WO2016155745A1

WO2016155745A1 - Cross-reactive anti-pad antibodies

Info

Publication number: WO2016155745A1
Application number: PCT/DK2016/050091
Authority: WO
Inventors: Claus Henrik Nielsen; Dres DAMGAARD
Original assignee: Rigshospitalet
Priority date: 2015-03-27
Filing date: 2016-03-29
Publication date: 2016-10-06

Abstract

Disclosed herein are cross-reactive anti-peptidylarginine deiminase (PAD) antibodies and fragments and variants thereof capable of binding to at least PAD2 and PAD4 and use of such anti-PAD antibodies or fragment or variants thereof in the treatment of autoimmune diseases characterized by extracellular citrullination, in particular rheumatoid arthritis (RA).

Description

CROSS-REACTIVE ANTI-PAD ANTIBODIES

All patent and non-patent references cited in the present application, are incorporated by reference in their entirety.

Field of invention

The present invention relates to cross-reactive anti-peptidylarginine deiminase (PAD) antibodies and fragments and variants thereof capable of binding to at least PAD2 and PAD4 and use of such anti-PAD antibodies or fragment or variants thereof in the treatment of autoimmune diseases characterized by extracellular citrullination, in particular rheumatoid arthritis (RA).

Background of invention

Citrullination is a process wherein arginine residues in various proteins are deiminated into citrulline. The process, referred to as citrullination, is catalysed by enzymes of the peptidylarginine deiminase (PAD) family. After this conversion, the protein loses positive charge, changes conformation and becomes more susceptible to degradation.

Autoimmune diseases arise from an inappropriate immune response of the body against substances and tissues normally present in the body. In a number of autoimmune diseases citrullination has been suggested to play an important role in the pathogenesis. Such diseases include e.g. rheumatoid arthritis, multiple sclerosis and psoriasis. Rheumatoid Arthritis (RA) affects 0.5-1 % of the adult population worldwide. RA is caused by an autoimmune attack on the synovium followed by chronic inflammation in the synovial joints. A role for the PAD isoforms PAD2 and PAD4 has previously been suggested in RA. PAD2 and PAD4 are both present in the inflamed joint. In RA, PAD enzymes are thought to citrullinate extracellular proteins such as fibrinogen, which may contribute to the pathogenesis and/or result in further disease progression by generation of citrullinated proteins that may induce production of anti-citrullinated protein antibodies (ACPAs) and stimulation of T cells reactive with citrullinated peptides. ACPAs are detectable in the serum years before the onset of arthritis symptoms, and ACPA-positive patients have more extensive joint erosion than ACPA- negative patients. ACPAs have proved a useful diagnostic marker for RA. Thus approximately 88-96% of ACPA-positive individuals will clinically present as RA patients, while approximately 70- 80% of RA patients are ACPA-positive. ACPA-positive and ACPA-negative RA are often considered two distinct disease entities with similar symptoms. Associated with ACPA-positive RA are human leukocyte A (HLA) antigens containing the "shared epitope", capable of comprising citrullinated peptides. Thus, proteins citrullinated by PAD trigger antibody responses as well as T-cell responses in ACPA-positive RA. PAD2 and PAD4 enzymes have previously been detected in synovial tissue from RA patients and their expression levels were found to be correlated with the intensity of inflammation (Foulquier et al. 2007, Arthritis & Rheumatism 56, 1 1 : 3541 -53,

Makrygiannakis et al., 2012, Arthritis Research & Therapy 14:R20). WO 2010/005293 discloses a short peptide inhibitor capable of inhibiting PAD2 and PAD4 activity. The peptide inhibitor comprises 5-20 amino acids.

WO 2014/086365 discloses monoclonal antibodies specific for PAD2. WO 201 1/050357 discloses a small molecule inhibitor of PAD activity of PAD1 , PAD3 and PAD4.

WO 2009/127048 discloses a small molecule inhibitor capable of inhibiting PAD2 activity.

US 2014/127720 discloses identification of cross-reactive anti-PAD3-PAD4

autoantibodies in RA patient and use of these in diagnosis and monitoring treatment efficiency. WO 2012/026309 discloses antibodies directed against PAD4 for the treatment of rheumatoid arthritis. Summary of invention

The present disclosure generally relates to anti-PAD antibodies or fragments or variants thereof capable of binding to at least PAD2 and PAD4 and use of such anti- PAD antibodies or fragments or variant thereof in the treatment of an autoimmune disease characterized by extracellular citrullination. The autoimmune disease may e.g. be rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, inflammatory bowel disease, and psoriasis, in particular rheumatoid arthritis.

The cross-reactive anti-PAD antibodies or fragments or variants thereof disclosed herein preferably bind to the catalytic domain of PAD and are capable of inhibiting PAD-mediated citrullination of at least PAD2 and PAD4.

The invention further relates to a method of treatment of autoimmune diseases characterized by extracellular citrullination comprising the administration of a suitable amount of a cross-reactive anti-PAD antibody or fragment or variant thereof to a subject in need thereof.

The advantage of using a cross-reactive anti-PAD antibody or fragment or variant thereof for inhibition of PAD activity is that the antibody will specifically inhibit extracellular citrullination mediated by several different PAD isoforms and not intracellular citrullination, thus preserving the cells' ability to citrullinate intracellular targets of PAD. Small molecule inhibitors of PAD may also bind to a range of non-PAD proteins, which may be the cause of the unspecific effects observed in response to small molecule inhibitors of PAD such as chloramidin. As a consequence, it is expected that a drug comprising a cross-reactive anti-PAD antibody or fragment or variant thereof will have fewer side-effects than a small molecule inhibitor of PAD activity, thus leading to higher patient compliance and safety. In addition, an antibody directed at PAD has the further advantage of being able to inhibit PAD activity at at least two levels; both by direct inhibition of enzyme activity and by stimulating clearance of PAD. Description of Drawings

Fig. 1. Culture supernatants from 36 mAbs (reacting with three different peptides) were tested on full-length human recombinant PAD2 or PAD4-coated plates (100 ng/mL). Shown is the absorbance at a dilution of 1 :20 in wash buffer of each of the culture supernatants. Horse radish peroxidase (HRP)-conjugated rabbit anti-mouse antibodies, diluted 1 :1000 in wash buffer, were added for 1 hour at RT, and the plates were developed with ortho-phenylenediamine (OPD) substrate. The levels are given as OD490-650 nm-units. Fig. 2. Four selected mAbs were purified and tested on plates coated with full-length human recombinant PAD2, PAD3 or PAD4-coated plates (100 ng/mL). Shown is the absorbance at a dilution of 1 :100 in wash buffer of purified mAbs. HRP-conjugated rabbit anti-mouse antibodies, diluted 1 :1000 in wash buffer, were added for 1 hour at RT, and the plates were developed with OPD substrate. The levels are given as OD490-650 nm-units. All antibodies are capable of binding to PAD2, PAD3 and PAD4, however, the tested antibodies differ in the affinity for the different PAD variants.

Fig. 3. Culture supernatants from 4 selected mAbs were tested on human recombinant PAD2 or murine recombinant PAD2 (mPAD2)-coated plates (100 ng/mL). Shown is the absorbance at a dilution of 1 :20 in wash buffer of the culture supernatants. HRP- conjugated rabbit anti-mouse antibodies, diluted 1 :1000 in wash buffer, were added for 1 hour at RT, and the plates were developed with OPD substrate. The levels are given as OD490-650 nm-units. All tested antibodies are capable of binding to both human and murine PAD2.

Fig. 4. Inhibitory capacity of cross-reacting anti-PAD mAbs. The ability of 4 selected mAbs to inhibit citrullination of fibrinogen was tested using recombinant human PAD2 (rhPAD2) as example of catalyst. Test of the inhibitory capacity of mSoU , mSol2, mSol3, mSol4, and a control mAb (anti-SCUBE1 ) was performed. Human fibrinogen (200 ug/mL) was incubated in citrullination buffer (100 mM Tris-HCI, 10 mM CaCI2, 1 mM DTT, pH 7.5) with 200 ng/mL rhPAD2 for 4 hours at 37 ^eC in the presence of the listed mAbs (10 μg mL). The citrullinated fibrinogen (incubated with each listed mAbs) was applied to ELISA plates, coated with 1 μg/mL rabbit polyclonal fibrinogen for 2 hours at room temperature. Following three washes, biotinylated anti-citrullinated fibrinogen antibody (clone 20B2, Cat No. MQ13.102, ModiQuest, Oss, Netherlands) was applied (0.5 μς/ηιΙ.) for 1 hour at room temperature. HRP-conjugated streptavidin, diluted 1 :3000 in wash buffer, was added for 1 hour at RT, and the plates were developed with OPD substrate. The levels are given as OD490-650 nm-units. The results show that the tested antibodies are capable of at least partially inhibiting PAD2 mediated citrullination of fibrinogen.

Fig. 5. Potential for humanization of four selected mAbs. Deamidation sites, important for the potential for humanization, were identified in sequences of variable heavy (VH) and variable light (VL) in mSoU , mSol2, mSol3 and mSol4. (÷) indicates no

deamidation sites and (X) indicates a deamidation site. Analysis of V gene sequences. *Framework mutations relative to nearest germ line V gene indicated in ( ). Alignment of Variable gene amino acid sequences at www.ebi.ac.uk.

Sequences

Definitions

Anti-citrullinated protein antibodies (ACPAs): Anti-citrullinated protein antibodies (ACPA), also known as anti-cyclic citrullinated peptide antibodies (anti-CCP), are autoantibodies that are frequently detected in the blood of rheumatoid arthritis patients. These antibodies recognize amino acid sequences containing citrulline in a variety of proteins. During inflammation, arginine residues in proteins such as fibrinogen and vimentin can be enzymatically converted into citrulline residues (a process referred to as citrullination), and, if their shapes are significantly altered, the proteins may be seen as antigens by the immune system, thereby generating an immune response. ACPAs have proved to be powerful biomarkers that allow the diagnosis of rheumatoid arthritis (RA) to be made at a very early stage.

Antibody: The term "antibody" or "antibody molecule" describes a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulin) or as one molecule (the antibody molecule or immunoglobulin molecule). An antibody is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. An individual antibody is usually regarded as monospecific, and a composition of antibodies may be monoclonal (i.e. consisting of identical antibody molecules) or polyclonal (i.e. consisting of two or more different antibodies reacting with the same or different epitopes on the same antigen or even on distinct, different antigens). Each antibody has a unique structure that enables it to bind specifically to its corresponding antigen, and all natural antibodies have the same overall basic structure of two identical light chains and two identical heavy chains.

Antibodies are also known collectively as immunoglobulins.

The terms "antibody" or "antibodies" as used herein are also intended to include fully murine, chimeric, humanized, fully human, bispecific and single chain antibodies, nanobodies, as well as binding fragments of antibodies, such as Fab, Fv fragments or single chain Fv (scFv) fragments, as well as multimeric forms such as dimeric IgA molecules or pentavalent IgM. An antibody may be of human or non-human origin, for example a murine or other rodent-derived antibody, or a chimeric, humanized or reshaped antibody based e.g. on a murine antibody. Each heavy chain of an antibody typically includes a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region typically includes three domains, referred to as CH1 , CH2 and CH3. Each antibody light chain typically includes a light chain variable region (VL) and a light chain constant region. The light chain constant region typically includes a single domain, referred to as CL. The VH and VL regions may be further subdivided into regions of hypervariability ("hypervariable regions", which may be hypervariable in sequence and/or in structurally defined loops). These are also referred to as complementarity determining regions (CDRs), which are interspersed with regions that are more conserved, termed framework regions (FRs).

Each VH and VL typically includes three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The amino acid residues in the variable regions are often numbered using a standardized numbering method known as the Kabat numbering scheme (Kabat et al. (1991 ) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, M D, USA).

The specificity of an antibody's interaction with a target antigen resides primarily in the amino acid residues located in the six CDRs of the heavy and light chains (three each; CDR1 , CDR2 and CDR3 of the heavy chain variable region (VH); and CDR1 , CDR2 and CDR3 of the light chain variable region (VL)). The amino acid sequences within CDRs are therefore much more variable between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody, or more generally any specific antibody with a given amino acid sequence, by constructing expression vectors that express CDR sequences from the specific antibody grafted into framework sequences from a different antibody. As a result, it is possible to "humanize" a non-human antibody and still substantially maintain the binding specificity and affinity of the original antibody.

It is well-known in the art that antibodies exist as different isotypes: IgA, IgD, IgE, IgG and IgM. The human IgG and IgA isotypes can be further divided into subclasses lgG1 , lgG2, lgG3, lgG4, IgAI and lgA2, whereas the murine IgG isotype can be subdivided into subclasses lgG1 , lgG2a, lgG2b, lgG3. Autoimmune disease is used interchangeably with the term "autoimmune disorder" and is characterized by an inappropriate immune response against own cells or tissue ("self"). In healthy circumstances, the immune system attacks only foreign

microorganisms or molecules, but in autoimmune diseases the immune system loses the ability to distinguish between self and non-self (loss of tolerance). Like adaptive immune responses against foreign antigens, autoimmune disorders are believed to be initiated by activation of antigen-specific T cells. The T cells may, in turn, activate self- reactive B cells with production of autoantibodies as a consequence. Both genetic and environmental risk factors contribute to breakage of self-tolerance in most autoimmune diseases.

Bispecific antibodies: A bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein that is composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigen. For example in cancer immunotherapy, where BsMAbs are engineered that simultaneously bind to a cytotoxic cell (using a receptor like CD3) and a target like a tumour cell to be destroyed.

Bispecific antibodies include trifunctional antibodies, chemically linked F(ab')₂ and bispecific T-cell engager (BiTE) herein. Chimeric antibody: A "chimeric antibody" refers in its broadest sense to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. As used herein, a "chimeric antibody" is generally an antibody that is partially of human origin and partially of non-human origin, i.e. derived in part from a non-human animal, for example a mouse or other rodent, or an avian species such as a chicken.

Cross-reactivity: The ability of an antibody to react with similar antigenic sites on different proteins or different isoforms of a protein. In the present context, a cross- reactive anti-PAD antibody is capable of binding to at least two different isoforms of PAD, in particular to PAD2 and PAD4.

Fab: The fragment antigen-binding (Fab fragment) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the paratope— the antigen-binding site— at the amino terminal end of the monomer. The two variable domains bind the epitope on their specific antigens. In an experimental setting, Fc and Fab fragments can be generated in the laboratory. The enzyme papain can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment. The enzyme pepsin cleaves below hinge region, so a F(ab')₂ fragment and a pFc' fragment is formed. The F(ab')₂ fragment can be split into two Fab' fragments by mild reduction.

The variable regions of the heavy and light chains can be fused together to form a single-chain variable fragment (scFv), which is only half the size of the Fab fragment, yet retains the original specificity of the parent immunoglobulin.

Nanobody: A nanobody is also known as a single-domain antibody (sdAb) and is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies (150-160 kDa), and even smaller than Fab fragments (-50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (-25 kDa, two variable domains, one from a light and one from a heavy chain).

Recombinant antibody: The term "recombinant antibody" refers to an antibody that is expressed from a cell or cell line transfected with an expression vector (or possibly more than one expression vector, typically two expression vectors) comprising the coding sequence of the antibody, where said coding sequence is not naturally associated with the cell. scFv: A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (V_H) and light chains (V_L) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N- terminus of the V_H with the C-terminus of the V_L, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. Unlike monoclonal antibodies, which are often produced in mammalian cell cultures, scFvs are more often produced in bacteria cell cultures such as E. coli. Symptom: A symptom is a departure from normal function or feeling which is noticed by a patient, indicating the presence of disease or abnormality. A symptom is subjective, observed by the patient, and cannot be measured directly. For example, the classical symptoms of RA include swollen, warm, painful and stiff joints, particularly early in the morning on waking or following prolonged inactivity.

Detailed description of the invention

Citrullination

Posttranslational modifications, e.g. phosphorylation, glycosylation and citrullination, are normal processes that occur after protein synthesis and are crucial for the enormous diversity of the proteome. Citrullination (also termed deimination) is a posttranslational modification whereby the amino acid arginine is modified to the non- standard residue citrulline. This reaction is catalyzed by a group of peptidylarginine deiminase (PAD) enzymes. Citrullination is important for many intracellular processes (reviewed by e.g. Gyorgy et al. 2006, Int J Biochem Cell Biol 38: 1662-77). Listed are a few examples:

- Citrullination of keratin and filaggrin is crucial for the final stage of keratinocyte differentiation.

Citrullination is involved in maturation of hair cuticle cells with importance in the formation of the rigid structures.

Citrullination of myelin basic protein (MBP) is important for ensuring electrical insulation of myelin sheaths.

- PAD citrullinates arginine and methylarginine of histone H3 and H4. This

disables histone methylation which regulates transcription of DNA.

- Regulation of gene expression

- apoptosis Arginine contains a positively charged guanido group in the side chain, before citrullination, which is replaced by a neutral citrulline ureido group, thus reducing the net-charge of a protein. This might prevent the ability to make ionic interactions with negatively charged side chains resulting in different structure of the protein. Loss of intramolecular interactions allowing a protein to unfold thus makes it more susceptible to proteolytic cleavage. This structural change can for example be seen experimentally for fibrinogen which migrates differently in SDS-PAGE after citrullination.

The function of proteins may be altered by citrullination as seen with citrullinated fibrinogen, which markedly impairs the function of thrombin-catalysed fibrin

polymerization and also inhibits fibrin formation. The structural changes in proteins can be so dramatic that they are recognized as non-self-protein by the immune system, which is the case in RA. Diseases in which citrullination has been shown or suggested to play a role include not only RA but also multiple sclerosis (MS), Sjogren's syndrome as well as psoriasis.

PAD catalyzes the citrullination reaction in a Ca²⁺ dependent manner. There are five members of the human PAD enzyme family (PAD1 , PAD2, PAD3, PAD4 and PAD6). They have a molecular mass of around 75 kDa. These PAD enzymes differ with respect to cellular expression patterns and substrate specificities. For example, PAD2 and PAD4 expression has been shown in RA synovium, synovial fluid cells and has further been detected extracellularly in synovial fluid in RA patients. None of the other family members (PAD1 , PAD3 and PAD6) have been detected in the synovial joints among patients with RA (Foulquier et al. 2007, Arthritis Rheum 56; 3541 -53).

PAD2 and PAD4 isoforms are likewise expressed in the brain and are present in myelin. It has been hypothesized that PAD2 contributes to destabilization of myelin in Multiple Sclerosis (Musse et al. 2008, Disease Models & Mechanisms 1 , 229-240). PAD1 is mainly expressed in epidermis and uterus and is important for the terminal differentiation of keratinocytes, keratins and filaggrin. PAD2 has been widely detected, notably in brain astrocytes, sweat glands, skeletal muscles, epidermis and in leukocytes, such as macrophages and neutrophils. PAD3 is co-expressed and co- localized with its natural substrate, thrichohyalin, which is a major structural protein of inner root sheath cells of hair follicles. PAD4 expression has so far been detected only in leucocytes e.g. monocytes, eosinophils and neutrophils. PAD4 is present in the nucleus. PAD6 is expressed in male and female germ cells.

PAD2 is also known as protein-arginine deiminase type-2 and peptidylarginine deiminase II. The human protein sequence ((UniProt no. Q9Y2J8 (PADI2 HUMAN)) comprises 665 amino acids (aa); cf. SEQ ID NO:41 . The mouse sequence comprises 673 aa (Q08642 (PADI2_MOUSE)). The protein sequence is highly conserved amongst species, e.g. between human mouse (mus musculus). The human protein sequence of PAD4 comprises 663 amino acids ((UniProt no.

Q9UM07 (PADI4 HUMAN)), cf. SEQ ID NO:42. The protein sequence of PAD4 is also highly conserved amongst species, e.g. between human and mouse PAD4.

Autoimmune diseases

The antibodies disclosed herein may be used in the treatment of an autoimmune disease. The autoimmune disease is in particular an autoimmune disease

characterized by hyper-citrullination and/or wherein citrullination plays a role in the pathogenesis of the disease.

Autoimmune diseases arise from an inappropriate immune response of the body against substances and tissues normally present in the body or against structurally modified derivatives of said substances and tissues. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells.

A number of autoimmune diseases are characterized by elevated levels of citrullinated proteins, which are believed to play an important role in the pathogenesis of the autoimmune disease. Such diseases include rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, inflammatory bowel disease and psoriasis, more preferred rheumatoid arthritis and multiple sclerosis, in particular rheumatoid arthritis.

Targeting extracellular citrullination with an anti-PAD antibody avoids the simultaneous inhibition of intracellular processes where citrullination is important, thus reducing or avoiding the risk of adverse effects associated with such general inhibition of citrullination.

Targeting extracellular citrullination with an anti-PAD antibody avoids toxicity associated with small-molecule inhibitors of PAD, due to presumably low specificity for PAD. In one embodiment of the present disclosure, there is provided a cross-reactive anti- PAD antibody or fragment or variant thereof for use in the treatment of an autoimmune disease, such as an autoimmune disease characterized by extracellular citrullination, such as extracellular hyper-citrullination.

In one embodiment of the present disclosure, use of a cross-reactive anti-PAD antibody or fragment or variant thereof for the manufacture of a medicament for the treatment of an autoimmune disease, such as an autoimmune disease characterized by extracellular citrullination, such as extracellular hyper-citrullination, is provided.

Rheumatoid arthritis

Rheumatoid Arthritis (RA) is a systemic autoimmune disease affecting 0.5-1 % of the adult population worldwide. RA is caused by an autoimmune attack on the synovium followed by chronic inflammation in the synovial joints. Systemic effects are mainly seen as inflammation in lungs, heart and eyes. Like many autoimmune diseases, RA occurs more frequently in women than in men (3:1 ratio) and disease onset is mostly seen at middle age (40-60 years old).

RA normally affects joints symmetrically. Wrists, fingers, feet, ankles and knees are the most commonly affected joints. The first symptoms to appear include warm and tender joints, morning stiffness, and stiffness in the affected joints if not used for an hour or even less. Later on RA patients may lose range of motion in joints and these may become deform, as a result of long-term inflammation and irreversible bone digestion in the synovial joints.

Genetic factors are estimated to be responsible for at least 50% of the risk of RA development, while environmental factors account for the remainder. RA is a polygenic disease and particularly genes of the major histocompatibility complex (MHC) class II provide a strong risk factor in RA as in many other autoimmune diseases. Certain MHC class II types within the HLA-DR region, are thus linked to RA, and 80 % of patients with RA carry the so-called shared epitope, variants of a motif (EQKRAA) which is present in the third hypervariable region of the HLA-DR beta chain with structural effect on the binding cleft in the MCH class II molecule. This confers binding of specific citrullinated peptides and thus affects antigen presentation to T-cell receptors. The motif is present in the DRB1 ^* variants ^*0101 , ^*0102, ^*0401 , ^*0404, ^*0405, ^*0408, ^*1001 and *1402, which have been associated with RA. The different alleles are associated with mild or a more erosive disease; thus they are likely to present antigens differently, leading to different phenotypes of RA - all with presence of anti-citrullinated protein antibodies (ACPAs). DR1 is associated with a relatively mild disease, whereas DR4 is associated with more severe RA.

A number of single nucleotide polymorphisms (SNPs) have also been associated with RA. Some of these, including TRAF1 -C5-, PTPN22- and PAD4 polymorphisms, are also associated with the presence of ACPAs. These factors constitute a smaller risk compared to the MHC-associated risk factors, however. The "non-MHC" risk factors may indicate some of the mechanisms associated with ACPA-negative RA.

Cigarette smoking is the best known environmental risk factor for RA. Several studies link smoking and the shared epitope together as a major combined risk factor. A recent study has shown that smoking increases the expression of PAD2 in bronchoalveolar lavage (BAL) cells along with an elevated level of citrullinated proteins (Makrygiannakis et al., Ann Rheum Dis 67; 1488-92).

The pathogenesis of RA is not clear, but over the last two decades, more insight into pathogenic pathways of RA has accumulated. Autoantigens, e.g. citrullinated peptides, are presented to T cells by antigen presenting cells (APCs) such as dendritic cells, macrophages or activated B cells. This autoantigen-presentation triggers the stimulation and expansion of antigen-specific T cells present in the joints and lymph nodes. Co-stimulatory signals, e.g. CD80 and CD86 presented on APCs, are needed for full activation of the T cells. These bind to surface expressed CD28 on T cells.

T cells localized to the synovial membrane secrete lnterleukin-2 (IL-2) and interferon-γ (INF-γ). These cytokines induce activation of macrophages, B cells, fibroblasts and osteoclasts. B cells differentiate into (auto)antibody-secreting plasma cells. Immune complexes containing autoantibodies and citrullinated self-antigens induce the secretion of proinflammatory cytokines, such as tumor necrosis factor a (TNF-a) via complement- and Fc-receptor mediated activation on human monocytes. Activated B cells also serve as APCs, leading to additional T-cell activation, which enhances the autoimmune response. T- and B cell activation result in increased production of cytokines and chemokines, leading to a feedback loop for additional activation of T cells, macrophages and B cells. Th1 cells activate monocytes and macrophages by cell-cell contact and/or by activation of different cytokines, such as INF-γ, TNF-a and IL-17. The macrophages and fibroblasts then overproduce proinflammatory cytokines, mainly TNF-a, IL-1 and IL-6, which activate osteoclasts (leading to bone destruction) and synovial fibroblasts (leading to production of matrix metalloproteinases and consequent cartilage destruction). A broad range of cytokines are present in the synovium, secreted by various cell populations. The cytokines that have been established to be most directly implicated in RA pathogenesis are TNF-a, IL-6, IL-1 , IL- 15, IL-18, IL-33 and IL-17.

There is no known cure for rheumatoid arthritis, but many different types of treatment can alleviate symptoms and/or modify the disease process. Pharmacological treatment of RA can be divided into disease-modifying antirheumatic drugs (DMARDs), biologies, anti-inflammatory agents and analgesics. Treatment also includes rest and physical activity.

In order to be effective, DMARDs must be administered before the deformities appear or the erosive disease occurs. Usually, Rheumatologists do not wait for the fulfilment of the criteria for classification of RA as published by the American College of

Rheumatology (ACR) and start treatment with this type of drugs if the pain and synovitis persist and the function is compromised.

DMARDs include but are not limited to: azathioprine, ciclosporin (cyclosporine A), D- penicillamine, gold salts, hydroxychloroquine, leflunomide, methotrexate (MTX), minocycline, sulfasalazine (SSZ) and cyclophosphamide.

Biological agents (biologies) for RA treatment include but are not limited to: tumor necrosis factor alpha (TNFa) blockers (etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi)), Interleukin 1 (IL-1 ) blockers (anakinra (Kineret)), monoclonal antibodies against B cells (rituximab (Rituxan)), T-cell costimulation blockers (abatacept (Orencia)), blockers of IL-6 signaling (tocilizumab (an anti-IL-6 receptor antibody) (RoActemra, Actemra)).

Anti-inflammatory agents include but are not limited to glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs, most also act as analgesics), and classical analgesics include but are not limited to paracetamol, opiates, diproqualone and lidocaine topical.

NSAIDs used in the treatment of RA include ibuprofen, naproxen, meloxicam, etodolac, nabumetone, sulindac, tolementin, choline magnesium salicylate, diclofenac, diflusinal, indomethicin, ketoprofen, oxaprozin, and piroxicam.

A role for PAD2 and PAD4 has previously been suggested in the pathogenesis of RA, see e.g. Damgaard et al. Arthritis Res Ther. 2014 Dec 5;16(6):498. PAD2 and PAD4 are both present at sites of inflammation, e.g. an inflamed joint. PAD2 and PAD4 differ with respect to citrullination efficiency of different substrates. Most proteins can be citrullinated by more than one PAD isoform, but they seem to have substrate- preferences and differ with respect to conditions required for efficient catalysis. For instance, fibrinogen can be citrullinated by either one of PAD2 or PAD4. However, PAD2 is able to citrullinate additional arginine residues compared to PAD4, and the two isoforms differ in enzymatic activity with regard to optimal pH and calcium

concentrations. It seems that the amino acid context is more important for PAD4 activity than for PAD2. Furthermore, PAD2 is able to citrullinate fibrinogen much more efficiently than PAD4. Both of PAD2 and PAD4 are able to citrullinate histone H3, however PAD4 is more efficient than PAD2, which is likely a result of PAD4 being present in the nucleus.

Inflammation in the joints leads to infiltration of inflammatory cells. Inflammation is strictly controlled and does not normally lead to production of autoantibodies or auto- reactive T cells. Inflammation is linked with apoptosis of cells, and the apoptotic cells are normally cleared by phagocytes. Massive apoptosis due to toxins, infections or defects in the clearing system, e.g. genetic defects, may result in necrosis of some cells, however. In a joint with inflammation and apoptosis, the intracellular

concentration of calcium may be elevated enough to activate PAD enzymes in PAD- containing cells, e.g. monocytes, granulocytes and macrophages. Cytosolic proteins like vimentin, which undergo citrullination, will normally not be exposed to the immune system. When cells become necrotic, i.e. when inflammation is uncontrolled, intracellular components like citrullinated proteins can be found extracellularly in the synovial joints. PAD has likewise been detected extracellularly in synovial joints. PAD enzymes are thought to citrullinate extracellular proteins such as fibrinogen, which may contribute to the pathogenesis of RA and/or result in further disease progression by generation of extracellular citrullinated proteins that may lead to the production of anti- citrullinated protein antibodies (ACPAs) and further inflammation. ACPAs are detectable in the serum years before the onset of arthritis symptoms, and a significant positive correlation exists between the serum titer and clinical, biologic, and radiologic data related to RA activity and severity. ACPA-positive patients have a more erosive disease than those patients that are ACPA-negative. Moreover, ACPA-positive and ACPA-negative RA patients differ with respect to environmental risk factors. A strong association between RA and HLA types containing the shared epitope exists in ACPA-positive RA, particularly in smokers. Taken together, these findings support the notion that ACPA-positive and ACPA-negative RA are actually two different disease entities. In one embodiment, the present disclosure relates to a cross-reactive anti-PAD antibody or fragment or variant thereof as disclosed herein for use in the treatment of rheumatoid arthritis, in particular ACPA-positive RA. The treatment may be

prophylactic, ameliorative and/or curative. In one embodiment the treatment is prophylactic and can be initiated before symptoms of RA appear. For example, the treatment may be initiated upon detection of ACPAs in a blood sample obtained from a patient.

In one embodiment an anti-PAD antibody or fragment or variant thereof of the present disclosure is co-administered with another RA drug, such as a DMARD, a biological agent, an anti-inflammatory agent and/or analgesics. The co-administration may be simultaneous, sequential and/or separate.

Multiple sclerosis

Multiple sclerosis (MS), also known as "disseminated sclerosis" or "encephalomyelitis disseminata", is an autoimmune disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms. Disease onset usually occurs in young adults, and it is more common in women. It has a prevalence that ranges between 2 and 150 per 100,000. MS affects the ability of nerve cells in the brain and spinal cord to communicate with each other effectively. In MS, the body's own immune system attacks and damages the myelin. When myelin is lost, the axons can no longer effectively conduct signals.

Although much is known about the mechanisms involved in the disease process, the cause remains unknown. Theories include genetics or infections. Different

environmental risk factors have also been found.

There is no known cure for multiple sclerosis. Treatments attempt to return function after an attack, prevent new attacks, and prevent disability. MS medications can have severe adverse effects or be poorly tolerated. The prognosis is difficult to predict; it depends on the subtype of the disease, the individual's disease characteristics, the initial symptoms and the degree of disability the person experiences as time advances. PAD2 and PAD4 are expressed in the brain, and de-regulated citrullination has been suggested to play a role in the pathogenesis of multiple sclerosis; hence the present authors suggest inhibition of PAD activity by administration of a cross-reactive anti- PAD antibody to a subject in need thereof. In one embodiment of the present disclosure a cross-reactive anti-PAD antibody or fragment or variant thereof as disclosed herein is used in the treatment of multiple sclerosis.

In one embodiment of the present disclosure a cross-reactive anti-PAD antibody or fragment or variant thereof as disclosed herein is co-administered with another drug for the treatment of multiple sclerosis. The co-administration may be simultaneous, sequential and/or separate.

An advantage of using an antibody to inhibit PAD activity compared to e.g. a small molecule inhibitor is that intracellular citrullination essential for production of functional myelin is not affected.

However, for an antibody drug to be able to reach the sclerotic lesions in the brain it is essential that the drug is able to cross the blood brain barrier. It has been reported that the blood-brain barrier is weakened in MS, wherefore it is likely that even an unmodified antibody drug will be able to cross the blood brain barrier in MS patients.

To treat MS in its early stages, it may however be advantageous to genetically engineer the antibody to ensure that the antibody is capable of crossing the blood-brain barrier, e.g. by making a bispecific antibody with one "arm" directed against a receptor which transports the antibody across the blood-brain barrier and the other "arm" directed against the target itself, as previously described (Pardridge et al. 2012, Methods Enzymol 503:269-92).

Another strategy which can be undertaken to make antibody drugs capable of crossing the blood-brain barrier is to link the antibody to a peptide capable of crossing the blood- brain barrier, such as viral Tat. In one embodiment the present invention relates to a modified cross-reactive anti-PAD antibody or fragment or variant thereof capable of crossing the blood-brain barrier, such as a bispecific anti-PAD antibody or a cross-reactive anti-PAD antibody linked to Tat. Methods for developing bispecific antibodies capable of crossing the blood-brain barrier are known in the art as are methods for linking antibodies to other

proteins/peptides.

Psoriasis

Psoriasis is an autoimmune disease that affects the skin. It occurs when the immune system mistakes the skin cells for a pathogen, and sends out faulty signals that speed up the growth cycle of skin cells. There are five types of psoriasis: plaque, guttate, inverse, pustular, and erythrodermic. The most common form, plaque psoriasis, is commonly seen as red and white hues of scaly patches appearing on the top first layer of the epidermis (skin). The cause and pathogenesis of psoriasis is not fully

understood.

Hyper-citrullination has been suggested to play a role in the pathogenesis of psoriasis; hence the present authors suggest inhibition of PAD according to the present disclosure, i.e. via a cross-reactive anti-PAD antibody. In one embodiment of the present disclosure a cross-reactive anti-PAD antibody or fragment or variant thereof as disclosed herein is used in the treatment of psoriasis.

In one embodiment of the present disclosure, a cross-reactive anti-PAD antibody or fragment or variant thereof as disclosed herein is co-administered with another drug for the treatment of psoriasis. The co-administration may be simultaneous, sequential and/or separate.

Sjogren's Syndrome

Sjogren's syndrome is a systemic autoimmune disease in which immune cells attack and destroy the exocrine glands that produce tears and saliva. It is estimated to affect as many as 4 million people in the United States alone, making it the second most common rheumatic disease. Sjogren's syndrome can exist as a disorder in its own right (primary Sjogren's syndrome) or may develop years after the onset of an associated rheumatic disorder, such as rheumatoid arthritis, systemic lupus erythematosus, scleroderma, primary biliary cirrhosis etc. (secondary Sjogren's syndrome). Sjogren's syndrome frequently occurs secondary to rheumatoid arthritis.

Blood tests can be done to determine if a patient has high levels of antibodies that are indicative of the condition, such as anti-nuclear antibody (ANA) and rheumatoid factor (because SS frequently occurs secondary to rheumatoid arthritis), which are associated with autoimmune diseases. Around 10 % of the patients produce ACPAs.

The pathogenesis of Sjogren's Syndrome is not well understood. At present, there is no cure for Sjogren's syndrome, nor does a specific treatment exist to permanently restore gland secretion. Increased levels of PAD2 and citrullinated proteins have been detected in salivary glands from patients with Sjogrens Syndrome, as compared to healthy controls.

Citrullination may be a determining factor in the autoimmune response in Sjogrens syndrome, at least in a proportion of the patients with ACPAs. Hence, inhibition of PAD by means of a cross-reactive anti-PAD antibody is potentially a therapeutic approach applicable for Sjogrens syndrome. In one embodiment an anti-PAD antibody or fragment or variant thereof as disclosed herein is used in the treatment of Sjogren's syndrome. Comorbidity is the presence of one or more additional disorders or diseases co- occurring with a primary disease or disorder; or the effect of such additional disorders or diseases. Sjogren's syndrome frequently occurs secondary to rheumatoid arthritis. Thus, in one embodiment the cross-reactive anti-PAD antibody of the present disclosure is used in the treatment of rheumatoid arthritis and Sjogren's syndrome.

In one embodiment, the present disclosure relates to the use of a cross-reactive anti- PAD antibody or fragment or variant thereof as disclosed herein for the manufacture of a medicament for the treatment of Sjogren's syndrom. In one embodiment the cross-reactive anti-PAD antibody or fragment or variant thereof as disclosed herein is co-administered with another drug for the treatment of Sjogren's syndrome. The co-administration may be simultaneous, sequential and/or separate.

Inflammatory bowel disease

PAD levels have been found to be elevated in mouse and human colitis and PADs have previously been suggested as therapeutic target for the treatment of inflammatory bowel diseases such as Chrohn's disease and ulcerative colitis (Chumanevich et al., Am J Physiol Gastrointest Liver Physiol. 201 1 Jun; 300(6): G929-G938). In one embodiment an anti-PAD antibody or fragment or variant thereof as disclosed herein is used in the treatment of inflammatory bowel disease.

In one embodiment, the present disclosure relates to the use of a cross-reactive anti- PAD antibody or fragment or variant thereof as disclosed herein for the manufacture of a medicament for the treatment of inflammatory bowel disease.

In one embodiment the cross-reactive anti-PAD antibody or fragment or variant thereof as disclosed herein is co-administered with another drug for the treatment of inflammatory bowel disease. The co-administration may be simultaneous, sequential and/or separate. In one embodiment the inflammatory bowel disease is Chrohn's disease and/or ulcerative colitis. Cross-reactive anti-PAD antibodies

The cross-reactive anti-PAD antibodies or fragments or variants thereof disclosed herein are preferably capable of binding to human PAD2 and PAD4, i.e. they are cross- reactive. In one embodiment, the antibody is further capable of binding to PAD3. In some embodiments, the antibody of the present disclosure has a higher affinity for PAD2 and/or PAD4 than for PAD3 or other PAD isoforms such as PAD1 .

The antibodies disclosed herein were raised against amino acid sequences located in the catalytic domain of PAD2 and PAD4. In one embodiment, the cross-reactive anti- PAD antibodies or fragment or variant thereof thereof disclosed herein are capable of inhibiting citrullination by direct inhibition of PAD catalytic activity.

In one embodiment, the cross-reactive anti-PAD antibodies or fragments or variants thereof disclosed herein lead to an increased clearance of PAD enzymes. Clearance of PAD from blood and extracellular fluid can occur via Fc receptor-mediated endocytosis by phagocytic cells, or (as a consequence formation of complement-activating immune complexes with PAD) via binding to complement receptors CD35, CD1 1 b/CD18, CD1 1 c/CD18, on phagocytic cells. In one embodiment the cross-reactive anti-PAD antibodies or fragments or variants thereof disclosed herein inhibit the catalytic activity of at least one PAD isoform, such as at least PAD2 and/or PAD4.

In one embodiment, the cross-reactive anti-PAD antibodies or fragments or variants thereof disclosed herein increase clearance of at least one PAD isoform, such as at least PAD2 and/or PAD4.

In one embodiment, the cross-reactive anti-PAD antibodies or fragments or variants thereof disclosed herein inhibit catalytic activity of PAD and increase clearance of PAD. In the present context, the term "variant" refers to an antibody or a fragment thereof comprising one or more peptide sequences having a certain degree of sequence identity to certain partial antibody sequences, such as a certain degree of sequence identity to a VH or a VL region or a CDR. Variants also include affinity matured antibody variants. The variant antibodies disclosed herein preferably retain their ability to bind to human PAD2 and PAD4.

In the present context, the term "fragment" refers to a polypeptide comprising one or more partial immunoglobulin sequences for example a VH or a VL region or one or more CDRs. An antibody fragment may e.g. be a Fab fragment, a nanobody or a single-chain Fv fragment (scFv). The fragments disclosed herein preferably retain their ability to bind to human PAD2 and PAD4.

A "CDR" or a "CDR sequence" may also be referred to as a "CDR binding domain" and the terms are used interchangeably.

It is well-known in the art that antibodies exist as different isotypes: IgA, IgD, IgE, IgG and IgM. The human IgG and IgA isotypes can be further divided into subclasses lgG1 , lgG2, lgG3, lgG4, IgAI and lgA2, whereas the murine IgG isotype can be subdivided into subclasses lgG1 , lgG2a, lgG2b, lgG3.

In one embodiment, the antibody isotype is IgA, IgD, IgG and IgM, preferably IgG or IgA, even more preferred IgG. In one embodiment, the antibody isotype is not IgE.

The four IgG subclasses (lgG1 , 2, 3, and 4) in humans are named in order of their abundance in serum (lgG1 being the most abundant).

In one embodiment of the present disclosure the cross-reactive anti-PAD antibody is lgG1 .

In one embodiment of the present disclosure the cross-reactive anti-PAD antibody is lgG3.

The advantage of using lgG1 and lgG3 isotypes is that they efficiently activate the complement system and bind to Fc receptors on phagocytic cells with high affinity. As a result, said isotype subclasses lead to efficient clearance of target molecule, i.e. PAD isoforms and in particular PAD2 and PAD4. The Fc-part of an IgG antibody allows salvage of the antibody through the neonatal Fc receptor in the pathway of endocytosis in endothelial cells. Fc receptors in the acidic endosomes bind to IgG internalized through pinocytosis, recycling it to the cell surface, releasing it at the basic pH of blood, thereby preventing it from undergoing lysosomal degradation. This mechanism prolongs the half-life of IgG in the blood compared to other isotypes, and conjugation of some drugs to the Fc domain of IgG significantly increases their half-life.

The cross-reactive anti-PAD antibody of the present disclosure is in one embodiment a theraupeutic antibody drug for use in the treatment of an autoimmune disease, such as RA. The therapeutic antibody drug may e.g. be a monoclonal therapeutic antibody or a polyclonal therapeutic antibody.

In one embodiment the cross-reactive anti-PAD antibody of the present disclosure is a recombinant antibody.

In other embodiments of the present disclosure, the cross-reactive anti-PAD antibody or fragment or variant thereof is selected from the group consisting of: a fully non- human (e.g. murine) antibody, a chimeric (e.g. human-mouse) antibody, a humanized antibody, a fully human antibody, a drug comprising one or more Fab fragments, a nanobody, a single-chain Fv fragment (scFv), and a bispecific antibody.

In one embodiment of the present disclosure the cross-reactive anti-PAD antibody is a fully non-human antibody, such as a murine monoclonal antibody. Methods for producing antibodies in e.g. mice, rabbits and other animals are well-known in the art. In one embodiment of the present disclosure the cross-reactive anti-PAD antibody is a chimeric antibody. Chimeric antibodies are generally preferred over non-human antibodies in order to reduce the risk of a human anti-antibody response, e.g. a human anti-mouse antibody response in the case of a murine antibody. An example of a typical chimeric antibody is one in which the variable region sequences are murine sequences derived from immunization of a mouse, while the constant region

sequences are human. In the case of a chimeric antibody, the non-human parts, i.e. typically the framework regions of the variable region sequences, may be subjected to further alteration in order to humanize the antibody. Methods for producing chimeric antibodies based on the sequence of a non-human antibody are well-known in the art and are described by e.g. Chintalacharuvu et al. 1995. Chimeric Antibodies: Production and Applications. Methods: 8(2); 73-82.

In one embodiment of the present disclosure, the cross-reactive anti-PAD antibody is a humanized antibody. Humanized antibodies are approximately 90-95% human and 5- 10% non-human, e.g. mouse. The term "humanize" refers to the fact that where an antibody is wholly or partially of non-human origin, for example a murine antibody obtained from immunization of mice with an antigen of interest or a chimeric antibody based on such a murine antibody, it is possible to replace certain amino acids, in particular in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize an immune response in humans. It is known that all antibodies have the potential for eliciting a human anti-antibody response, which correlates to some extent with the degree of "humanness" of the antibody in question. Although it is not possible to precisely predict the immunogenicity and thereby the human anti-antibody response of a particular antibody, non-human antibodies tend to be more immunogenic than human antibodies. Chimeric antibodies, where the foreign (usually rodent) constant regions have been replaced with sequences of human origin, have been shown to be generally less immunogenic than antibodies of fully foreign origin, and the trend in therapeutic antibodies is towards humanized or fully human antibodies. For chimeric antibodies or other antibodies of non-human origin, it is therefore preferred that they be humanized to reduce the risk of a human anti-antibody response.

For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of a complementarity determining region (CDR) will typically not be altered in connection with humanization, although in certain cases it may be desirable to alter individual CDR amino acid residues, for example to remove a glycosylation site, a deamidation site or an undesired cysteine residue. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of an N- glycosylation site may be achieved by mutating either the Asn or the Ser/Thr residue to a different residue, preferably by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, in particular Asn-Gly, is present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues.

Numerous methods for humanization of an antibody sequence are known in the art; see e.g. the review by Aim agro & Fransson (2008) Front Biosci. 13 : 1619-1633. One commonly used method is CDR grafting, which for e.g. a murine-derived chimeric antibody involves identification of human germline gene counterparts to the murine variable region genes and grafting of the murine CDR sequences into this framework. CDR grafting may be based on the Kabat CDR definitions. Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR grafted non-human antibody, back mutations may be introduced at selected positions of the CDR grafted antibody in order to retain the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for back mutations are typically those that are located at the surface of an antibody molecule, while residues that are buried or that have a low degree of surface exposure will not normally be altered. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are altered to human residues.

In a particular or even preferred embodiment, the anti-PAD antibodies disclosed herein are humanized antibodies, such as fully human monoclonal antibodies, i.e. they have undergone the further process of antibody humanization of non-human monoclonal antibodies. This process is currently well-known to the skilled person, and also commercially available. For instance, software platforms which allows a robust, rapid and accurate modernized version of the traditional CDR grafting technique are developed and available, whereby the CDRs from the murine antibody sequences are identified and grafted into antibody frameworks to produce a panel of high quality, full length, humanized antibodies for expression. Antibody humanization and/or chimerization services are commercially available from i.a. Genscript (see e.g. US 61 /494,593), Fusion antibodies, and PX herapeutics.

Typical procedures for antibody humanization include one or more of steps 1 -5:

1 ) Production and characterization of the reference murine antibody and determination of its affinity constant, e.g. by BIACORE,

2) Determination of the specific murine variable region sequences,

3) Structural modeling of the mAb variable regions and construct of a panel of variants,

4) Affinity characterization and analysis of the humanized variants followed by recombinant expression in mammalian cells, and/or

5) If required, generation of additional variants to optimize antibody affinity. In certain cases, it may also be desirable to alter one or more CDR amino acid residues in order to improve binding affinity to the target epitope. This is known as "affinity maturation" and may optionally be performed in connection with humanization, for example in situations where humanization of an antibody leads to reduced binding specificity or affinity and it is not possible to sufficiently improve the binding specificity or affinity by back mutations alone. Various affinity maturation methods are known in the art, for example the in vitro scanning saturation mutagenesis method described by Burks et al. (1997) PNAS USA, vol. 94, pp. 412-417 and the stepwise in vitro affinity maturation method of Wu et al. (1998) PNAS USA, vol. 95, pp. 6037-6042. In one embodiment of the present disclosure, the anti-PAD antibody is an affinity- matured antibody.

In one embodiment of the present disclosure, the cross-reactive anti-PAD antibody is a fully human antibody. In one embodiment of the present disclosure, the anti-PAD antibody is a fully human antibody capable of inhibiting catalytic activity of at least PAD2 and PAD4.

In one embodiment of the present disclosure the cross-reactive anti-PAD antibody is a bispecific antibody, e.g. an antibody capable of binding to two different epitopes simultaneously, wherein said epitopes are present on the same or on different antigens.

A bispecific antibody according to the present disclosure may also comprise one antigen-binding region capable of binding to a PAD epitope and another antigen- binding region capable of binding to a different antigen e.g. to target the bispecific antibody to a preferred site of action. For example the other antigen may e.g. be a synovial membrane protein in case of RA or a blood-brain barrier protein in case of MS, as described previously.

Nanobodies (single-domain antibodies (sdAbs)) are antibody fragments consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single- domain antibodies are much smaller than common antibodies (150-160 kDa), and even smaller than Fab fragments (-50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (-25 kDa, two variable domains, one from a light and one from a heavy chain). These peptides have similar affinity to antigens as whole antibodies, but are more heat-resistant and stable towards detergents and high concentrations of urea. The comparatively low molecular mass leads to a better permeability in tissues, and to a short plasma half-life since they are eliminated renally. Unlike whole antibodies, they do not show complement system triggered cytotoxicity because they lack an Fc region. Camelid and fish derived sdAbs are able to bind to hidden antigens that are not accessible to whole antibodies, for example to the active sites of enzymes. This property has been shown to result from their extended CDR3 loop, which is able to penetrate such sites.

In one embodiment the cross-reactive anti-PAD antibody of the present disclosure is a fragment of an antibody, such as a Fab-fragment, a single-chain variable fragment (scFv) or a nanobody. Knowing the sequence of the monoclonal antibodies is the first step towards antibody engineering and function optimization. Sequencing of monoclonal antibodies is state of the art and automated antibody-sequencing services are performed by several companies. Sequencing of monoclonal antibodies may e.g. be performed by a method comprising the steps of: mRNA isolation, reverse transcription, PCR amplification of heavy and light chains, cloning into a standard sequencing vector; sequencing. Full- length antibody sequencing, CDR sequencing and characterization of antibodies may also be performed using a combination of N-terminal and internal Edman protein sequencing, capillary LC with microfraction collector, MS and MSMS mass

spectrometry and de novo peptide sequencing. LC and HC fragments of purified antibody are separately digested by several cleavage methods into peptides to get overlapping amino acid peptide sequences of the protein. The peptide mixtures are analyzed directly by nanoLC-ESI-MSMS and after capLC separation and fractionation of peptides by MALDI-MS / MSMS, nanoESI-MSMS and/or Edman protein sequencing. MSMS peptide fragmentation data are evaluated by de novo peptide sequencing and/or protein database search using available antibody sequencing software and relevant databases. The full protein amino acid sequence is derived by putting together the peptide puzzle of overlapping peptide sequences. Once the sequence of an antibody is known, the variable regions, such as the CDR regions, can easily by identified by state of the art bioinformatics tools known to a person of skill.

Cross-reactive anti-PAD antibody mSoU and fragments and variants thereof mSol1 1 was generated against anti-PAD4 629-645 (SEQ ID NO:46) as explained in Example 1 . mSoU comprises a VH amino acid sequence according to SEQ ID NO:1 and a VL amino acid sequence according to SEQ ID NO:6 and VH CDR sequences according to SEQ ID NOs: 2-4 and VL CDR sequences according to SEQ ID NOs: 7-9.

In one embodiment of the present disclosure there is provided an anti-PAD antibody or a fragment or variant thereof capable of binding to a PAD4 epitope consisting of SEQ ID NO:46, wherein said antibody or fragment thereof is capable of binding to at least PAD2 and PAD4. In one embodiment, the anti-PAD antibody or a fragment or variant thereof is further capable of binding to PAD3.

In one embodiment of the present disclosure there is provided an anti-PAD antibody or a fragment or variant thereof, wherein said anti-PAD antibody or fragment or variant thereof comprises a Heavy chain variable region (VH) comprising

a) a first CDR consisting of SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 75% sequence identity to SEQ ID NO:2, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:2,

b) a second CDR consisting of SEQ ID NO:3 or a variant of SEQ ID NO:3 having at least 75% sequence identity to SEQ ID NO:3, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:3,

c) a third CDR consisting of SEQ ID NO:4 or a variant of SEQ ID NO:4 having at least 75% sequence identity to SEQ ID NO:4, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:4,

In one embodiment of the present disclosure there is provided an anti-PAD antibody or fragment or variant thereof, wherein said anti-PAD antibody or fragment or variant thereof comprises a Light chain variable region (VL) comprising

a) a first CDR consisting of SEQ ID NO:7 or a variant of SEQ ID NO:7 having at least 75% sequence identity to SEQ ID NO:7, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:7,

b) a second CDR consisting of SEQ ID NO:8 or a variant of SEQ ID NO:8 having at least 75% sequence identity to SEQ ID NO:8, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:8,

c) a third CDR consisting of SEQ ID NO:9 or a variant of SEQ ID NO:9 having at least 75% sequence identity to SEQ ID NO:9, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:9,

In one embodiment of the present disclosure, the anti-PAD antibody or fragment or variant thereof comprises

a) a Heavy chain variable region (VH) comprising

i) a first CDR consisting of SEQ ID NO:2 or a variant of SEQ ID NO:2

having at least 75% sequence identity to SEQ ID NO:2, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:2,

ii) a second CDR consisting of SEQ ID NO:3 or a variant of SEQ ID NO:3 having at least 75% sequence identity to SEQ ID NO:3, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID

NO:3,

iii) a third CDR consisting of SEQ ID NO:4 or a variant of SEQ ID NO:4

having at least 75% sequence identity to SEQ ID NO:4, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:4,

b) a Light chain variable region (VL) comprising

i) a first CDR consisting of SEQ ID NO:7 or a variant of SEQ ID NO:7

having at least 75% sequence identity to SEQ ID NO:7, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:7,

ii) a second CDR consisting of SEQ ID NO:8 or a variant of SEQ ID NO:8 having at least 75% sequence identity to SEQ ID NO:8, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:8,

iii) a third CDR consisting of SEQ ID NO:9 or a variant of SEQ ID NO:9 having at least 75% sequence identity to SEQ ID NO:9, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:9.

In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises a Heavy chain variable region (VH) consisting of SEQ ID NO:1 , or a variant of SEQ ID NO:1 having at least 75% sequence identity to SEQ ID NO:1 , such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 .

In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises a Light chain variable region (VL) consisting of SEQ ID NO:6, or a variant of SEQ ID NO:6 having at least 75% sequence identity to SEQ ID NO:6, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:6. In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises

a) a Heavy chain variable region (VH) consisting of SEQ ID NO:1 , or a variant of SEQ ID NO:1 having at least 75% sequence identity to SEQ ID NO:1 , such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 , and

b) a Light chain variable region (VL) consisting of SEQ ID NO:6, or a variant of SEQ ID NO:6 having at least 75% sequence identity to SEQ ID NO:6, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:6.

In one embodiment, the sequence variation is outside the CDRs, i.e. the sequence variation is restricted to the framework regions and the CDRs are unaltered. Thus, in one embodiment, the sequence variation is not in the CDRs.

In one embodiment, the cross-reactive antibody or fragment or variant thereof is capable of at least partially inhibiting the enzymatic activity of one or more PAD isoforms, such as PAD2 and/or PAD4.

In one embodiment, the cross-reactive antibody or fragment or variant thereof is capable of increasing clearance of one or more PAD isoforms, such as at least PAD2 and/or PAD4. In one embodiment, the cross-reactive antibody is a monoclonal antibody. In one embodiment, the cross-reactive antibody is a chimeric antibody. In one embodiment, the cross-reactive antibody is a humanized antibody.

In one embodiment, the cross-reactive antibody is a fully human antibody.

In one embodiment, the cross-reactive antibody is an antibody of an IgG isotype selected from the group consisting of lgG1 and lgG3. In one embodiment, the cross-reactive antibody or fragment of variant thereof is a fragment of an antibody, such as a Fab-fragment, a single-chain variable fragment (scFv) or a nanobody.

In one embodiment, the cross-reactive antibody is an affinity matured variant of an antibody, wherein the affinity matured variant has a higher affinity for PAD2 and/or PAD4, in particular human PAD2 and/or PAD4, as compared to an antibody comprising a) a Heavy chain variable region (VH) consisting of SEQ ID NO:1 , and

b) a Light chain variable region (VL) consisting of SEQ ID NO:6

In one embodiment, the cross-reactive antibody is an affinity matured variant of an antibody, wherein the affinity matured variant has a higher affinity for PAD2 and/or PAD4, in particular human PAD2 and/or PAD4, as compared to an antibody comprising a) a Heavy chain variable region (VH) comprising a first CDR consisting of SEQ ID NO:2,

b) a Heavy chain variable region (VH) comprising a second CDR consisting of SEQ ID NO:3,

c) a Heavy chain variable region (VH) comprising a third CDR consisting of SEQ ID NO:4,

d) a Light chain variable region (VL) comprising a first CDR consisting of SEQ ID NO:7,

e) a Light chain variable region (VL) comprising a second CDR consisting of SEQ ID NO:8,

f) a Light chain variable region (VL) comprising a third CDR consisting of SEQ ID NO:9.

Cross-reactive anti-PAD antibody mSol2 and fragments and variants thereof mSol2 was generated against anti-PAD2 462-477 (SEQ ID NO:48) as explained in Example 1 . mSol2 comprises a VH amino acid sequence according to SEQ ID NO:1 1 and a VL amino acid sequence according to SEQ ID NO:16 and VH CDR sequences according to SEQ ID NOs:12-14 and VL CDR sequences according to SEQ ID NOs:17- 19. In one embodiment of the present disclosure there is provided an anti-PAD antibody or a fragment or variant thereof capable of binding to a PAD2 epitope consisting of SEQ ID NO:48, wherein said antibody or fragment thereof is capable of binding to at least PAD2 and PAD4.

In one embodiment, the anti-PAD antibody or a fragment or variant thereof is further capable of binding to PAD3.

a) a first CDR consisting of SEQ ID NO:12 or a variant of SEQ ID NO:12 having at least 75% sequence identity to SEQ ID NO:12, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:12,

b) a second CDR consisting of SEQ ID NO:13 or a variant of SEQ ID NO:13

having at least 75% sequence identity to SEQ ID NO: 13, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:13, c) a third CDR consisting of SEQ ID NO:14 or a variant of SEQ ID NO:14 having at least 75% sequence identity to SEQ ID NO:14, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:14,

a) a first CDR consisting of SEQ ID NO:17 or a variant of SEQ ID NO:17 having at least 75% sequence identity to SEQ ID NO:17, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:17,

b) a second CDR consisting of SEQ ID NO:18 or a variant of SEQ ID NO:18

having at least 75% sequence identity to SEQ ID NO: 18, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:18, c) a third CDR consisting of SEQ ID NO:19 or a variant of SEQ ID NO:19 having at least 75% sequence identity to SEQ ID NO:19, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:19, In one embodiment of the present disclosure, the anti-PAD antibody or fragment or variant thereof comprises

a) a Heavy chain variable region (VH) comprising

i) a first CDR consisting of SEQ ID NO:12 or a variant of SEQ ID NO:12 having at least 75% sequence identity to SEQ ID NO: 12, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:12,

ii) a second CDR consisting of SEQ ID NO:13 or a variant of SEQ ID NO:13 having at least 75% sequence identity to SEQ ID NO: 13, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:13,

iii) a third CDR consisting of SEQ ID NO:14 or a variant of SEQ ID NO:14 having at least 75% sequence identity to SEQ ID NO: 14, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:14,

b) a Light chain variable region (VL) comprising

i) a first CDR consisting of SEQ ID NO:17 or a variant of SEQ ID NO:17 having at least 75% sequence identity to SEQ ID NO: 17, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:17,

ii) a second CDR consisting of SEQ ID NO:18 or a variant of SEQ ID NO:18 having at least 75% sequence identity to SEQ ID NO: 18, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:18,

iii) a third CDR consisting of SEQ ID NO:19 or a variant of SEQ ID NO:19 having at least 75% sequence identity to SEQ ID NO:19, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:19.

In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises a Heavy chain variable region (VH) consisting of SEQ ID NO:1 1 , or a variant of SEQ ID NO:1 1 having at least 75% sequence identity to SEQ ID NO:1 1 , such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 1 . In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises a Light chain variable region (VL) consisting of SEQ ID NO:16, or a variant of SEQ ID NO:16 having at least 75% sequence identity to SEQ ID NO:16, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:16.

In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises

a) a Heavy chain variable region (VH) consisting of SEQ ID NO:1 1 , or a variant of SEQ ID NO:1 1 having at least 75% sequence identity to SEQ ID NO:1 1 , such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 1 , and

b) a Light chain variable region (VL) consisting of SEQ ID NO:16, or a variant of SEQ ID NO:16 having at least 75% sequence identity to SEQ ID NO:16, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:16.

In one embodiment, the cross-reactive antibody or fragment or variant thereof is capable of increasing clearance of one or more PAD isoforms, such as at least PAD2 and/or PAD4. In one embodiment, the cross-reactive antibody is a monoclonal antibody. In one embodiment, the cross-reactive antibody is a chimeric antibody. In one embodiment, the cross-reactive antibody is a humanized antibody. In one embodiment, the cross-reactive antibody is a fully human antibody.

In one embodiment, the cross-reactive antibody is an antibody of an IgG isotype selected from the group consisting of lgG1 and lgG3.

In one embodiment, the cross-reactive antibody or fragment of variant thereof is a fragment of an antibody, such as a Fab-fragment, a single-chain variable fragment (scFv) or a nanobody.

In one embodiment, the cross-reactive antibody is an affinity matured variant of an antibody, wherein the affinity matured variant has a higher affinity for PAD2 and/or PAD4, in particular human PAD2 and/or PAD4, as compared to an antibody comprising a) a Heavy chain variable region (VH) consisting of SEQ ID NO:1 1 , and b) a Light chain variable region (VL) consisting of SEQ ID NO:16

In one embodiment, the cross-reactive antibody is an affinity matured variant of an antibody, wherein the affinity matured variant has a higher affinity for PAD2 and/or PAD4, in particular human PAD2 and/or PAD4, as compared to an antibody comprising a) a Heavy chain variable region (VH) comprising a first CDR consisting of SEQ ID NO:12,

b) a Heavy chain variable region (VH) comprising a second CDR consisting of SEQ ID NO:13,

c) a Heavy chain variable region (VH) comprising a third CDR consisting of SEQ ID NO:14,

d) a Light chain variable region (VL) comprising a first CDR consisting of SEQ ID NO:17,

e) a Light chain variable region (VL) comprising a second CDR consisting of SEQ ID NO:18,

f) a Light chain variable region (VL) comprising a third CDR consisting of SEQ ID NO:19.

Cross-reactive anti-PAD antibody mSol3 and fragments and variants thereof mSol3 was generated against anti-PAD4 629-645 (SEQ ID NO: 46) as explained in Example 1 . mSol3 comprises a VH amino acid sequence according to SEQ ID NO:21 and a VL amino acid sequence according to SEQ ID NO:26 and VH CDR sequences according to SEQ ID NOs:22-24 and VL CDR sequences according to SEQ ID NOs:27- 29. In one embodiment of the present disclosure there is provided an anti-PAD antibody or a fragment or variant thereof capable of binding to a PAD4 epitope consisting of SEQ ID NO:46, wherein said antibody or fragment thereof is capable of binding to at least PAD2 and PAD4. In one embodiment, the anti-PAD antibody or a fragment or variant thereof is further capable of binding to PAD3.

a) a first CDR consisting of SEQ ID NO:22 or a variant of SEQ ID NO:22 having at least 75% sequence identity to SEQ ID NO:22, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:22,

b) a second CDR consisting of SEQ ID NO:23 or a variant of SEQ ID NO:23

having at least 75% sequence identity to SEQ ID NO:23, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:23, c) a third CDR consisting of SEQ ID NO:24 or a variant of SEQ ID NO:24 having at least 75% sequence identity to SEQ ID NO:24, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:24,

a) a first CDR consisting of SEQ ID NO:27 or a variant of SEQ ID NO:27 having at least 75% sequence identity to SEQ ID NO:27, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:27,

b) a second CDR consisting of SEQ ID NO:28 or a variant of SEQ ID NO:28

having at least 75% sequence identity to SEQ ID NO:28, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:28, c) a third CDR consisting of SEQ ID NO:29 or a variant of SEQ ID NO:29 having at least 75% sequence identity to SEQ ID NO: 29, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:29,

a) a Heavy chain variable region (VH) comprising

i) a first CDR consisting of SEQ ID NO:22 or a variant of SEQ ID NO:22 having at least 75% sequence identity to SEQ ID NO:22, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:22,

ii) a second CDR consisting of SEQ ID NO:23 or a variant of SEQ ID NO:23 having at least 75% sequence identity to SEQ ID NO:23, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:23,

iii) a third CDR consisting of SEQ ID NO:24 or a variant of SEQ ID NO:24 having at least 75% sequence identity to SEQ ID NO:24, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:24,

b) a Light chain variable region (VL) comprising

i) a first CDR consisting of SEQ ID NO:27 or a variant of SEQ ID NO:27 having at least 75% sequence identity to SEQ ID NO:27, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:27,

ii) a second CDR consisting of SEQ ID NO:28 or a variant of SEQ ID NO:28 having at least 75% sequence identity to SEQ ID NO:28, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:28,

iii) a third CDR consisting of SEQ ID NO:29 or a variant of SEQ ID NO:29 having at least 75% sequence identity to SEQ ID NO:29, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:29.

In one embodiment of the present disclosure, there is provided an anti-PAD antibody fragment or variant thereof, wherein said antibody comprises a Heavy chain variable region (VH) consisting of SEQ ID NO:21 , or a variant of SEQ ID NO:21 having at least 75% sequence identity to SEQ ID NO:21 , such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:21 .

In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises a Light chain variable region (VL) consisting of SEQ ID NO:26, or a variant of SEQ ID NO:26 having at least 75% sequence identity to SEQ ID NO:26, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:26.

a) a Heavy chain variable region (VH) consisting of SEQ ID NO:21 , or a variant of SEQ ID NO:21 having at least 75% sequence identity to SEQ ID NO:21 , such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:21 , and

b) a Light chain variable region (VL) consisting of SEQ ID NO:26, or a variant of SEQ ID NO:26 having at least 75% sequence identity to SEQ ID NO:26, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:26.

In one embodiment, the sequence variation is outside the CDRs, i.e. the sequence variation is restricted to the framework regions and the CDRs are unaltered. Thus, i one embodiment, the sequence variation is not in the CDRs.

In one embodiment, the cross-reactive antibody or fragment or variant thereof is capable of increasing clearance of one or more PAD isoforms, such as at least PAD2 and/or PAD4.

In one embodiment, the cross-reactive antibody is a monoclonal antibody. In one embodiment, the cross-reactive antibody is a chimeric antibody.

In one embodiment, the cross-reactive antibody is a humanized antibody. In one embodiment, the cross-reactive antibody is a fully human antibody.

In one embodiment, the cross-reactive antibody is an affinity matured variant of an antibody, wherein the affinity matured variant has a higher affinity for PAD2 and/or PAD4, in particular human PAD2 and/or PAD4, as compared to an antibody comprising a) a Heavy chain variable region (VH) consisting of SEQ ID NO:21 , and b) a Light chain variable region (VL) consisting of SEQ ID NO:26

In one embodiment, the cross-reactive antibody is an affinity matured variant of an antibody, wherein the affinity matured variant has a higher affinity for PAD2 and/or PAD4, in particular human PAD2 and/or PAD4, as compared to an antibody comprising a) a Heavy chain variable region (VH) comprising a first CDR consisting of SEQ ID NO:22,

b) a Heavy chain variable region (VH) comprising a second CDR consisting of SEQ ID NO:23,

c) a Heavy chain variable region (VH) comprising a third CDR consisting of SEQ ID NO:24,

d) a Light chain variable region (VL) comprising a first CDR consisting of SEQ ID NO:27,

e) a Light chain variable region (VL) comprising a second CDR consisting of SEQ ID NO:28,

f) a Light chain variable region (VL) comprising a third CDR consisting of SEQ ID NO:29. Cross-reactive anti-PAD antibody mSol4 and fragments and variants thereof mSol4 was generated against anti-PAD2 462-477 (SEQ ID NO:48) as explained in Example 1 . mSol4 comprises a VH amino acid sequence according to SEQ ID NO:31 and a VL amino acid sequence according to SEQ ID NO:36 and VH CDR sequences according to SEQ ID NOs:32-34 and VL CDR sequences according to SEQ ID NOs:37- 39.

In one embodiment of the present disclosure there is provided an anti-PAD antibody or a fragment or variant thereof capable of binding to a PAD2 epitope consisting of SEQ ID NO:48, wherein said antibody or fragment thereof is capable of binding to at least PAD2 and PAD4.

a) a first CDR consisting of SEQ ID NO:32 or a variant of SEQ ID NO:32 having at least 75% sequence identity to SEQ ID NO:32, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:32,

b) a second CDR consisting of SEQ ID NO:33 or a variant of SEQ ID NO:33

having at least 75% sequence identity to SEQ ID NO: 33, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:33, c) a third CDR consisting of SEQ ID NO:34 or a variant of SEQ ID NO:34 having at least 75% sequence identity to SEQ ID NO:34, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:34, In one embodiment of the present disclosure there is provided an anti-PAD antibody or fragment or variant thereof, wherein said anti-PAD antibody or fragment or variant thereof comprises a Light chain variable region (VL) comprising

a) a first CDR consisting of SEQ ID NO:37 or a variant of SEQ ID NO:37 having at least 75% sequence identity to SEQ ID NO:37, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:37, a second CDR consisting of SEQ ID NO:38 or a variant of SEQ ID NO:38 having at least 75% sequence identity to SEQ ID NO:38, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:38, a third CDR consisting of SEQ ID NO:39 or a variant of SEQ ID NO:39 having at least 75% sequence identity to SEQ ID NO:39, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:39,

a) a Heavy chain variable region (VH) comprising

i) a first CDR consisting of SEQ ID NO:32 or a variant of SEQ ID NO:32 having at least 75% sequence identity to SEQ ID NO:32, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:32,

ii) a second CDR consisting of SEQ ID NO:33 or a variant of SEQ ID NO:33 having at least 75% sequence identity to SEQ ID NO:33, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:33,

iii) a third CDR consisting of SEQ ID NO:34 or a variant of SEQ ID NO:34 having at least 75% sequence identity to SEQ ID NO:34, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:34,

b) a Light chain variable region (VL) comprising

i) a first CDR consisting of SEQ ID NO:37 or a variant of SEQ ID NO:37 having at least 75% sequence identity to SEQ ID NO:37, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:37,

ii) a second CDR consisting of SEQ ID NO:38 or a variant of SEQ ID NO:38 having at least 75% sequence identity to SEQ ID NO:38, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to

SEQ ID NO:38,

iii) a third CDR consisting of SEQ ID NO:39 or a variant of SEQ ID NO:39 having at least 75% sequence identity to SEQ ID NO:39, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:39. In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises a Heavy chain variable region (VH) consisting of SEQ ID NO:31 , or a variant of SEQ ID NO:31 having at least 75% sequence identity to SEQ ID NO:31 , such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:31 .

In one embodiment of the present disclosure, there is provided an anti-PAD antibody or fragment or variant thereof, wherein said antibody comprises a Light chain variable region (VL) consisting of SEQ ID NO:36, or a variant of SEQ ID NO:36 having at least 75% sequence identity to SEQ ID NO:36, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:36.

a) a Heavy chain variable region (VH) consisting of SEQ ID NO:31 , or a variant of SEQ ID NO:31 having at least 75% sequence identity to SEQ ID NO:31 , such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:31 , and

b) a Light chain variable region (VL) consisting of SEQ ID NO:36, or a variant of

SEQ ID NO:36 having at least 75% sequence identity to SEQ ID NO:36, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:36. In one embodiment, the sequence variation is outside the CDRs, i.e. the sequence variation is restricted to the framework regions and the CDRs are unaltered. Thus, in one embodiment, the sequence variation is not in the CDRs.

In one embodiment, the cross-reactive antibody or fragment or variant thereof is capable of increasing clearance of one or more PAD isoforms, such as at least PAD2 and/or PAD4. In one embodiment, the cross-reactive antibody is a monoclonal antibody.

In one embodiment, the cross-reactive antibody is a chimeric antibody.

In one embodiment, the cross-reactive antibody is a humanized antibody.

In one embodiment, the cross-reactive antibody is a fully human antibody.

In one embodiment, the cross-reactive antibody is an affinity matured variant of an antibody, wherein the affinity matured variant has a higher affinity for PAD2 and/or PAD4, in particular human PAD2 and/or PAD4, as compared to an antibody comprising a) a Heavy chain variable region (VH) consisting of SEQ ID NO:31 , and b) a Light chain variable region (VL) consisting of SEQ ID NO:36

In one embodiment, the cross-reactive antibody is an affinity matured variant of an antibody, wherein the affinity matured variant has a higher affinity for PAD2 and/or PAD4, in particular human PAD2 and/or PAD4, as compared to an antibody comprising a) a Heavy chain variable region (VH) comprising a first CDR consisting of SEQ ID NO:32,

b) a Heavy chain variable region (VH) comprising a second CDR consisting of SEQ ID NO:33,

c) a Heavy chain variable region (VH) comprising a third CDR consisting of SEQ

ID NO:34,

d) a Light chain variable region (VL) comprising a first CDR consisting of SEQ ID NO:37,

e) a Light chain variable region (VL) comprising a second CDR consisting of SEQ ID NO:38, f) a Light chain variable region (VL) comprising a third CDR consisting of SEQ ID NO:39.

Therapeutic compositions

Another aspect of the disclosure is a pharmaceutical composition comprising as an active ingredient a cross-reactive anti-PAD antibody or a fragment or variant thereof. Such compositions are intended for amelioration, prevention and/or curative treatment of autoimmune diseases characterized by extracellular citrullination, preferably hyper- citrullination. The pharmaceutical composition may be administered to a human subject or to a domestic animal or pet, but will typically be administered to humans.

In addition to at least one antibody of the invention, the pharmaceutical composition will usually further comprise at least one pharmaceutically acceptable diluent, carrier or excipient. These may for example include preservatives, stabilizers, surfactants/wetting agents, emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers. Solutions or suspensions may further comprise viscosity-increasing substances, such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin. A suitable pH value for the pharmaceutical composition will generally be in the range of about 5.5 to 8.5, such as about 6 to 8, e.g. about 7, maintained where appropriate by use of a buffer.

Conventional pharmaceutical practice may be employed to provide suitable

formulations or compositions. The administration may be prophylactic; meaning that treatment is initiated before clinical symptoms of the disease appears. The treatment will, however, typically be therapeutic, meaning that it is administered after a particular autoimmune disease has been diagnosed due to the manifestation of clinical symptoms. Any appropriate route of administration may be employed, for example parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, intranasal, aerosol, suppository or oral administration. Pharmaceutical compositions of the invention will typically be administered in the form of liquid solutions or

suspensions, more typically aqueous solutions or suspensions, in particular isotonic aqueous solutions or suspensions. The pharmaceutical compositions of the invention are prepared in a manner known per se, for example, by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, for example, Remington : The Science and Practice of Pharmacy (21 st edition), ed . A. R. Gennaro, 2005, Lippincott Williams & Wilkins, Philadelph ia, PA, USA; and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, 3rd edition, 2006, Informa Healthcare, New York, NY, USA). As an alternative to a liquid formulation, the compositions of the invention may be prepared in lyophilized form comprising at least one antibody alone or together with a carrier, for example mannitol, in which case the composition is reconstituted with a liquid such as sterile water prior to use. The pharmaceutical compositions comprise from approximately 1 % to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may e.g. be produced in unit dose form, such as in the form of ampoules, vials, suppositories, tablets or capsules. The formulations can be administered to human individuals in therapeutically or prophylactically effective amounts (e.g., amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for an autoimmune disease or other condition. The preferred dosage of therapeutic agent to be administered is likely to depend on such variables as the severity of the disease, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration.

The antibodies or fragments or variants thereof and compositions of the invention will be administered in an effective amount for treatment of the condition in question, i.e. at dosages and for periods of time necessary to achieve a desired result. The dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, the effect desired, and whether the cross-reactive antibodies of the present disclosure are being administered as a stand-alone treatment or in combination with one or more additional treatments. An effective amount for therapy may be measured by its ability to inhibit disease development, to stabilize disease progression and/or ameliorate symptoms in a patient, and preferably to reverse disease progression. The ability of an antibody or

composition of the invention to inhibit the autoimmune disease of the present invention may be evaluated in suitable animal models that are predictive of the efficacy in human patients. Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a single bolus or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case.

Usually a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight, such as about 0.1 to 80 milligrams per kilogram of body weight, for example 1 to 50 milligrams per kilogram of body weight, such as 1 to 30 milligrams per kilogram of body weight, for example 1 to 20 milligrams per kilogram of body weight, such as 1 to 10 milligrams per kilogram of body weight, for example 1 to 5 milligrams per kilogram of body weight.

The cross-reactive anti-PAD antibody or fragment or variant thereof may be

administered once a day or several times a day. The cross-reactive anti-PAD antibody or fragment or variant thereof may also be administered at intervals such as once a week, twice a week, three times a week, once every other week, once every three weeks, once every four weeks or once a month. Dosage forms suitable for administration generally contain from about 0.01 milligram to about 1000 milligrams of cross-reactive anti-PAD antibody or fragment or variant thereof per dose, such as about 0.05 milligram to about 500 milligrams of cross- reactive anti-PAD antibody or fragment or variant thereof per dose, for example about 0.1 milligram to about 500 milligrams of cross-reactive anti-PAD antibody or fragment or variant thereof per dose, such as about 1 milligram to about 500 milligrams of cross- reactive anti-PAD antibody or fragment or variant thereof per dose, for example about 5 milligram to about 500 milligrams of cross-reactive anti-PAD antibody or fragment or variant thereof per dose, such as about 10 milligram to about 500 milligrams of cross- reactive anti-PAD antibody or fragment or variant thereof per dose, for example about 20 milligram to about 500 milligrams of cross-reactive anti-PAD antibody or fragment or variant thereof per dose, such as about 30 milligram to about 500 milligrams of cross- reactive anti-PAD antibody or fragment or variant thereof per dose, for example about 40 milligram to about 500 milligrams of cross-reactive anti-PAD antibody or fragment or variant thereof per dose such as about 50 milligram to about 500 milligrams of cross- reactive anti-PAD antibody or fragment or variant thereof per dose.

In one embodiment the cross-reactive anti-PAD antibody or fragment or variant thereof of the present disclosure is administered in doses of about 0.01 to 10 mg/kg/dose, such as about 0.05 to 5.0 mg/kg/dose, for example about 0.1 to 5 mg/kg/dose, such as about 0.2 to 5.0 mg/kg/dose, for example about 0.3 to 5 mg/kg/dose, such as about 0.4 to 5.0 mg/kg/dose, for example about 0.5 to 5 mg/kg/dose.

The cross-reactive anti-PAD antibody or fragment or variant thereof may e.g. be administered in a similar manner as described for other biologies for treatment of RA e.g. the anti-TNF medications etanercept, adalimumab, certolizumab and golimumab are usually administered by injection under the skin (e.g. injected into the thigh or abdomen) and infliximab is administered by intravenous infusion over several hours.

In one embodiment the cross-reactive anti-PAD antibodies or fragments or variants thereof of the present disclosure are administered as a stand-alone treatment or in combination with one or more additional treatments.

In one embodiment the cross-reactive anti-PAD antibodies or fragments or variants thereof of the present disclosure are administered essentially as described in the below table for anti-TNF drugs for treatment of RA.

In one embodiment the cross-reactive anti-PAD antibody of the present disclosure is used in the treatment of an autoimmune disease. The autoimmune disease is preferably a disease where extracellular citrullination plays a role in the pathogenesis of said disease, such as a disease selected from the group consisting of rheumatoid arthritis, multiple sclerosis and psoriasis. The treatment may be prophylactic, ameliorative and/or curative.

In one embodiment the present disclosure relates to the use of a cross-reactive anti- PAD antibody as disclosed herein for the manufacture of a medicament for the treatment of an autoimmune disease characterized by extracellular citrullination, such as RA.

In one embodiment the present disclosure relates to the use of a cross-reactive anti- PAD antibody as disclosed herein for use as a medicament.

In one embodiment the present disclosure relates to the use of a cross-reactive anti- PAD antibody as disclosed herein for use in the treatment of an autoimmune disease characterized by extracellular citrullination. Preferably, the extracellular citrullination is involved in the pathogenesis of the autoimmune disease and the citrullination may be hyper-citrullination.

In one embodiment the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, inflammatory bowel disease and psoriasis.

In one embodiment the present disclosure relates to a method of treating a subject suffering from an autoimmune disease characterized by extracellular citrullination comprising the administration of a suitable amount of the anti-PAD antibody or fragment or variant thereof as disclosed herein to said subject.

The subject is preferably a human.

Examples

The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

Example 1. Generation of anti-PAD cross-reactive monoclonal antibodies

The catalytic site of PAD2 and PAD4, comprises the following sequences, respectively: PAD4; 400-410 GISGLDSFGNL (SEQ ID NO:43)

PAD4; 462-477 LYSDWLSVGHVDEFLS (SEQ ID NO:44)

PAD4: 569-589 QLFKLKEFSKAEAFFPNMVNM (SEQ ID NO:45)

PAD4: 629-645 FINDFFTYHIRHGEVHC (SEQ ID NO:46)

PAD2: 400-410 ESVTSLDSFGNL (SEQ ID NO:47)

PAD2: 462-477 LYSDWLTVGHVDEFMS (SEQ ID NO:48)

PAD2; 569-589 ALFKMDEDHRARAFFPNMVNM (SEQ ID NO:49)

PAD2; 629-645 FIDDISAYHKFLGEVHC (SEQ ID NO:50)

Four peptides in total were selected for the development of cross-reacting anti-PAD antibodies against the catalytic site on PAD2 and PAD4. Two peptides were chosen from the human PAD2 catalytic site (PAD2; 400-410 and PAD2; 462-477) and two from the human PAD4 catalytic site (PAD4; 569-589 and PAD4; 629-645) - these are underlined above. For immunisation, a cysteine residue was added at the C-terminal end of the peptides according to standard immunisation techniques. The monoclonal murine antibodies were generated using standard techniques known in the art and essentially as described previously in WO 2014/086365.

After preliminary screening, the PAD2; 400-410 peptide was abandoned due to inferior results.

Figure 1 shows the binding profile of the generated antibodies (36 in total) against full length human recombinant PAD2 or PAD4-coated plates (100 ng/mL). Shown is the absorbance at a dilution of 1 :20 in wash buffer of each of the culture supernatants. HRP-conjugated rabbit anti-mouse antibodies, diluted 1 :1000 in wash buffer, were added for 1 hour at RT, and the plates were developed with OPD substrate. The levels are given as OD490-650 nm-units.

Several of the generated antibodies showed cross-reactivity with PAD2 and PAD4 and were capable of binding to both PAD2 and PAD4 with high affinity. Eight of the high affine cross-reacting antibodies were selected for sequencing. Four of the eight sequenced antibodies showed good potential for humanization and were selected for further in vitro studies. The four selected antibodies were designated mSol1 -4:

The affinity of the four selected mAbs mSol1 -4 were purified and tested on plates coated with full-length human recombinant PAD2, PAD3 or PAD4-coated plates (100 ng/mL). Shown is the absorbance at a dilution of 1 :100 in wash buffer of purified mAbs. HRP-conjugated rabbit anti-mouse antibodies, diluted 1 :1000 in wash buffer, were added for 1 hour at RT, and the plates were developed with OPD substrate. The levels are given as OD490-650 nm-units. All antibodies are capable of binding to PAD2, PAD3 and PAD4, however, the tested antibodies differ in the affinity for the different PAD isoforms. Affintiy of mSol1 -4 against PAD2, PAD3 and PAD4 is shown in figure 2.

To demonstrate that mSol1 -4 were also capable of binding to murine PAD, the affinity of the mAbs for murine PAD2 was investigated and compared to the affinity for human PAD2. The results are shown in figure 3. As predicted, the results confirm that the mAbs bind murine and human PAD2 with similar affinity, presumably due to high interspecies homology between the murine and human catalytic sites. To test the inhibitory effect of mSol1 -4 on PAD enzyme activity, the mAbs were tested in an in vitro setup using PAD2 as an exemplary PAD isoform. The results are depicted in figure 4. The ability of the 4 selected mAbs to inhibit citrullination of fibrinogen was tested using recombinant human PAD2 (rhPAD2) as example of catalyst. Test of the inhibitory capacity of mSoU , mSol2, mSol3, mSol4, and a control mAb (anti-SCUBE1 ) was performed. Human fibrinogen (200 ug/mL) was incubated in citrullination buffer (100 mM Tris-HCI, 10 mM CaCI2, 1 mM DTT, pH 7.5) with 200 ng/mL rhPAD2 for 4 hours at 37 ^eC in the presence of the listed mAbs (10 μg/mL). The citrullinated fibrinogen (incubated with each listed mAbs) was applied to ELISA plates, coated with 1 μg/mL rabbit polyclonal fibrinogen for 2 hours at room temperature. Following three washes, biotinylated anti-citrullinated fibrinogen antibody (clone 20B2, Cat No.

MQ13.102, ModiQuest, Oss, Netherlands) was applied (0.5 μg/mL) for 1 hour at room temperature. HRP-conjugated streptavidin, diluted 1 :3000 in wash buffer, was added for 1 hour at RT, and the plates were developed with OPD substrate. The levels are given as OD490-650 nm-units.

The results show that the tested antibodies are capable of at least partially inhibiting PAD2 mediated citrullination of fibrinogen. In conclusion, we have generated four cross-reactive anti-PAD antibodies displaying good affinity for both PAD2 and PAD4 and which are capable of at least partially inhibiting PAD catalytic activity.

Example 2. DNA sequencing of anti-PAD2 antibodies

Selected antibodies were sequenced and CDR analysis performed according to standard methods in the art essentially as described previously in WO 2014/086365.

The DNA sequencing was successful and provided 1 heavy and 1 light chain variable region for each hybridoma. CDR sequence analysis tools (www.ebi.ac.uk) provided the CDR sequences.

Results - mSol1 :

Heavy chain variable region (VH): amino acid sequence (SEQ ID NO:1 ) LPQVKLEQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWIRQSPGKGLEWLGVIWSRG STDYNAAFISRLSISKDNSKSQVFFKMNGLQANDTAIYYCARIRGELGPYWYFDVRGA GTTVTVSS Heavy chain variable region (VH): nucleotide sequence (SEQ ID NO:5)

GGAAGGTGTGCACACCGCTGGACAGGGATCCAGAGTTCCAGGTCAAGGTCACTG GCTCAGGGAAATAACCCTTGACCAGGCATCCTAGAGTCACCGAGGAGCCAGTTGT ATCTCCACACACAGGGGCCAGTGGATAGACCGATGGGGCTGTTGTTTTGGCTGA GGAGACGGTGACCGTGGTCCCTGCGCCCCTGACATCGAAGTACCAGTAAGGTCC CAGTTCCCCCCGAATTCTGGCACAGTAATATATGGCTGTGTCATTAGCTTGCAGAC CGTTCATTTTAAAGAAAACTTGGCTCTTGGAATTGTCCTTGCTGATGCTCAGTCTG GATATGAAAGCTGCATTATAGTCTGTGCTTCCACGACTCCATATCACTCCCAGCCA CTCCAGACCCTTTCCTGGAGACTGGCGAATCCAGTGTACACCATAGCTAGTTAAT GAGAAACCAGAGACTGTGCAGGTGATGGACAGGCTCTGTGAGGGCTGCACTAGG CCAGGTCCTGACTGCTCCAGCTTAACCTGCGGAAG

Heavy chain variable region (VH) - CDR analysis:

CDR1 (VH): GFSLTSYG (SEQ ID NO:2)

CDR2 (VH): IWSRGST (SEQ ID NO:3)

CDR3 (VH): ARIRGELGPYWYFDV (SEQ ID NO:4)

Light chain variable region (VL): amino acid sequence (SEQ ID NO:6)

DIVLXQSPASLAVSLGQRATISCRASESVEYYGSSLMQWYQQKPGQPPKLLIYAASNV ESGVPARFSGSGSGTDFSLNIRPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRA

Light chain variable region (VL): nucleotide sequence (SEQ ID NO:10)

GGTGGATACAGTTGGTGGAGCATCAGCCCGTTTGATTTCCAGCTTGGTGCCTCCA CCGAACGTCCACGGAACCTTCCTACTTTGCTGACAGAAATACATTGCAATATCATC CTCCTCCACAGGACGGATGTTGAGGCTGAAGTCTGTCCCAGACCCACTGCCACTA AACCTGGCAGGGACCCCAGATTCTACGTTGGATGCAGCATAGATGAGGAGTTTGG GTGGCTGTCCTGGTTTCTGTTGGTACCACTGCATTAAACTTGAGCCATAATATTCA ACACTTTCACTGGCTCTGCAGGAGATGGTGGCTCTCTGCCCTAGAGACACAGCCA AAGAAGCTGGAGACTGGNTCAGCACAATGTCCC

Light chain variable region (VL) - CDR analysis:

CDR1 (VL): ESVEYYGSSL (SEQ ID NO:7)

CDR2 (VL): AAS (SEQ ID NO:8)

CDR3 (VL): QQSRKVPWT (SEQ ID NO:9)

Results - mSol2:

Heavy chain variable region (VH): amino acid sequence (SEQ ID NO:1 1 )

EVKXXESGAELARPGASVKLSCKASGYTGNTFTKYWMQWIKQRPGQGLEWIGAVYP GNGDTRYTQDFKDKATLTADISSSTAYMQLNNLAPEDSAVYYCVRGGSGGSWGQGT LVTVSA Heavy chain variable region (VH): nucleotide sequence (SEQ ID NO:15)

GGAAGGTGTGCACACCGCTGGACAGGGATCCAGAGTTCCAGGTCACTGTCACTG GCTCAGGGAAATAGCCCTTGACCAGGCATCCCAGGGTCACCATGGAGTTAGTTTG GGCAGCAGATCCAGGGGCCAGTGGATAGACAGATGGGGGTGTCGTTTTGGCTGC AGAGACAGTGACCAGAGTCCCTTGGCCCCAGGAGCCGCCGGAACCTCCCCTCAC ACAATAATAGACCGCAGAGTCTTCAGGTGCCAAGTTGTTGAGTTGCATGTAGGCT GTGCTGGAGGATATATCTGCAGTCAATGTGGCCTTGTCCTTGAAATCCTGAGTATA CCTAGTATCACCATTTCCAGGATAAACAGCCCCAATCCATTCCAGACCCTGTCCAG GCCTCTGTTTTATCCACTGCATCCAGTACTTGGTAAAGGTGTTGCCAGTGTAGCCA GAGGCCTTGCAGGACAACTTCACTGAAGCCCCAGGTCTTGCCAGCTCAGCCCCA GACTCCTNCNGCTTCACCTCCGGAAG

Heavy chain variable region (VH) - CDR analysis:

CDR1 (VH): GYTGNTFTKYW (SEQ ID NO:12)

CDR2 (VH): VYPGNGD (SEQ ID NO:13)

CDR3 (VH): VRGGSGGS (SEQ ID NO:14)

Light chain variable region (VL): amino acid sequence (SEQ ID NO:16)

DIVLTQSPASLAVSLGQRATISCRASESVDNFGKSFLHWYQQKPGQPPRLLIYLASNL ESGVPARFSGSGSRTDFTLSIDPVEADDAATYYCQQNNDDLYTFGGGTKLEIKRA

Light chain variable region (VL): nucleotide sequence (SEQ ID NO:20)

GGTGGATACAGTTGGTGGAGCATCAGCCCGTTTTATTTCCAGCTTGGTCCCCCCT CCGAACGTGTACAGATCGTCATTATTTTGCTGACAGTAATAGGTTGCAGCATCATC AGCCTCCACAGGATCAATGGAAAGGGTGAAGTCTGTCCTAGACCCACTGCCACTG AACCTGGCAGGGACCCCAGATTCTAGGTTGGATGCAAGATAGATGAGGAGTCTG GGTGGTTGTCCTGGTTTCTGCTGGTACCAGTGTAGAAAACTTTTGCCAAAGTTATC AACACTTTCACTGGCTCTGCAGGATATGGTGGCCCTCTGCCCTAGAGACACAGCC AAAGAAGCTGGAGATTGGGTCAGCACAATATCCC

Light chain variable region (VL) - CDR analysis:

CDR1 (VL): ESVDNFGKSF (SEQ ID NO:17)

CDR2 (VL): LAS (SEQ ID NO:18)

CDR3 (VL): QQNNDDLYT (SEQ ID NO:19)

Results - mSol3:

Heavy chain variable region (VH): amino acid sequence (SEQ ID NO:21 )

EVQLEESGADLVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEIIPSNG HTNYNEKFKSKATLTVDKSSSTAYMQLSSLTPEDSAVYYCARGRDFFYGFGDYWGQ GTTLTVSS

Heavy chain variable region (VH): nucleotide sequence (SEQ ID NO:25)

GGAAGGTGTGCACACCGCTGGACAGGGATCCAGAGTTCCAGGTCAAGGTCACTG GCTCAGGGAAATAACCCTTGACCAGGCATCCTAGAGTCACCGAGGAGCCAGTTGT ATCTCCACACACAGGGGCCAGTGGATAGACCGATGGGGCTGTTGTTTTGGCTGA GGAGACTGTGAGAGTGGTGCCTTGGCCCCAGTAGTCACCGAAACCATAAAAGAA GTCCCTCCCTCTTGCACAGTAATAGACCGCAGAGTCCTCAGGTGTCAGGCTGCTG AGTTGCATGTAGGCTGTGCTGGAGGATTTGTCTACAGTCAGTGTGGCCTTGCTCT TGAACTTCTCATTGTAGTTAGTATGACCATTGCTAGGAATAATCTCTCCAATCCACT CAAGGCCTTGTCCAGGCCTCTGCTTCACCCAGTGCATCCAGTAGCTGGTGAAGGT GTAGCCAGAAGCCTTGCAGGACAGCTTCACTGAAGCCCCAGGCTTCACCAGGTC AGCCCCAGACTCCTCCAGCTGTACCTCCGGAAG Heavy chain variable region (VH) - CDR analysis:

CDR1 (VH): GYTFTSYWM (SEQ ID NO:22)

CDR2 (VH): IIPSNGHT (SEQ ID NO:23)

CDR3 (VH): ARGRDFFYGFGDYW (SEQ ID NO:24)

Light chain variable region (VL): amino acid sequence (SEQ ID NO:26)

DIVMTQSPSSLAVSVGEKVTMRCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYRYPRTFGGGTNLEIKRA

Light chain variable region (VL): nucleotide sequence (SEQ ID NO:30)

GGTGGATACAGTTGGTGGAGCATCAGCCCGTTTGATTTCCAGGTTGGTGCCTCCA CCGAACGTCCGAGGATACCTATAATATTGCTGACAGTAATAAACTGCCAGGTCTTC AGCCTTCACACTGCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCTGTG AAGCGATCAGGGACCCCAGATTCCCTAGTGGATGCCCAGTAAATCAGCAGTTTAG GAGACTGCCCTGGTTTCTGCTGGTACCAGGCCAAGTAGTTCTTTTGATTGCTACTA TATAAAAGGCTCTGACTGGACTTGCACCTCATAGTAACCTTCTCTCCAACTGACAC AGCTAGGGAGGATGGAGACTGGGTCATCACAATGTCCC

Light chain variable region (VL) - CDR analysis:

CDR1 (VL): QSLLYSSNQKNY (SEQ ID NO:27)

CDR2 (VL): WAS (SEQ ID NO:28)

CDR3 (VL): QQYYRYPRT (SEQ ID NO:29)

Results - mSol4:

Heavy chain variable region (VH): amino acid sequence (SEQ ID NO:31 )

LEESGGDLVKLGGSLKLSCAASGFTFSSYYMSWVRQTPEKRLELVAAIHSDGGNTYY PDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALYYCARHSYYGSSYDYWGQGTTLTV SS

Heavy chain variable region (VH): nucleotide sequence (SEQ ID NO:35)

GGAAGGTGTGCACACCGCTGGACAGGGATCCAGAGTTCCAGGTCAAGGTCACTG GCTCAGGGAAATAACCCTTGACCAGGCATCCTAGAGTCACCGAGGAGCCAGTTGT ATCTCCACACACAGGGGCCAGTGGATAGACCGATGGGGCTGTTGTTTTGGCTGA GGAGACTGTGAGAGTGGTGCCTTGGCCCCAGTAGTCGTAACTACTACCGTAGTAA CTATGTCTTGCACAGTAATATAAGGCTGTGTCCTCAGACTTCAGACTGCTCATTTG CAGGTACAGGGTGTTCTTGGCATTGTCTCTGGAGATGGTGAATCGGCCCTTCACA GTGTCTGGATAGTAGGTGTTACCACCATCACTATGAATGGCTGCGACCAACTCCA GCCTCTTCTCTGGAGTCTGGCGAACCCAAGACATGTAATAGCTACTGAAAGTGAA TCCAGAGGCTGCACAGGAGAGTTTCAAGGACCCTCCAAGTTTCACTAAGTCTCCC CCAGACTCCTCCAGCTGAACTTCCGGAAG

Heavy chain variable region (VH) - CDR analysis:

CDR1 (VH): GFTFSSYY (SEQ ID NO:32)

CDR2 (VH): IHSDGGNT (SEQ ID NO:33)

CDR3 (VH): ARHSYYGSSYDY (SEQ ID NO:34)

Light chain variable region (VL): amino acid sequence (SEQ ID NO:36)

DIVLTQSPLSLPVSLGDQASISCESSQSLVHSNGITYLHWYLQKPGQSPKLLIYKVSHR FSGVPDRFSGSGSGTDFTLKISRVEAVDLGVYFCSQSTHVPFTFGSGTKLEIKRA

Light chain variable region (VL): nucleotide sequence (SEQ ID NO:40)

GGTGGATACAGTTGGTGGAGCATCAGCCCGTTTTATTTCCAACTTTGTCCCCGAG CCGAACGTGAATGGAACATGTGTACTTTGAGAGCAGAAATAAACTCCCAGATCCA CAGCCTCCACTCTGCTGATCTTGAGTGTGAAATCTGTCCCTGATCCACTGCCACT GAACCTGTCTGGGACCCCAGAAAATCGGTGGGAAACTTTGTAGATCAGGAGCTTT GGAGACTGGCCTGGCTTCTGCAGGTACCAATGTAAATAGGTGATTCCATTACTGT GTACAAGGCTCTGACTAGATTCGCAAGAGATGGAGGCTTGATCTCCAAGACTGAC AGGCAGGGAGAGTGGAGACTGGGTGAGCACAATATCCC

Light chain variable region (VL) - CDR analysis:

CDR1 (VL): QSLVHSNGITY (SEQ ID NO:37)

CDR2 (VL): KVS (SEQ ID NO:38)

CDR3 (VL): SQSTHVPFT (SEQ ID NO:39)

Example 3. In vivo efficacy testing of cross-reactive anti-PAD antibodies

The antibodies described herein are tested for in vivo efficacy in the collagen-induced arthritis (CIA) mouse model.

Phase I:

In 10 DBA-1 mice per group arthritis is induced with collagen type II to generate the CIA mouse model as previously described. Compounds (e.g. 25-500

ug/mouse/injection) are given i.p, e.g. by daily injections or three times a week. Group 1 : CIA

Group 2: CIA + Vehicle

Group 3: CIA + Therapeutic test compound

Group 4: CIA + Negative control compound Group 5: CIA + Cl-amidine

Group 6: CIA + positive control compound

Inflammation is scored continually through the study, such as 3 times a week, starting from day 21 .

Claims

1 . An anti-PAD antibody or fragment or variant thereof, wherein said antibody or fragment or variant thereof is capable of binding to the catalytic domain of at least PAD2 and PAD4, or an antibody or fragment or variant thereof which competes for binding to the catalytic domain of PAD2 and PAD4 with said anti-PAD antibody or fragment or variant thereof.

2. The anti-PAD antibody or fragment or variant thereof according to claim 1 , wherein said antibody or fragment or variant thereof is an antibody or fragment or variant thereof capable of binding to a PAD peptide selected from the group consisting of a) a PAD4 peptide consisting of SEQ ID NO:46,

b) a PAD2 peptide consisting of SEQ ID NO:48, and

c) a PAD4 peptide consisting of SEQ ID NO:45,

or an antibody or fragment or variant thereof which competes for binding to SEQ ID NO:46, SEQ ID NO:48 or SEQ ID NO:45,

wherein said antibody or fragment or variant thereof is capable of binding to at least PAD2 and PAD4.

3. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein said antibody comprises

i) a Heavy chain variable region (VH) comprising

a) a first CDR consisting of SEQ ID NO:22 or a variant thereof having at least 75% sequence identity to SEQ ID NO:22,

b) a second CDR consisting of SEQ ID NO:23 or a variant thereof having at least 75% sequence identity to SEQ ID NO:23,

c) a third CDR consisting of SEQ ID NO: 24 or a variant thereof having at least 75% sequence identity to SEQ ID NO:24,

and/or comprises

ii) a Light chain variable region (VL) comprising a) a first CDR consisting of SEQ ID NO:27 or a variant thereof having at least 75% sequence identity to SEQ ID NO:27,

b) a second CDR consisting of SEQ ID NO:28 or a variant thereof having at least 75% sequence identity to SEQ ID NO:28

c) a third CDR consisting of SEQ ID NO: 29 or a variant thereof having at least 75% sequence identity to SEQ ID NO:29.

4. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein said antibody comprises

a) a Heavy chain variable region (VH) consisting of SEQ ID NO:21 , or a variant thereof having at least 75% sequence identity thereto;

and/or comprises

b) a Light chain variable region (VL) consisting of SEQ ID NO:26, or a variant thereof having at least 75% sequence identity thereto.

5. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein said antibody comprises

i) a Heavy chain variable region (VH) comprising

a) a first CDR consisting of SEQ ID NO:12 or a variant thereof having at least 75% sequence identity to SEQ ID NO:12,

b) a second CDR consisting of SEQ ID NO:13 or a variant thereof having at least 75% sequence identity to SEQ ID NO:13,

c) a third CDR consisting of SEQ ID NO:14 or a variant thereof having at least 75% sequence identity to SEQ ID NO:14,

and/or comprises

ii) a Light chain variable region (VL) comprising

a) a first CDR consisting of SEQ ID NO:17 or a variant thereof having at least 75% sequence identity to SEQ ID NO:17,

b) a second CDR consisting of SEQ ID NO:18 or a variant thereof having at least 75% sequence identity to SEQ ID NO:18 c) a third CDR consisting of SEQ ID NO:19 or a variant thereof having at least 75% sequence identity to SEQ ID NO:19.

6. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein said antibody comprises

a) a Heavy chain variable region (VH) consisting of SEQ ID NO:1 1 , or a variant thereof having at least 75% sequence identity thereto;

and/or comprises

b) a Light chain variable region (VL) consisting of SEQ ID NO:16, or a variant thereof having at least 75% sequence identity thereto.

7. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein said antibody or fragment or variant thereof comprises i) a Heavy chain variable region (VH) comprising

a) a first CDR consisting of SEQ ID NO:2 or a variant thereof having at least

75% sequence identity to SEQ ID NO:2,

b) a second CDR consisting of SEQ ID NO:3 or a variant thereof having at least 75% sequence identity to SEQ ID NO:3,

c) a third CDR consisting of SEQ ID NO:4 or a variant thereof having at least 75% sequence identity to SEQ ID NO:4,

and/or comprises

ii) a Light chain variable region (VL) comprising

a) a first CDR consisting of SEQ ID NO:7 or a variant thereof having at least 75% sequence identity to SEQ ID NO:7,

b) a second CDR consisting of SEQ ID NO:8 or a variant thereof having at least 75% sequence identity to SEQ ID NO:8,

c) a third CDR consisting of SEQ ID NO:9 or a variant thereof having at least 75% sequence identity to SEQ ID NO:9.

8. The anti-PAD antibody or fragment or variant thereof according to any of the preceding claims, wherein said antibody comprises

a) a Heavy chain variable region (VH) consisting of SEQ ID NO:1 , or a variant thereof having at least 75% sequence identity thereto;

and/or comprises

b) a Light chain variable region (VL) consisting of SEQ ID NO:6, or a variant thereof having at least 75% sequence identity thereto.

9. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein said antibody comprises

i) a Heavy chain variable region (VH) comprising

a) a first CDR consisting of SEQ ID NO:32 or a variant thereof having at least 75% sequence identity to SEQ ID NO:32,

b) a second CDR consisting of SEQ ID NO:33 or a variant thereof having at least 75% sequence identity to SEQ ID NO:33,

c) a third CDR consisting of SEQ ID NO:34 or a variant thereof having at least 75% sequence identity to SEQ ID NO:34,

and/or comprises

ii) a Light chain variable region (VL) comprising

a) a first CDR consisting of SEQ ID NO:37 or a variant thereof having at least 75% sequence identity to SEQ ID NO:37,

b) a second CDR consisting of SEQ ID NO:38 or a variant thereof having at least 75% sequence identity to SEQ ID NO:38

c) a third CDR consisting of SEQ ID NO:39 or a variant thereof having at least 75% sequence identity to SEQ ID NO:39.

10. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein said antibody comprises a) a Heavy chain variable region (VH) consisting of SEQ ID NO:31 , or a variant thereof having at least 75% sequence identity thereto;

and/or comprises

b) a Light chain variable region (VL) consisting of SEQ ID NO:36, or a variant thereof having at least 75% sequence identity thereto.

1 1 . The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein the sequence variation is outside the CDRs.

12. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein the antibody is capable of at least partially inhibiting the enzymatic activity of one or more PAD isoforms, such as PAD2 and/or PAD4.

13. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein the antibody or fragment or variant thereof is capable of increasing clearance of one or more PAD isoforms, such as at least PAD2 and/or PAD4.

14. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein the antibody is a monoclonal antibody.

15. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein the antibody is a chimeric antibody.

16. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein the antibody is a humanized antibody.

17. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein the antibody is a fully human antibody.

18. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims, wherein the antibody is an IgG isotype selected from the group consisting of lgG1 and lgG3.

19. The anti-PAD antibody or fragment or variant thereof according to any of the preceding claims, wherein the fragment is a Fab-fragment, a single-chain variable fragment (scFv) or a nanobody.

20. An affinity matured variant of an antibody according to any of the preceding claims, said affinity matured variant having a higher affinity for PAD2 and/or PAD4 as compared to the antibody or fragment or variant thereof according to any of the preceding claims.

21 . The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims for use as a medicament.

22. The anti-PAD antibody or fragment or variant thereof according to any of the

preceding claims for use in the treatment of an autoimmune disease characterized by extracellular citrullination.

23. The anti-PAD antibody or fragment or variant thereof for use according to any of the preceding claims, wherein the extracellular citrullination is involved in the pathogenesis of the autoimmune disease.

24. The anti-PAD antibody or fragment or variant thereof for use according to any of the preceding claims, wherein the citrullination is hyper-citrullination.

25. The anti-PAD antibody or fragment or variant thereof for use according to any of the preceding claims, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, inflammatory bowel disease and psoriasis.

26. The anti-PAD antibody or fragment or variant thereof for use according to any of the preceding claims, wherein the autoimmune disease is rheumatoid arthritis.

27. A pharmaceutical composition comprising an anti-PAD antibody or fragment or variant thereof according to any of claims 1 -20 and at least one pharmaceutically acceptable diluent, carrier or excipient.

28. A method of treating a subject suffering from an autoimmune disease characterized by extracellular citrullination comprising the administration of a suitable amount of the anti-PAD antibody or fragment or variant thereof according to any of claims 1 - 20 to said subject.

29. The method according to claim 28, wherein said autoimmune disease is selected from the group consisting of rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis and psoriasis.

30. The method according to any of claims 28 to 29, wherein said autoimmune disease is rheumatoid arthritis.

31 . The method according to claim 30, wherein the rheumatoid arthritis is ACPA- positive rheumatoid arthritis.

32. The method according to any of claims 28 to 31 , wherein said treatment is

prophylactic, ameliorative or curative.

33. The method according to any of claims 28 to 32, wherein said treatment is a

prophylactic treatment initiated upon detection of ACPAs in said subject.

34. The method according to any of claims 28 to 33, wherein said antibody is coadministered with another drug suitable for treating said autoimmune disease.

35. The method according to any of claims 28 to 34, wherein the subject is human.