L, Ki-67, CTLA-4 intracellularly) to determine Treg activation. It was determined that 0.1 g / ml was sufficient based on analysis of, inter alia, down-modulation of CD4 expression on CD4+ T cells, and the increase in 4- IBB expression by Tregs as a result of activation. (Down-modulation of CD4 expression was also confirmed during subsequent assays using FACS analysis (data not shown).) The optimisation showed that the presence of IL-2 was not required for the assay. It was noted that the PBMCs used should be freshly prepared for the assay(or any preculture restricted to a minimum).

[0105] Purified human PBMC of healthy blood donors (2 x 105cells / well; 96 well U-bottom plate) were pre-incubated with 0.1 pg / ml Tregalizumab (starting day 0) for one day, and a parallel cell culture without Tregalizumab was used as a control.

[0106] Restimulation was then performed (3 wells per condition); the following reagents were added to an amount of 10% of the total volume: medium only (-ve control), anti-CD3 mAb in solution (+ve control; clone: HIT3a, LEAF purified; Biolegend Cat.# 300314; final concentration: 1 pg / ml), PPD (Mycobacterial antigen; Pharmore Cat.# 302956; final concentration: 10 pg / ml), and A. fitmigalus-dcvAcd peptide pool (Miltenyi Biotec; PepTivator A. fumigatus crfl, Aspf22, SOD, Cat.# 130-096-775, 130-099-776, 130-097-288; final concentration: 0.3 nmol / ml each); set-up in triplicates (4 x 3 x 2 = 24 wells per donor).

[0107] On day 4 the cell culture supernatants were collected and stored at -80°C. Analysis of culture supernatants to determine cytokine levels was performed using LEGENDplex assay: IFNy, IL- 2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL- 22 and TNFa. For further analyses, median values of cytokine concentrations of the triplicate cultures were used and values obtained after incubation of PBMC with medium only for four days were substracted ('Delta medium control (pg / ml)'). In total 11 donors were assayed.

[0108] Results

[0109] The results are shown in Tables 2 and 3 below, and in Figures 7A to 71.

[0110] Table 2 shows the number of PBMC culture (from 11 different donors) that produced a cytokine response after pre-incub ation with culture medium only (i.e. no Tregalizumab) followed by (re)stimulation with PPD or A.fumigatus antigen, or after stimulation with anti-CD3 monoclonal antibodies. The table shows that responses across all cytokine types were detectable. The donors whose PBMC cultures show a response (responder donors) are taken to have had previous exposure to Mycobacteria / A.fumigatus antigens such that the exposure to these antigens in the assay can be considered a restimulation.

[0111] Table 2. Number of donors (and % of total 11 donors) producing a cytokine response.

[0112] Figures 7 A to 71 show the cytokine responses (of the cytokines listed in Table 1) from the responder donors after pre-incub ation with Tregalizumab or culture medium followed by restimulation with PPD or A.fumigatus antigen, or after stimulation with anti-CD3 monoclonal antibodies. The results show that in the presence of Tregalizumab, effector cytokine release by PBMC after antigenic recall (Mycobacteria: PPD, A. fumigatus antigen) was reduced. Specifically, after antigenic recall with the Mycobacterial antigen PPD, release of Thl, Th2 and Thl7 cytokines was detected and all were reduced by the preincubuation of the PBMC cultures with Tregalizumab. The same holds true for IL- 17 after antigenic recall against A. fumigatus,' for Thl responses there was a similar trend; Th2 responses against A. fumigatus antigens were not detected in enough donors for further analyses.

[0113] The data from Figures 7 A to 71 is summarised below in Table 3, showing that the presence of Tregalizumab is able to significantly reduce the release of a number of different cytokines.

[0114] Table 3. Summary of data from Figures 7A to 71 showing reduction in cytokine release after preincubation with Tregalizumab across the different cytokines measured. (Paired one-tailed Mann-Whitney U-test; * p < 0.05; ** p < 0.01; "- " too few responders for statistical signficance; "x“ no reduction by Tregalizumab.

[0115] Together, the results in these examples support the medical uses of Tregalizumab and other Treg activators that are described herein.

[0116] 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.

[0117] SEQUENCES:

[0118] SEQ ID No: 1 - BT-061 Heavy chain sequence

[0119] 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

[0120] SEQ ID No: 2 - BT-061 Light chain sequence

[0121] DIVMTQSPDS LAVSLGERAT INCRASKSVS TSGYSYIYWY QQKPGQPPKL LIYLASILES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQHSRELPW TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

[0122] SEQ ID No: 3 - BT-061 Heavy chain variable sequence

[0123] EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWLRQAPGKGLEWIGVISVKSEN YGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAWFA YWGQGTLVTVSS

[0124] SEQ ID No: 4 - BT-061 Light chain variable sequence

[0125] DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILE

[0126] SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPWTFG QGTKVEIK

[0127] SEQ ID No: 5 - BT-061 Heavy chain CDR1 sequence

[0128] DCRMY

[0129] SEQ ID No: 6 - BT-061 Heavy chain CDR2 sequence

[0130] VISVKSENYGANYAESVRG SEQ ID No: 7 - BT-061 Heavy chain CDR3 sequence SYYRYDVGAWFAY

[0131] SEQ ID No: 8 - BT-061 Light chain CDR1 sequence

[0132] RASKSVSTSGYSYIY

[0133] SEQ ID No: 9 - BT-061 Light chain CDR2 sequence

[0134] LASILES

[0135] SEQ ID No: 10 - BT-061 Light chain CDR3 sequence QHSRELPWT

[0136] SEQ ID No: 11 - A. fumigatus Enol sequence

[0137] >sp|Q96X30|ENO_ASPFU Enolase OS=Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC Al 100) OX=330879 GN=enoA PE=1 SV=3

[0138] MPISKIHARSVYDSRGNPTVEVDVVTETGLHRAIVPSGASTGQHEAHELRDGDKTQW GGKGVLKAVKNVNETIGPALIKENIDVKDQSKVDEFLNKLDGTANKSNLGANAILGV SLAVAKAGAAEKGVPLYAHISDLAGTKKPYVLPVPFQNVLNGGSHAGGRLAFQEFM

[0139] IVPDSAPSFSEALRQGAEVYQKLKALAKKKYGQSAGNVGDEGGVAPDIQTAEEALDL ITEAIEQAGYTGKIKIAMDVASSEFYKADVKKYDLDFKNPESDPSKWLTYEQLADLY KSLAAKYPIVSIEDPFAEDDWEAWSYFYKTSDFQIVGDDLTVTNPGRIKKAIELKSCN

[0140] ALLLKVNQIGTLTESIQAAKDSYADNWGVMVSHRSGETEDVTIADIAVGLRSGQIKT GAPCRSERLAKLNQILRIEEELGENAVYAGSKFRTAVNL

[0141] SEQ ID No: 12 - A. flavus Enol sequence

[0142] >tr|A0A5N6GF46|A0A5N6GF46_ASPFL Enolase OS=Aspergillus flavus OX=5059

[0143] GN=BDV35DRAFT_371256 PE=3 SV=1

[0144] MPISKIHARSVYDSRGNPTVEVDVVTETGLHRAIVPSGASTGQHEAHELRDGDKTHW

[0145] GGKGVLKAVENVNKTIAPAVLKENLDVKDQSKVDEFLKKLDGSANKSNLGANAILG

[0146] VSLAIAKAGAAEKGVPLYAHISDLAGTKKPYVLPVPFQNVLNGGSHAGGRLAFQEF

[0147] MIVPSAAPSFSEALRQGAEVYQKLKTLAKKKYGQSAGNVGDEGGVAPDIQTAEEAL

[0148] DLITEAIEQAGYTGKMKIAMDVASSEFYKADVKKYDLDFKNPDSDSSKWLTYEQLA

[0149] DLYKTLASKYPIVSIEDPFAEDDWEAWSYFYKTSDFQIVGDDLTVTNPLRIKKAIETK

[0150] ACNALLLKVNQIGTLTESIQAAKDSYADNWGVMVSHRSGETEDVTIADIAVGLRSGQ

[0151] IKTGAPARSERLAKLNQILRIEEELGNNAIYAGEKFRTSVNL

[0152] SEQ ID No: 13 - A. niger Enol sequence

[0153] >tr|A0A100INJ9|A0A100INJ9_ASPNG Enolase OS=Aspergillus niger OX=5061

[0154] GN=ABL_07151 PE=3 S V= 1

[0155] MPISKIHARSVYDSRGNPTVEVDVVTETGLHRAIVPSGASTGQHEAHELRDGDKTMW

[0156] GGKGVLKAVKNVNETIAPAVIKENLDVKDQSKVDEFLNKLDGTANKSNLGANAILG

[0157] VSLAIAKAGAAEKGIPLYAHISDLAGTKKPYVLPVPFQNVLNGGSHAGGRLAFQEFMI VPNTASSFSEGLRQGAEVYQKLKALAKKKYGQSAGNVGDEGGVAPDIQTAEEALDL

[0158] ITESIEQAGYTGKISIAMDVASSEFYKADVKKYDLDFKNPESDSSKWLTYEQLADLYK

[0159] SLASKYPIVSIEDPFAEDDWEAWSYFYKTSDFQIVGDDLTVTNPLRIKKAIELKSCNAL

[0160] LLKVNQIGTLTESIQAAKDSYADNWGVMVSHRSGETEDVTIADIAVGLRSGQIKTGA

[0161] PARSERLAKLNQILRIEEELGDNAVYAGEKFRTAVNL

[0162] SEQ ID No: 14 - A. terreus Enol sequence

[0163] >tr|H9BAU9|H9BAU9_ASPTE Enolase OS=Aspergillus terreus OX=33178 GN=enoA PE=2 SV=1

[0164] MPISKIHARSVYDSRGNPTVEVDVVTETGLHRAIVPSGASTGQHEACELRDGDKTHW

[0165] GGKGVLKAVKNVNETIGPAVIKENLDVKDQSKVDEFLNKLDGSANKSNLGANAILG

[0166] VSLAIAKAGAAEKGVPLYAHISDLAGTKKPYVLPVPFQNVLNGGSHAGGRLAFQEF

[0167] MIVPDSAPSFSEALRQGAEVYQKLKALAKKKYGQSAGNVGDEGGVAPDIQTAEEAL

[0168] DLISEAIEQAGYTGKMHIAMDVASSEFYKADVKKYDLDFKNPESDPSKWLTYEQLA

[0169] DLYKSLASKYPIVSIEDPFAEDDWEAWSYFYKTSDFQIVGDDLTVTNPLRIKKAIELKS

[0170] CNALLLKVNQIGTLTESIQTAKDSYADNWGVMVSHRSGETEDVTIADIAVGLRSGQI

[0171] KTGAPARSERLAKLNQILRIEEELGDNAVYAGEKFRTAVNL

[0172] SEQ ID No: 15 -A. nidulans Enol sequence

[0173] >sp|Q5B135|ENO_EMENI Enolase OS=Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) OX=227321 GN=enoA PE=1 SV=1

[0174] MPISKIHARSVYDSRGNPTVEVDVVTETGLHRAIVPSGASTGQHEAHELRDGDKSKW

[0175] LGKGVLTAVKNVNETIGPAVIKENLDVKEQSKIDEFLNKLDGTPNKSNLGANAILGVS

[0176] LAIAKAGAAEKGVPLYAHISDLAGTKKPYVLPVPFQNVLNGGSHAGGRLAFQEFMIV

[0177] PDTASSFSEGLRQGAEVYHKLKALAKKKYGQSAGNVGDEGGVAPDIQTAEEALDLIT

[0178] EAIEQAGYTGKIHIAMDVASSEFYKPEEKKYDLDFKNPDSDPSKWLTYEQLADLYKS

[0179] LAAKYPIVSIEDPFAEDDWEAWSYFYKTSDFQIVGDDLTVTNPLRIKKAIELKSCNAL

[0180] LLKVNQIGTLTESIQAAKDSYADGWGVMVSHRSGETEDVTIADISVGLRSGQIKTGAP

[0181] ARSERLAKLNQILRIEEELGENAVYAGQNFRKSVNL

[0182] SEQ ID NO: 16 - human CD4

[0183] >sp|P01730|CD4_HUMAN T-cell surface glycoprotein CD4 OS=Homo sapiens OX=9606

[0184] GN=CD4 PE=1 SV=1

[0185] MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNS

[0186] NQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQK

[0187] EEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQL

[0188] ELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKL

[0189] TGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQAL

[0190] PQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKL

[0191] ENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALI

[0192] VLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI

Claims

CLAIMS1. An activator of CD4+Foxp3+regulatory T cells for use in treating or preventing an allergy to Aspergillus or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response in a subject, wherein the activator of CD4+Foxp3+regulatory T cells binds to domain 2 of CD4.

2. The activator for use according to claim 1, wherein the disease or condition associated with an allergy o Aspergillus is allergic bronchopulmonary aspergillosis (AB PA), Farmer’s lung, or Aspergillus- vAcn aggravation of chronic obstructive pulmonary disease (COPD).

3. The activator for use according to claim 2, wherein the disease or condition associated with an allergy to Aspergillus is ABPA.

4. The activator for use according to any of claims 1 to 3, wherein the disease or condition associated with a pathogen-driven immune response is pathogen-driven aggravation of COPD.

5. The activatory for use according to any one of claims 1 to 4, wherein the subject has an underlying respiratory illness selected from asthma, cystic fibrosis, or COPD.

6. The activator for use according to any one of claims 1 to 5, wherein the Aspergillus is selected from A. fumigatus, A. flavus, A. niger, A. terreus and A. nidulans, and preferably wherein the Aspergillus is A. fumigatus.

7. The activator for use according to any one of claims 1 to 6, wherein the activator is or comprises an antibody, an antibody fragment, a bispecific antibody, or a multispecific antibody.

8. The activator for use according to any one of claims 1 to 7, wherein the activator is or comprises an anti-CD4 antibody or a fragment thereof.

9. The activator for use according to claim 8, wherein the anti-CD4 antibody or fragment thereof comprises a variable heavy chain polypeptide sequence having at least 85% sequence identitywith SEQ ID NO: 3 and a variable light chain polypeptide sequence having at least 85% sequence identity with SEQ ID NO: 4.

10. The activator for use according to claim 8 or claim 9, 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.

11. The activator for use according to any one of claims 8 to 10, 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.

12. The activator for use according to any one of claims 7 to 11, wherein the antibody is an IgG antibody.

13. The activator for use according to any one of claims 7 to 12, wherein the antibody is an IgGl antibody.

14. The activator for use according to any one of claims 7 to 13, wherein the antibody comprises a modified Fc region with a reduced or ablated effector function.

15. The activator for use according to any one of claims 7 to 13, wherein the antibody is an anti-CD4 antibody having a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2.

16. The activator for use according to any preceding claim wherein the activator is comprised in a pharmaceutical composition.

17. The activator for use according to any preceding claim, wherein the subject is a human subject.

18. The activator for use according to any preceding claim, wherein the activator is to be administered to the subject intravenously, intramuscularly, subcutaneously, or via inhalation.

19. The activator for use according to claim 18, wherein the activator is to be administered subcutaneously.

20. An anti-CD4 antibody or fragment thereof for use in treating or preventing an allergy to Aspergillus or a lung disease or lung condition associated with an allergy to Aspergillus or associated with a pathogen-driven immune response in a subject, wherein the 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.

21. An anti-CD4 antibody or fragment thereof for use in treating or preventing allergic bronchopulmonary aspergillosis (AB PA), Farmer’s lung, or Aspergillus- v'wcn aggravation of chronic obstructive pulmonary disease (COPD), in a subject, wherein the 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.

22. An anti-CD4 antibody or fragment thereof for use in treating or preventing a pathogen- driven aggravation of COPD in a subject, wherein the 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.

23. The anti-CD4 antibody or fragment thereof for use according to any one of claims 20 to22, wherein the antibody is an IgG antibody, or an IgGl antibody.

24. The anti-CD4 antibody or fragment thereof for use according to any one of claims 20 to23, wherein the antibody comprises a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2.

25. The anti-CD4 antibody or fragment thereof for use according to any one of claims 20 to 23, wherein the antibody comprises a modified Fc region with a reduced or ablated effector function.

26. The anti-CD4 antibody or fragment thereof for use according to any one of claims 20 to 25 wherein the antibody or fragment thereof is comprised in a pharmaceutical composition.

27. The anti-CD4 antibody or fragment thereof for use according to any one of claims 20 to 26, wherein the subject is a human subject.

28. The anti-CD4 antibody or fragment thereof for use according to any one of claims 20 to 27, wherein the antibody or fragment thereof is to be administered to the subject intravenously, intramuscularly, subcutaneously, or via inhalation.

29. The anti-CD4 antibody or fragment thereof for use according to claim 28, wherein the anti- CD4 antibody or fragment thereof is to be administered subcutaneously.

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