CD-52 ANTIBODIES AND THEIR USE IN DETERMINING AND ENHANCING AN IMMUNE RESPONSE IN A SUBJECT

FIELD OF THE INVENTION

The present disclosure generally relates to antibodies that are capable of enhancing an immune response to an antigen and to methods of using such antibodies. In one example, the present disclosure generally relates to antibodies that are capable of binding to the cell surface membrane-anchored glycoprotein CD52 or a soluble fragment thereof. Exemplary methods of using such antibodies include enhancing a subject's immune response to an antigen, determining the level of a subject's immune response to an antigen, and other methods disclosed herein.

BACKGROUND OF THE INVENTION

Regulatory T cells (Treg cells; also known as suppressor T cells) are subpopulations of T cells that maintain immune homeostasis and help avert autoimmune disease (Sakaguchi et al, 2008; Shevach, 2006; Vignali et al, 2008). Interest in Treg cells is focused predominantly on prototypic CD4 ⁺ CD25 ⁺ Treg cells that are programmed by the transcription factor FoxP3 (Fontenot et al, 2003; Hori et al., 2003). In resting polyspecific populations these Treg cells are characterized in the mouse both as 'natural', thymus-derived and induced 'adaptive' cells that suppress the activation, proliferation and functions of other T cells (Sakaguchi et al, 2008; Shevach, 2006). However, in human blood CD4 ⁺ Treg cells are not as reliably distinguished by FoxP3 expression (Roncarolo and Gregori, 2008; Allan et al., 2007; Gavin et al, 2006). Thus, CD4 ⁺ T cells with markers of either naive or memory cells were shown to have similar suppressor functions despite low and high expression, respectively, of FoxP3 (Miyara et al, 2009). Other surface markers of human CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Treg cells such as decreased expression of the IL-7 receptor, CD 127 (Liu et al, 2006; Seddiki et al, 2006), are not specific for Treg cells.

Aside from the paucity of specific cell surface markers, the mechanisms underlying suppression by CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Treg cells remain controversial. In the mouse, suppression ex vivo has been shown to require cell-cell contact but has been attributed to multiple mechanisms (Vignali et al, 2008; Shevach, 2009; Sakaguchi et al, 2009); even less is known about the function of similar human Treg cells. Furthermore, other types of both CD4 ⁺ and CD8 ⁺ Treg cells that differ in proposed mechanisms of suppressor function have been described in the context of various tissue sites or diseases (Vignali et al, 2008). Treg cells induced by administration of autoantigens have been shown to protect against some autoimmune diseases in certain animal models (reviewed by von Herrath and Harrison, 2003). For example, in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D) CD4 ⁺ Treg cells induced by administered pancreatic islet autoantigens such as insulin (Bergerot et al., 1994) or glutamic acid decarboxylase 65 (GAD65) (Tisch et al., 1999), or transfer of CD4 ⁺ Treg cells induced by proinsulin (Every et al., 2006) or a putative pancreatic islet antigen (Tang et al., 2004), have been shown to protect against autoimmune diabetes. However, in these models Treg cells have been studied in resting, polyspecific populations and have not been studied during the host's response to a particular antigen. Recently, proinsulin- and GAD65 -specific human CD4 ⁺ T cell clones were generated and Treg clones were distinguished by their suppressor function in vitro (Dromey et al., 2011). The cell surface membrane- anchored glycoprotein CD52 was shown to be upregulated in these expanded CD4 ⁺ Treg clones. However, the mechanism of Treg cell suppression has not previously been characterized. Accordingly, ways of modulating the process of Treg cell suppression remain unclear.

Antibodies that bind the cell surface membrane-anchored glycoprotein CD52 have been described. For example, Campath-IH (alemtuzumab) is a monoclonal antibody that binds the extracellular peptide portion of CD52 (Riechmann et al, 1988). Campath-IH is used as an immunosuppressant (resulting from its ability to induce lysis of human lymphocytes) in the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma. Other antibodies that bind to an epitope comprising part of the GPI anchor of CD52, or that bind to an epitope on the carbohydrate moiety of CD52 have been described (Hale, 2001); all of these have been described has having strong lytic activity with human complement (Hale, 2001), suggesting their use as an immunosuppressant.

SUMMARY OF THE INVENTION

The present inventors have now demonstrated that Treg cells exert their immunosuppressive effect at least in part by secretion of a soluble CD52 glycoprotein fragment and that antibodies which are capable of binding to the cell surface membrane-anchored glycoprotein CD52 or a soluble fragment thereof enhance a subject's immune response to an antigen. The inventors have therefore demonstrated, for the first time, that such antibodies can be used to enhance a subject's immune response. Accordingly, in a first aspect, the present disclosure provides a method of enhancing an immune response in a subject, the method comprising administering to the subject an antibody or fragment thereof which specifically binds a CD52 glycoprotein.

n one example, the antibody or fragment thereof specifically binds a carbohydrate moiety of a CD52 glycoprotein. Thus, the antibody or fragment may specifically bind to an epitope on the carbohydrate moiety of a CD52 glycoprotein.

The antibody or fragment thereof may specifically bind either a soluble CD52 glycoprotein (such as the soluble fragment of CD52 which can be cleaved from the full, cell surface membrane-bound CD52 protein), or the full, cell surface membrane-bound CD52 glycoprotein itself.

In one example, the antibody or fragment thereof specifically binds a CD52 glycoprotein comprising an amino acid sequence at least 60% identical to the amino acid sequence of any one or more of GQNDTSQTSSPS (SEQ ID NO: 3), SQNATSQSSPS (SEQ ID NO: 4), GQATTAASGTNKNSTST TPLKS (SEQ ID NO: 5), GQNSTAVTTPANKAATTAAATTKAAATTATKTTTAVRKTPGKPPKA (SEQ ID NO: 6) or GNSTTPRMTTKKVKSATPA (SEQ ID NO: 7), and a carbohydrate. In other examples, the CD52 glycoprotein may comprise an amino acid sequence which is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, or is 100% identical, to any one or more of the amino acid sequences identified in SEQ ID NOs: 3, 4, 5, 6 or 7.

The CD52 glycoprotein may comprise any carbohydrate known to be attached to the extracellular portion of CD52 in a host cell, such as a host lymphocyte. Thus, the antibody or fragment thereof may specifically bind to any carbohydrate known to be attached to the extracellular portion of CD52 in a host cell, such as a host lymphocyte.

In one example, the CD52 glycoprotein may comprise a carbohydrate comprising one or more bi-, tri- or tetra-antennary sugars, which may be terminally sialylated.

Any known antibody which specifically binds a CD52 glycoprotein may be used in the methods disclosed herein. For example, any known antibody which specifically binds a carbohydrate moiety of a CD52 glycoprotein may be used in the methods disclosed herein. Examples of such known antibodies include the antibodies CF1D12, HI-186 (Hale, 2001), 2C6 (Kameda et al, 1991), H6-C34, or S19 (Diekman et al, 1999), and others. In addition, any number of the CDRs of antibodies which are known to specifically bind a CD52 glycoprotein may be used in the methods disclosed herein. For example, the antibody or fragment may comprise any one or more of the CDRs set out in SEQ ID NOs: 8, 9, 10, 11, 12 or 13. In one example, the antibody or fragment comprises all of the CDRs set out in SEQ ID NOs: 8, 9, 10, 1 1 , 12 or 13.

The antibody or fragment thereof which specifically binds a CD 52 glycoprotein may be used to enhance any immune response in a subject. Thus, the antibody or fragment thereof can be used as an adjuvant.

In one example, the immune response is an immune response to an antigen. The methods disclosed herein may further comprise administering the antigen to the subject. Such methods may, for example, be used as a method for vaccinating a subject against a particular antigen. The effect of the antibody or fragment disclosed herein in reducing Treg cell-mediated suppression of the subject's T cells results in the production of an enhanced immune response by the subject to the antigen, thereby enhancing the potency of the vaccine. Accordingly, the present disclosure provides a method of vaccinating a subject, comprising administering an antigen and an antibody which specifically binds a CD52 glycoprotein as disclosed herein to the subject.

The antibody or fragment thereof which specifically binds a CD52 glycoprotein may, also be used as a therapeutic adjuvant. Thus, the antibody or fragment may be administered to a subject together with a therapeutic agent. It will be appreciated that the antibody or fragment thereof may be administered simultaneously, sequentially, or subsequent to the therapeutic agent.

The antibody or fragment may be administered to a subject together with any therapeutic agent. In one example, the therapeutic agent is a chemotherapeutic agent. Thus, the antibody or fragment thereof may be administered as an adjuvant in the treatment of cancer.

The present disclosure also provides an antibody or fragment thereof which specifically binds a CD52 glycoprotein, for use in enhancing an immune response in a subject. It will be understood that the antibody or fragment can comprise any of the specific features of the antibody or fragment thereof described herein in the context of a method of treatment.

The present disclosure also provides the use of an antibody or fragment thereof which specifically binds a CD52 glycoprotein in the manufacture of a medicament for enhancing an immune response in a subject. Again, it will be understood that the antibody or fragment can comprise any of the specific features of the antibody or fragment thereof described herein in the context of a method of treatment.

In another aspect, the present disclosure provides a method of determining a subject's immune response to an antigen, the method comprising: i) contacting immune cells of the subject with an antigen in the presence of an antibody or fragment thereof which specifically binds a CD52 glycoprotein; and

ii) determining the level of an activity of the immune cells.

The antibody or fragment thereof disclosed herein provides the advantage of amplifying a subject's immune response to an antigen. The inventors believe that this effect is achieved by the antibody or fragment thereof reducing Treg cell-mediated suppression of the subject's T cells. By reducing Treg-mediated suppression, the subject's T cells are able to exert a greater immune response. This effect renders the present methods particularly suitable for determining a subject's immune response to a particular antigen, since they negate the need for costly and lengthy intermediary steps of expanding a subject's T cell population to an amount which is large enough to produce a detectable result. For example, whereas previous methods of detecting T cell response in a blood sample taken from a patient may require T-cell expansion over a period of three or four days or more in order to produce enough T cells to provide a detectable response, the methods disclosed herein allow a much faster analysis to be performed by significantly enhancing the subject's immune response. Thus, the methods disclosed herein can be performed to provide a measurement of a subject's immune response over a much shorter timescale than previously used methods.

In addition, by enhancing a subject's immune response to an antigen, the methods disclosed herein have the additional advantage of providing a more sensitive assay of a subject's immune response. Whereas previous methods may have detected only a low immune response to a given antigen, the methods disclosed herein allow a more sensitive determination of a subject's immune response to be made by enhancing the host's immune response. Accordingly, the increased sensitivity of the methods disclosed herein may reduce the likelihood of incorrectly determining that a subject does not respond significantly to an antigen (i.e., the methods disclosed herein may reduce the occurrence of false negative diagnoses). Thus, the present disclosure provides a method for diagnosing or predicting a subject's response to an antigen. The inventors have shown that an antibody or fragment thereof which specifically binds a CD52 glycoprotein is capable of enhancing a subject's immune response to any antigen. Thus, any antigen may be used in the methods disclosed herein.

In one example, the antigen is an auto-antigen and the method allows a diagnosis of an autoimmune disease or of the likelihood of a subject developing an autoimmune disease. The greater the subject's immune response to an auto antigen, the greater the risk of that subject developing the autoimmune disease. One example of such an auto antigen is the protein glutamic acid decarboxylase 65 (GAD65). Thus, in one example, the methods disclosed herein may be performed to determine the risk of a subject developing type 1 diabetes. Many other examples of auto antigens are known in the art, and can equally be used in the methods disclosed herein.

In another example, the antigen is gliadin or a gluten peptide, the target of the immune response in coeliac disease. The gliadin may be any known form of gliadin. For example, the gliadin may be a gliadin α, β, γ, Y, or ω gliadin. The gluten peptide may be any gluten peptide capable of triggering a host immune response. Suitable gluten peptides are known in the art. In one example, the gluten peptide may be a peptide comprising or consisting of the amino acid sequence set out in SEQ ID NO: 65 or SEQ ID NO: 66. Thus, the method allows a diagnosis of coeliac disease or of the likelihood of a subject developing coeliac disease. It will be appreciated that an antibody or fragment thereof which specifically binds a CD52 glycoprotein has been shown herein to be capable of enhancing a subject's immune response to any antigen and that any antigen may be used in the methods disclosed herein.

The methods disclosed herein can also be used to monitor a subject's immune response over the course of a therapeutic or prophylactic treatment. Thus, the present disclosure provides a method of determining a subject's immune response to an antigen, the method comprising:

i) contacting immune cells of the subject with an antigen in the presence of an antibody or fragment thereof which specifically binds a CD52 glycoprotein; and

ii) determining the level of an activity of the immune cells,

which is performed at a first time point and again at a second, later time point, wherein a greater level of activity of the subject's immune cells at the second time point indicates an enhancement in the subject's immune response to the antigen since the first time point.

The antibody or fragment thereof disclosed herein can also be used to identify a particular epitope of any molecule (e.g., protein, glycoprotein, etc.) that evokes the strongest immune response in a subject. Again, because the antibody or fragment thereof reduces Treg cell-mediated suppression of the subject's T cells, it effectively unmasks the level of a subject's immune response and can therefore be used to rapidly determine the level of a subject's immune response to a potentially antigenic epitope.

Thus, in another aspect, the present disclosure provides a method of identifying an antigenic epitope, the method comprising: i) contacting immune cells of a subject with one or more potentially antigenic epitopes and an antibody or fragment thereof which specifically binds a CD52 glycoprotein; and

ii) determining the level of an activity of the immune cells,

wherein an increased level of an activity of the immune cells in the presence of a potentially antigenic epitope identifies that epitope as an antigenic epitope.

In one example, the methods comprise contacting immune cells of a subject with a plurality of peptide fragments derived from a polypeptide. The plurality of peptide fragments may comprise partially overlapping amino acid sequences. For example, peptide fragments sequentially scanning the length of a given polypeptide can be used to identify an antigenic epitope within the full length epitope.

Any of the methods disclosed herein may further comprise a step of comparing the level of activity of the immune cells of a subject with a reference level. The reference level may be predetermined. Alternatively, the methods disclosed herein may further comprise contacting the immune cells of one or more subjects with the antigen or one or more potentially antigenic epitopes in the presence of an antibody or fragment thereof which specifically binds a CD52 glycoprotein, determining the level of activity of the immune cells of the one or more subjects, and determining a reference level of immune cell activity.

When comparing the level of activity of a subject's immune cells to a reference level, a level of activity of the subject's immune cells which is greater than the reference level may indicate that the subject is responding to therapy, or has an increased likelihood of having or developing a disease or condition, or that the potentially antigenic epitope is likely to be an antigenic epitope. Conversely, a level of activity of the subject's immune cells which is less than the reference level may indicate that the subject is not responding to therapy, or has a decreased likelihood of having or developing a disease or condition, or that the potentially antigenic epitope is unlikely to be an antigenic epitope.

The features of any embodiment described herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1: Monoclonal antibody CF1D12 abrogates CD52 ^h 'CD4 ⁺ T cell suppression of TT-stimulated CD52 ^lo CD4 ⁺ T cells.

lFN-γ spots per ELISpot plate well for CD52 ^hi and/or CD52 ¹⁰ CD4 ⁺ T cells incubated separately, together, or together with an isotype control antibody or the monoclonal antibody CF1D12, in the presence (black bars) or absence (white bars) of TT antigen. Figure 2: CF1D12 enhances responses ofT cells in PBMCs to antigens.

IFN-γ spots per ELISpot plate well for PBMCs pre-incubated with either an isotype control antibody or CF1D12 for 1 h at 37°C and subsequently exposed to no antigen, TT or GAD65 in the presence (black bars) or absence (white bars, labelled 'Antibody pre-bound only') of additional antibody.

Figure 3: CF1D12 exhibits a dose-dependent effect in enhancing responses of T cells in PBMCs to antigens.

IFN-γ spots per ELISpot plate well for PBMCs pre-incubated with CF1D12 for 1 h at 37°C and subsequently exposed to TT in the presence (black bars) or absence (white bars, labelled 'Antibody pre-bound') of additional CF 1 D 12 antibody .

Figure 4: CF1D12 enhances T cell response to antigens in people at risk for type 1 diabetes.

IFN-γ spots per ELISpot plate well for PBMCs incubated with isotype control antibody or CF 1 D 12 in the presence of no antigen, TT or GAD65.

Figure 5: DNA constructs for expression in lentivirus vector.

SigP = signal peptide; ECD = extracellular domain; Strep2 = purification tag encoding 8 amino acids that binds to Strep-Tactin, a specifically engineered streptavidin. Figure 6: Soluble CD52 fusion protein directly suppresses T cell proliferation and effector function.

Suppression of T cell proliferation by recombinant CD52-Fc. PBMCs (200,000) were cultured with TT for 7 days (A) and purified CD4 ⁺ T cells (20,000) with anti-CD3 (100 ng/ml) and anti-CD28 (200 ng/ml) antibody for 48 hr (B), with 4 times the number of irradiated PBMCs in 200μ1 round bottom wells, in the presence of recombinant CD52- Fc or Fc protein control protein at the indicated concentrations. ³ H-thymidine uptake was measured over the final 16 hr of incubation. Results (mean ± sem of triplicates) are representative of six independent experiments.

(C) Suppression of cytokine secretion by recombinant CD52-Fc. Media from PBMCs activated with TT in (C) ± 3.3 μΜ CD52-Fc or Fc proteins were sampled after 48 hr incubation and assayed for cytokines by multiplex bead array. (D) Impaired suppression by CD52-Fc after cleavage of N-linked carbohydrate. CD52- Fc (0.66 μΜ) was incubated with PNGase F (10 ³ units) or reaction buffer only in 20 ml overnight at 37°C, as recommended by the supplier, and the reaction terminated by heating at 75°C for 10 min. The decrease in size of CD52-Fc after treatment, determined by SDS-PAGE and Coomassie staining (at top of figure), is consistent with loss of the carbohydrate moiety. PBMCs were incubated with TT and either PNGase F- treated or untreated CD52-Fc (final 0.33 μΜ), in triplicate, for 7 days at 37°C, before measurement of ³ H -thymidine uptake as in (C).

Figure 7: CD52-Fc does not affect the T cell stimulatory capacity of dendritic cells.

FACS-sorted human blood CDlb/c ⁺ DC were pre-incubated with CD52-Fc or Fc protein, washed twice and co-cultured with allogeneic CFSE-labeled CD4+ T cells for 6 days. The frequency of dividing CD4 ⁺ T cells identified as CFSE ¹⁰ was determined by flow cytometry. The result is representative of two independent experiments with different donors. Similar results were obtained for CD304+ plasmacytoid DC and for CD 14+ monocytes (data not shown).

Figure 8: CF1D12 enhances responses of T cells in PBMCs to coeliac disease antigens.

IFN-γ responses were determined by ELISpot assay in two representative HLA- DQ2.5-positive coeliac disease subjects (Figure 8A: subject #0537; Figure 8B: subject #0564) following oral wheat challenge. PBMCs were incubated with either deamidated chymotrypsin-digested gliadin, Peptide 1 or Peptide 2, in medium alone or with CF1D12 antibody or IgG3 isotype control (final concentration of each 20 g/ml). Results show mean spot forming units (SFU) per million PBMCs plated following subtraction of mean Nil response. SEM is also shown. The horizontal dotted line (at 10 SFU/10 ⁶ PBMCs on the y axis) is the cut-off of 10 SFU/million PBMCs for a significant result.

Figure 9: CF1D12 enhances responses of T cells in PBMCs to coeliac disease antigens.

IFN-γ responses were determined by ELISpot assay in two HLA-DQ2.5-positive non- celiac control subjects (Figure 9A: subject #0562; Figure 9B: subject #0563) maintaining a normal diet. PBMCs were incubated with either deamidated chymotrypsin-digested gliadin, Peptide 1 or Peptide 2, in medium alone or with CF1D12 antibody or IgG3 isotype control (final concentration of each 20 μg/ml). Results show mean SFU per million PBMCs plated following subtraction of mean Nil response. SEM is also shown. The horizontal dotted line (at 10 SFU/10 ⁶ PBMCs on the y axis) is the cut-off of 10 SFU/million PBMC for a significant result. KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1 - Human CD52 mRNA transcript (NCBI Reference Sequence: ^' NM 001803.2)

SEQ ID NO: 2 - Amino acid sequence of human CD52

SEQ ID NO: 3 - 12 amino acid soluble peptide of human CD52

SEQ ID NO: 4 - Orthologous monkey soluble CD52 peptide

SEQ ID NO: 5 - Orthologous mouse soluble CD52 peptide

SEQ ID NO: 6 - Orthologous rat soluble CD52 peptide

SEQ ID NO: 7 - Orthologous dog soluble CD52 peptide

SEQ ID NO: 8 - CF 1 D 12 Light chain CDR- 1

SEQ ID NO: 9 - CF1D12 Light chain CDR-2

SEQ ID NO: 10 - CF1D12 Light chain CDR-3

SEQ ID NO: 11 - CF 1 D 12 Heavy chain CDR- 1

SEQ ID NO: 12 - CFl D12 Heavy chain CDR-2

SEQ ID NO: 13 - CF1D12 Heavy chain CDR-3

SEQ ID NO: 14 - CF1D12 Light chain variable region

SEQ ID NO: 15 - CF1D12 Heavy chain variable region SEQ ID NO: 16 - Alternat ve antibody CDR

SEQ ID NO: 17 - Alternat ve antibody CDR

SEQ ID NO: 18 - Alternat ve antibody CDR

SEQ ID NO: 19 - Alternati ive antibody CDR

SEQ ID NO: 20 - Alternat ve antibody CDR

SEQ ID NO: 21 - Alternat) ve antibody CDR

SEQ ID NO: 22 - Alternati ve antibody CDR

SEQ ID NO: 23 - Alternati ive antibody CDR

SEQ ID NO: 24 - Alternat ve antibody CDR

SEQ ID NO: 25 - Alternati ve antibody CDR

SEQ ID NO: 26 - Alternati ve antibody CDR'

SEQ ID NO: 27 - Alternati ve antibody CDR

SEQ ID NO: 28 - Alternati ve antibody CDR

SEQ ID NO: 29 - Alternati ve antibody CDR

SEQ ID NO: 30 - Alternati ve antibody CDR

SEQ ID NO: 31 - Alternati ve antibody CDR

SEQ ID NO: 32 - Alternati ve antibody CDR

SEQ ID NO: 33 - Alternati ve antibody CDR

SEQ ID NO: 34 - Alternati ve antibody CDR

SEQ ID NO: 35 - Alternati ve antibody CDR

SEQ ID NO: 36 - Alternati ve antibody CDR

SEQ ID NO: 37 - Alternati ve antibody CDR

SEQ ID NO: 38 - Alternati ve antibody CDR

SEQ ID NO: 39 — Alternati ve antibody CDR

SEQ ID NO: 40 - Alternati ve antibody CDR

SEQ ID NO: 41 - Alternati ve antibody CDR

SEQ ID NO: 42 - Alternati ve antibody CDR

SEQ ID NO: 43 - Alternati ve antibody CDR

SEQ ID NO: 44 - Alternati ve antibody CDR

SEQ ID NO: 45 - Alternati ve antibody CDR

SEQ ID NO: 46 - Alternat] ve antibody CDR

SEQ ID NO: 47 - Alternati ve antibody CDR

SEQ ID NO: 48 - Alternati ve antibody CDR

SEQ ID NO: 49 - Alternati ve antibody CDR

SEQ ID NO: 50 - Alternati ve antibody CDR

SEQ ID NO: 51 - Alternati ve antibody CDR SEQ ID NO: 52 - Alternative antibody CDR

SEQ ID NO: 53 - Alternative antibody CDR

SEQ ID NO: 54 - Alternative antibody CDR

SEQ ID NO: 55 - Alternative antibody CDR

SEQ ID NO: 56 - Alternative antibody CDR

SEQ ID NO: 57 - Alternative antibody CDR

SEQ ID NO: 58 1F1 primer

SEQ ID NO: 59 - 1R1 primer

SEQ ID NO: 60 1F2 primer

SEQ ID NO: 61 - 1R2 primer

SEQ ID NO: 62 - 2F primer

SEQ ID NO: 63 - 2R1 primer

SEQ ID NO: 64 2R2 primer

SEQ ID NO: 65 - Amino acid sequence of immunogenic peptide 1

SEQ ID NO: 66 - Amino acid sequence of immunogenic peptide 2

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al, (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present). The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term "about", unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, of the designated value. For the avoidance of doubt, the term "about" followed by a designated value is to be interpreted as also encompassing the exact designated value itself (for example, "about 10" also encompasses 10 exactly).

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein, the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of an agent sufficient to reduce or eliminate at least one symptom of disease.

As used herein, the terms "preventing", "prevent" or "prevention" include administering a therapeutically effective amount of an agent sufficient to prevent the manifestation of at least one symptom of disease.

As used herein, the term "suppressing" includes reducing by any quantifiable amount.

As used herein, the term "subject" refers to an animal, e.g., a mammal. In a preferred embodiment, the subject is mammalian, for example a human. Other preferred embodiments include livestock animals such as horses, cattle, sheep and goats, as well as companion animals such as cats and dogs.

As used herein, the term "host" refers to any organism. The host may be whole organism or may be a cell derived therefrom. The host may be an animal, e.g., a mammal. In a preferred embodiment, the host is mammalian, for example a human. Other preferred hosts include mice, rats, monkeys, hamsters, guinea-pigs, rabbits, and others.

As used herein, the terms "linked", "attached", "conjugated", "bound", "coupled" or variations thereof are used broadly to refer to any form of covalent or non-covalent association which joins one entity to another for any period of time.

As used herein, the terms "variable region" and "variable domain" refer to the portion of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region may comprise three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. "V _H " refers to the variable region of the heavy chain. "VL" refers to the variable region of the light chain.

As used herein, the term "competitively inhibits" shall be understood to mean that a candidate antibody reduces or prevents binding of an antibody disclosed herein (for example, the monoclonal antibody CF ID 12) to a CD52 glycoprotein. This may be due to the candidate antibody binding to the same or an overlapping epitope as the antibody disclosed herein. It will be apparent from the foregoing that the candidate antibody need not completely inhibit binding of the antibody, rather it need only reduce binding by a statistically significant amount, for example, by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Preferably, the candidate antibody reduces binding of the antibody disclosed herein by at least about 30%, more preferably by at least about 50%, more preferably, by at least about 70%, still more preferably by at least about 75%, even more preferably, by at least about 80% or 85% and even more preferably, by at least about 90%. Methods for determining competitive inhibition of binding are known in the art and/or described herein. In one example, the competitive inhibition is not due to steric hindrance.

CD52

CD52 is a surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein present on most lymphoid cells, initially recognised as the target of complement- binding CAMPATH monoclonal antibodies used therapeutically to deplete lymphocytes (Treumann et al, 1995; Xia et al, 1991; Hale, 2001). The mRNA transcript of the human CD52 gene is shown in SEQ ID NO: 1 and the translated amino acid sequence is shown in SEQ ID NO: 2. Mature CD52 tethered by its GPI anchor comprises only 12 amino acids and an asparagine (N)-linked terminal carbohydrate.

Membrane-anchored CD52 can be cleaved (for example, enzymatically) to release a soluble peptide fragment comprising the amino acid sequence GQNDTSQTSSPS (SEQ ID NO: 3). The term "CD52 glycoprotein" as used herein also encompasses any soluble CD52 glycoprotein fragment which can be cleaved (for example, enaymatically) from a membrane-anchored CD52 molecule. The soluble CD52 glycoprotein disclosed herein may comprise an amino acid sequence at least 60% identical to the amino acid sequence of SEQ ID NO: 3 or at least 60% identical to the amino acid sequence of other known, orthologous CD52 soluble fragment sequences. Such sequences include but are not limited to the monkey sequence SQNATSQSSPS (SEQ ID NO: 4), the mouse sequence GQATTAASGTNKNSTSTKKTPLKS (SEQ ID NO: 5), the rat sequence

GQNSTAVTTPAN AATTAAATTKAAATTATKTTTAVRKTPGKPPKA (SEQ ID NO: 6), the dog sequence GNSTTPRMTTKKVKSATPA (SEQ ID NO:7), and other orthologous sequences readily identifiable from known CD52 polypeptide and polynucleotide sequences.

Percentage identity to any of the amino acid or polynucleotide sequences disclosed herein may be determined by methods known in the art. For example, amino acid and polynucleotide sequences can be compared manually or by using computer- based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et at, 1993); see also www.ncbi.nlm.nih.gov/BLAST/), the Clustal method of alignment (Higgins and Sharp, 1989) and others, wherein appropriate parameters for each specific sequence comparison can be selected as would be understood by a person skilled in the art. The amino acid sequence of the peptide portion of the glycoprotein disclosed herein can be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, or at least 99% identical to any one of the amino acid sequences identified in SEQ ID NOs: 3, 4, 5, 6 or 7. For example, the amino acid sequence of the peptide portion of the glycoprotein disclosed herein can be 100% identical to any one of the amino acid sequences identified in SEQ ID NOs: 3, 4, 5, 6 or 7.

The soluble CD52 glycoprotein may be isolated for use in generating antibodies that bind thereto. In this context, term "isolated" is used to define the isolation of the soluble CD52 glycoprotein so that it is present in a form suitable for use in generating antibodies that bind to the soluble CD52 glycoprotein. In one example, the soluble CD52 glycoprotein may be isolated for use in generating antibodies that bind to a carbohydrate moiety of the soluble CD52 glycoprotein. Thus, the glycoprotein disclosed herein may be isolated from other components of a host cell or fluid or expression system to the extent that is required for subsequent production of antibodies. The isolated glycoprotein is therefore provided in a form which is substantially free of other components of a host cell (for example, proteins) which may hinder the production of antibodies. Thus, the isolated glycoprotein may be free or substantially free of material with which it is naturally associated such as other glycoproteins, polypeptides or nucleic acids with which it is found in its natural environment, or the environment in which it is prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Soluble glycoprotein can be isolated from a host cell or fluid or expression system by methods known in the art. The term "soluble" is used herein to define a peptide or glycoprotein which is not bound to a cell membrane. The soluble peptide or glycoprotein may be able to move freely in any solvent or fluid, such as a bodily fluid. For example, the soluble peptide or glycoprotein may be able to circulate in blood.

The carbohydrate may be any carbohydrate moiety attached to the soluble CD52 peptide fragment. For example, the carbohydrate may be any carbohydrate moiety found to be attached to the extracellular portion of the CD52 protein in a host: Thus, the carbohydrate may be any carbohydrate capable of being attached to the extracellular portion of the CD52 protein by a glycosylation reaction known to take place in a host.

Carbohydrate moieties present on a naturally occurring CD52 glycoprotein can be identified by known methods, such as those described in Schroter et al., (1999). Such carbohydrate moieties may be identified from CD52 glycoproteins present in any host cell expressing CD52, and particularly lymphocytes, such as CD4+ or CD8+ T cells. Thus, the precise structure of the carbohydrate moiety can be determined by applying methods such as mass spectrometry (e.g. Matrix-assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry (MALDI-TOF)), Mono-Q anion-exchange chromatography, high pH anion exchange chromatography (HPAEC- PAD), methylation analysis, endo-P-galactosidase digestion, and other methods. The N- glycans may be separated from naturally Occurring CD52 glycoprotein using known cleavage enzymes such as peptide-N4-( -acety-p-D-glucosaminyl)asparagines amidase F ('PNGase F' from Flavobacterium meningosepticum, recombinant from Escherichia coli; obtainable from commercial suppliers such as Roche). The N-glycans can be isolated for further characterisation using known chromatographic methods, such as C8-reversed phase chromatography. In one example, the carbohydrate may comprise one or more bi-, tri- or tetra-antennary sugars, which may be terminally sialylated. For example, the carbohydrate may comprise one or more tetra-antennary sugars. The sugars may be branched or unbranched. The sugars may comprise a proximal fucose. Thus, the carbohydrate may be fucosylated. The sugars may comprise one or more N- acetyllactosamine repeats. Thus, the sugars may comprise polylactosamine units. In addition, the sugars may comprise a mannose core.

The carbohydrate may have any one or more of the structures described in Treumann et al, (1995). Thus, for example, the carbohydrate may have any of the following structures:

Further examples of the N-linked CD52 carbohydrate which the glycoprotein disclosed herein may comprise are those derived or derivable from host lymphocyte CD52 glycoproteins or genital tract cell CD52 glycoproteins.

Due to the complex nature of many naturally occurring carbohydrate moieties known to be linked to the extracellular protein portion of human CD52 and the many variations in these structures that may arise from varying glycosylation patterns, it will be understood that the precise nature of the carbohydrate moiety present in the glycoprotein disclosed herein may vary. As stated above, methods are available to precisely identify particular carbohydrate moieties from naturally occurring CD52 glycoproteins. In addition, a number of different carbohydrate moieties can be added to the soluble peptide fragment of CD52 by expressing CD52 under varying glycosylation conditions. For example, the soluble glycoprotein disclosed herein may be expressed in and/or isolated from host lymphocyte cells or host genital tract cells (e.g. sperm cells) and may therefore comprise different carbohydrate groups as a result. Alternative host cells providing different glycosylation conditions may be selected for expression of soluble CD52 in order to provide alternative forms of carbohydrate on the soluble glycoprotein.

The carbohydrate may be attached to any one or more amino acid in the peptide which is capable of having a carbohydrate moiety attached thereto. For example, the carbohydrate may be attached to one or more asparagine, serine, threonine, tyrosine, hydroxylysine, hydroxyproline, phosphoserine or tryptophan residues if present in the amino acid sequence. In one example, the carbohydrate is attached to the asparagine (N) residue in a peptide portion having a sequence at least 60% identical to the amino acid sequence set out in SEQ ID NO: 3.

The present disclosure also provides variants, mutants, biologically active fragments, modifications, analogs and/or derivatives of the glycoprotein disclosed herein, which can all be used to generate an antibody or fragment thereof which specifically binds a CD52 glycoprotein. Such compounds can be identified by screening for compounds which mimic the structure and/or function of the polypeptide disclosed herein, using methods including any of the methods disclosed herein.

The CD52 glycoprotein or fragment thereof may be produced by any suitable method known in the art. The CD52 glycoprotein or fragment thereof may also be produced as a fusion protein, wherein the peptide portion of the CD52 glycoprotein is linked to another protein. The other protein may serve to improve the stability, function and/or ease of manufacture of the CD52 glycoprotein or fragment thereof. Antibodies

The CD52 glycoprotein or soluble CD52 glycoprotein fragment disclosed herein can be used to generate an antibody which binds specifically binds thereto, using any method known in the art for generating antibodies.

The CD52 glycoprotein or soluble CD52 glycoprotein fragment can be used alone, or can be used in combination with a carrier, such as an immunogenic carrier. Accordingly, the present disclosure provides a composition comprising a CD52 glycoprotein or soluble CD52 glycoprotein fragment as disclosed herein and a carrier. The carrier may be an immunogenic carrier or adjuvant.

Methods for producing antibodies are known in the art, and any suitable method can be used to produce an antibody which specifically binds the CD52 glycoprotein or soluble CD52 glycoprotein fragment. For example, such methods can include a step of immunizing an animal with the CD52 glycoprotein or soluble CD52 glycoprotein fragment. Any suitable animal can be immunized, for example, a mouse, rat, rabbit, and others.

The CD52 glycoprotein or soluble CD52 glycoprotein fragment may be administered in combination with an adjuvant. Adjuvants are useful for improving the immune response and/or increasing the stability of antigenic preparations. Adjuvants are typically described as non-specific stimulators of the immune system, but can also be useful for targeting specific arms of the immune system. One or more compounds which have this activity may be administered in combination with the CD 52 glycoprotein or soluble CD52 glycoprotein fragment. Examples of chemical compounds that can be used as adjuvants include, but are not limited to aluminium compounds (e.g., aluminium hydroxide), metabolizable and non-metabolizable oils, mineral oils including mannide oleate derivatives in mineral oil solution (e.g., MONTANIDE ISA 70 from Seppic SA, France), and light mineral oils such as DRAKEOL 6VR, block polymers, ISCOM's (immune stimulating complexes), vitamins and minerals (including but not limited to: vitamin E, vitamin A, selenium, and vitamin B 12) and CARBOPOL ^® .

Other suitable adjuvants, which sometimes have been referred to as immune stimulants, include, but are not limited to: cytokines, growth factors, chemokines, supematants from cell cultures of lymphocytes, monocytes, cells from lymphoid organs, cell preparations and/or extracts from plants, bacteria or parasites (e.g. Staphylococcus aureus or lipopolysaccharide preparations) or mitogens.

Generally, an adjuvant is administered at the same time as the CD52 glycoprotein or soluble CD52 glycoprotein fragment. However, adjuvants can also, or alternatively be administered within a two-week period prior to administration of the CD52 glycoprotein or soluble CD52 glycoprotein fragment, and/or for a period of time after administration of the CD52 glycoprotein or soluble CD52 glycoprotein fragment, i.e., so long as the CD52 glycoprotein or soluble CD52 glycoprotein fragment persists in the tissues.

Immunogenic compositions disclosed herein may include the CD52 glycoprotein or soluble CD52 glycoprotein fragment disclosed herein, or immunogenic fragments thereof, and may be administered using any form of administration known in the art or described herein. In some embodiments, the immunogenic composition may include a live bacterial pathogen, a killed bacterial pathogen, or components thereof. Live bacterial pathogens, which may be administered in the form of an oral vaccine, may be attenuated so as to reduce the virulence of the bacterial pathogen, but not its induction of an immune response. A live vaccine may be capable of colonizing the intestines of the inoculated animal, e.g., avian.

In some embodiments, the CD52 glycoprotein or soluble CD52 glycoprotein fragment described herein may be administered to poultry, e.g., chicken, ducks, turkeys, etc., so as to elicit an immune response e.g., raise antibodies, in the poultry. Eggs, or products thereof, obtained from such poultry, that exhibit an immune response against the CD52 glycoprotein or soluble CD52 glycoprotein fragment, or immunogenic fragments thereof, may be administered to an animal, e.g., humans, cattle, goats, sheep, etc., to elicit an immune response to the CD52 glycoprotein or soluble CD52 glycoprotein fragment in the animal. Methods of raising antibodies in poultry, and administering such antibodies, are described in for example, US 5,750,1 13 and US 6,730,822.

The immunogenic compositions disclosed herein may be administered as a liquid, emulsion, dried powder and/or in a mist through any parenteral route, intravenously, intraperitoneally, intradermally, by scarification, subcutaneously, intramuscularly, or inoculated by a mucosal route, e.g., orally, intranasally, as an aerosol, by eye drop, by in ovo administration, or implanted as a freeze dried powder.

The immunogenic compositions disclosed herein may further comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier includes a veterinarily acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

As used herein, the term "specifically binds" shall be taken to mean that the antibody or fragment thereof reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a CDS 2 glycoprotein or soluble CD52 glycoprotein fragment than it does with another protein or epitope. For example, an antibody or fragment that specifically binds to a CD52 glycoprotein or soluble CD52 glycoprotein fragment binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to another polypeptide or glycoprotein. In this regard, the degree of greater affinity, avidity, more readily, and/or with greater duration will depend on the application of the antibody or fragment. For example, for detection/diagnostic/prognostic purposes the degree of specificity should be sufficiently high to permit quantification (where required). For therapeutic/prophylactic applications, the degree of specificity should be sufficient to provide a therapeutic/prophylactic effect without serious adverse effects resulting from cross- reactivity of the antibody or fragment. It is also to be understood by reading this definition that the term "specifically binds" does not necessarily require exclusive binding or non-detectable binding of another molecule, this is encompassed by the term "selective binding". Generally, but not necessarily, reference to binding means specific binding.

It will be appreciated that the particular measurement of binding will depend on the method used to assess binding, and that the binding levels may vary depending on the experimental conditions used. Several different methods of determining the level of binding of an antibody of fragment thereof to a particular epitope are known. For examp e, a variety of immunoassay formats (such as solid-phase ELISA immunoassays) are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See Harlow and Lane (1988) Antibodies, a Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity .

Antibodies which are known in the art to bind specifically to a CD52 glycoprotein or soluble CD52 glycoprotein fragment can be used in the methods disclosed herein. In particular, antibodies which are known in the art to bind specifically to a carbohydrate moiety of a CD52 glycoprotein or soluble CD52 glycoprotein fragment can be used in the methods disclosed herein. Examples of such antibodies include CF1D12, HI-186 (Hale, 2001), 2C6 (Kameda et al, 1991), H6-C34, or S 19 (Diekman et al. , 1999), C AMPATH- 1 H, and others.

In addition, any antibody which competitively inhibits the binding of any antibody or fragment described herein to a CD52 glycoprotein or soluble CD52 glycoprotein fragment can be used in the methods disclosed herein. For example, an antibody which competitively inhibits the binding of the monoclonal antibody CF1D12 to a CD52 glycoprotein or soluble CD52 glycoprotein fragment can be used in the methods disclosed herein.

The antibodies disclosed herein may exist as intact immunoglobulins, or as modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH« as described for a camelid), a dimer of the light chain variable region (VLL), FV fragments containing only the light and heavy chain variable regions, or Fd fragments containing the heavy chain variable region and the CHI domain. For example, the antibody may be a domain antibody including either the V _H or VL domain of the monoclonal antibody CF1D12; a dimer of the heavy chain variable region (VHH, as described for a camelid) of the monoclonal antibody CF1D12; a dimer of the light chain variable region (V _L L) of the monoclonal antibody CF1D12; Fv fragments containing only the light and heavy chain variable regions of the monoclonal antibody CF1D12; or Fd fragments containing the heavy chain variable region and the CHI domain of the monoclonal antibody CF1D12.

A scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody (Bird et al., 1988; Huston et al., 1988) and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term "antibody". Thus, the antibody may be a scFv consisting of the variable regions of the heavy and light chains of the monoclonal antibody CF1D12 linked together to form a single-chain antibody, or may be an oligomer thereof (such as a diabody or triabody).

Also encompassed are fragments of antibodies such as Fab, (Fab')2 and FabFc ₂ fragments which contain the variable regions and parts of the constant regions. Thus, the antibody may be a fragment such as a Fab, (Fab') ₂ and FabFc ₂ fragment containing the variable regions and parts of the constant regions of the monoclonal antibody CF1D12.

CDR-grafted antibody fragments and oligomers of antibody fragments are also encompassed. For example, the antibody fragment or oligomers of antibody fragments may comprise any one or more of the CDRs present in the monoclonal antibody CF1D12.

The CDRs of the monoclonal antibody CF1D12 have been described in WO

2010/132659 as follows:

Light chain CDR-1 : KSSQSLLESDGRTYLN (SEQ ID NO: 8)

Light chain CDR-2: LVSNLDS (SEQ ID NO: 9)

Light chain CDR-3: WQGTHFPWT (SEQ ID NO: 10)

Heavy chain CDR-1 : GFTFSDAWMD (SEQ ID NO: 1 1)

Heavy chain CDR-2: EIRN AKNHVAYYAESVKG (SEQ ID NO: 12)

Heavy chain CDR-3: TTLDS (SEQ ID NO: 13)

The antibody or fragment disclosed herein may comprise any one or more of the CDRs of the monoclonal antibody CF1D12 (i.e., any one or more of the amino acid sequences set out in SEQ ID NOs: 8, 9, 10, 11, 12 and 13). Thus, the antibody or fragment thereof may comprise any 1, 2, 3, 4, 5 or 6 CDRs of the monoclonal antibody CF1D12. Preferably, the antibody or fragment thereof comprises all 6 CDRs of the monoclonal antibody CF1D12.

The light chain and heavy chain variable region sequences of the monoclonal antibody CF1D12 have been described in Figures 2 and 3 of WO 2010/1 2659 as follows:

Light chain variable region sequence:

DVVMTQTPLALSVTIGHPASISCKSSQSLLESDGRT YLN WLFQRPGQSPKRLI YL VSNLDSGVPDRFSGSGSGTDFTL ISRVEAEDLGVYYCWQGTHFPWTFGGGT LEIK (SEQ ID NO: 14).

Heavy chain variable region sequence:

EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIR NKAKNHVAYYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTTLDSW GQGTALTVSS (SEQ ID NO: 15).

The antibody or fragment disclosed herein may comprise the light chain variable region sequence of the monoclonal antibody CF1D12 (i.e., the amino acid sequence set out in SEQ ID NO: 14), or the heavy chain variable region sequence of the monoclonal antibody CF1D12 (i.e., the amino acid sequence set out in SEQ ID NO: 15), or both the heavy and light chain variable region sequences of the monoclonal antibody CF1D12. Preferably, the antibody or fragment disclosed herein comprises both the heavy and light chain variable region sequences of the monoclonal antibody CF 1 D 12.

The antibody or fragment disclosed herein may comprise any one or more of the CDRs identified in WO 2010/132659 as being present in antibodies known to bind to CD52. Thus, the antibody or fragment disclosed herein may comprise any one or more of the CDRs set out in Table 1 below, in any combination.

Table 1 : CD52 antibody CDR sequences

CDR CDR sequence (SEQ ID NO)

Light chain CDR- 1 ASQNID YLN (SEQ ID NO: 16)

KSSQSLLDSDGKTYLN (SEQ ID NO: 17) KSSQSLLDSDGRTYLN (SEQ ID NO: 18) SSQSLL YSNGKT YLN (SEQ ID NO: 19) RSSQSLVHTNGNSYLH (SEQ ID NO: 20) RSSQSLVHTNGNTYLH (SEQ ID NO: 21)

Light chain CDR-2 NTNNLQT (SEQ ID NO: 22)

LVS LDS (SEQ ID NO: 23)

LVSNLGS (SEQ ID NO: 24)

LVSALDS (SEQ ID NO: 25)

LVSNLNS (SEQ ID NO: 26)

LVSHLDS (SEQ ID NO: 27)

MVSNRFS (SEQ ID NO: 28)

Light chain CDR-3 LQHISRPRT (SEQ ID NO: 29)

VQGSHFHT (SEQ ID NO: 30)

VQGTRFHT (SEQ ID NO: 31)

VQGTHLHT (SEQ ID NO: 32)

SQSTHVPFT (SEQ ID NO: 33)

SQSAHVPPLT (SEQ ID NO: 34)

Heavy chain CDR-1 GFTFTDFYMN (SEQ ID NO: 35)

RFTFSDAWMD (SEQ ID NO: 36)

GLTFSDAWMD (SEQ ID NO: 37)

GFPFSNYW N (SEQ ID NO: 38)

GFTFNKYWMN (SEQ ID NO: 39)

GFTFNTYWMN (SEQ ID NO: 40)

GFTFTDYYMS (SEQ ID NO: 41)

Heavy chain CDR-2 FIRDKAKGYTTEYNPSVKG (SEQ ID NO: 42)

EIRNKANNHATYYAESVKG (SEQ ID NO: 43) EIRNKAKNHVKYYAESVKG (SEQ ID NO: 44) EIRNKAKNHATYYAESVKG (SEQ ID NO: 45) EIRKKVNNHATYYAESVKG (SEQ ID NO: 46) QIRLKSNNYATHYAESVKG (SEQ ID NO: 47) QIRLKSDN YATH Y AES VKG (SEQ ID NO: 48) FIRNKANGYTTEYNASVKG (SEQ ID NO: 49) FIRNKANGYTTEYSASVKG (SEQ ID NO: 50)

Heavy chain CDR-3 AREGHTAAPFDY (SEQ ID NO: 51)

TSLDY (SEQ ID NO: 52)

TGLDY (SEQ ID NO: 53)

TPIDY (SEQ ID NO: 54) TPVDF (SEQ ID NO: 55)

TRYIFFDY (SEQ ID NO: 56)

TRYIWFDY (SEQ ID NO: 57)

As used herein, the term "CDRs" refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or VL) typically has three CDR regions identified as CDR1, CDR2 and CDR3. In one example, the amino acid positions assigned to CDRs and framework regions (FRs) are defined according to Kabat (1987) and (1991) (also referred to herein as "the Kabat numbering system"). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http://www.bioinfo.org.uk/mdex.html). According to the numbering system of Kabat, V _H FRs and CDRs are positioned as follows: residues 1-30 (FRl), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 1 13 (FR4). According to the numbering system of Kabat, VL FRS and CDRs are positioned as follows: residues 1-23 (FRl), 24-34 (CDRl), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4).

The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk (1987); Chothia et al., (1989); and/or Al- Lazikani et al., (1997); the numbering system of Honnegher and Pliikthun (2001); or the IMGT system discussed in Giudicelli et al., (1997). In one example, the CDRs are defined according to the Kabat numbering system. Optionally, heavy chain CDR2 according to the Kabat numbering system does not comprise the five C-terminal amino acids in the CDR2 sequence of the monoclonal antibody CF1D12 or any one or more of those amino acids are substituted with another naturally-occurring amino acid. In an additional, or alternative, option, light chain CDRl does not comprise the four N- terminal amino acids in the CDRl sequence of the monoclonal antibody CF1D12 or any one or more of those amino acids are substituted with another naturally -occurring amino acid. In this regard, Padlan et al., (1995) established that the five C-terminal amino acids of heavy chain CDR2 and/or the four N-terminal amino acids of light chain CDRl are not generally involved in antigen binding.

Suitable methods for identifying CDRs within a given antibody (such as a monoclonal antibody) are well known in the art. The amino acid sequence of antibodies such as the monoclonal antibody CF1D12 can be determined by routine methods. For example, the encoding nucleotide sequence can be determined by routine methods and the amino acid sequence of the antibody can be deduced therefrom. The entire coding sequence may be determined. Alternatively, the nucleotide sequence encoding only the variable regions of the antibody may be determined. For example, primer sequences for the isolation of murine antibody variable region sequences are readily available in the art (e.g., as described in WO 2006/106331). In other examples, primer sequences for the isolation of antibody variable region sequences from rat (see, e.g., WO 2006/106336), sheep (see, e.g., WO 2006/106341), pig (see, e.g., WO 2006 106339), and other species are readily available. Thus, CDRs from antibodies disclosed herein that are raised in rats, sheep, pigs and other species can also be determined. Once the amino acid sequence of the antibody is determined, the sequences of the CDRs can be determined as described above. The CDR sequences can be determined manually. Alternatively, publicly available software can be used to identify CDRs automatically.

The heavy and light chain components of an Fv may be derived from the same antibody (e.g., the monoclonal antibody CF1D12) or different antibodies thereby producing a chimeric Fv region. The antibody may be of animal (for example mouse, rabbit or rat) or human origin or may be chimeric (Morrison et al., 1984) or humanized (published UK patent application No. 8707252). As used herein the term "antibody or fragment thereof includes these various forms. Using the guidelines provided herein and those methods well known to those skilled in the art which are described in the references cited above and in such publications as Harlow & Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory, (1988) the antibodies or fragments thereof for use in the methods disclosed herein can be readily made.

In addition, the antibodies may be Fv regions comprising a variable light (VL) and a variable heavy (V _H ) chain. In one example, the VL and/or VH chains are those of the monoclonal antibody CFID12. The light and heavy chains may be joined directly or through a linker. As used herein a linker refers to a molecule that is covalently linked to the light and heavy chain and provides enough spacing and flexibility between the two chains such that they are able to achieve a conformation in which they are capable of specifically binding the epitope to which they are directed. Protein linkers are particularly preferred as they may be expressed as an intrinsic component of the Ig portion of the fusion polypeptide.

Polyclonal antibodies The antibody disclosed herein may be a polyclonal antibody. Thus, the antibody may be comprised in antisera produced by immunisation of an animal with the glycoprotein disclosed herein. Any animal may be immunized in order to produce said antisera. In one embodiment, the antisera is rabbit antisera.

Monoclonal antibodies

Alternatively, the antibody disclosed herein may be a monoclonal antibody. Monoclonal antibodies can be produced by methods known in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody -producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against the CD52 glycoprotein disclosed herein can be screened for various properties; i.e. for isotype and epitope affinity.

The monoclonal antibody may, for example, be the monoclonal antibody

CF1D12.

Animal-derived monoclonal antibodies can be used for both direct in vivo and extracorporeal immunotherapy. However, it has been observed that when, for example, mouse-derived monoclonal antibodies are used in humans as therapeutic agents, the patient produces human anti-mouse antibodies. Thus, animal-derived monoclonal antibodies are not preferred for therapy, especially for long term use. With established genetic engineering techniques it is possible, however, to create chimeric or humanized antibodies that have animal-derived and human-derived portions. The animal can be, for example, a mouse or other rodent such as a rat.

If the variable region of the chimeric antibody is, for example, mouse-derived while the constant region is human-derived, the chimeric antibody will generally be less immunogenic than a "pure" mouse-derived monoclonal antibody. These chimeric antibodies would likely be more suited for therapeutic use, should it turn out that "pure" mouse-derived antibodies are unsuitable. Thus, the chimeric antibody may comprise one or more variable regions of the monoclonal antibody CF1D12 and a human-derived constant region.

Methodologies for generating chimeric antibodies are available to those in the art. For example, the light and heavy chains can be expressed separately, using, for example, immunoglobulin light chain and immunoglobulin heavy chains in separate plasmids. These can then be purified and assembled in vitro into complete antibodies; methodologies for accomplishing such assembly have been described (see, for example, Sun et al., 1986). Such a DNA construct may comprise DNA encoding functionally rearranged genes for the variable region of a light or heavy chain of an anti-CD52 glycoprotein antibody linked to DNA encoding a human constant region. Lymphoid cells such as myelomas or hybridomas transfected with the DNA constructs for light and heavy chain can express and assemble the antibody chains.

In vitro reaction parameters for the formation of IgG antibodies from reduced isolated light and heavy chains have also been described (see, for example, Beychok, 1979). Co-expression of light and heavy chains in the same cells to achieve intracellular association and linkage of heavy and light chains into complete H2L2 IgG antibodies is also possible. Such co-expression can be accomplished using either the same or different plasmids in the same host cell.

Humanising methodologies/techniques

In one embodiment disclosed herein, the anti-CD52 antibody is humanized, that is, an antibody produced by molecular modelling techniques wherein the human content of the antibody is maximised while causing little or no loss of binding affinity attributable to the variable region of, for example, a parental rat, rabbit or mouse antibody.

An antibody may be humanized by grafting the desired CDRs onto a human framework according to the method described in EP-A-0239400. For example, the CDRs may be selected from the monoclonal antibody CF1D12. Preferably all 6 CDRs are selected. A DNA sequence encoding the desired reshaped antibody can be made beginning with the human DNA whose CDRs it is wished to reshape. The animal- derived variable domain amino acid sequence containing the desired CDRs (e.g., the CDRs of the monoclonal antibody CF1D12) is compared to that of the chosen human antibody variable domain sequence. The residues in the human variable domain are marked that need to be changed to the corresponding residue in the animal to make the human variable region incorporate the animal-derived CDRs. There may also be residues that need substituting in, adding to or deleting from the human sequence.

Oligonucleotides are synthesized that can be used to mutagenize the human variable domain framework to contain the desired residues. Those oligonucleotides can be of any convenient size. One is normally only limited in length by the capabilities of the particular synthesizer one has available. The method of oligonucleotide-directed in vitro mutagenesis is well known.

Alternatively, humanisation may be achieved using the recombinant polymerase chain reaction (PCR) methodology of WO 92/07075. Using this methodology, a CDR (e.g., a CDR of the monoclonal antibody CF1D12) may be spliced between the framework regions of a human antibody. In general, the technique of WO 92/07075 can be performed using a template comprising two human framework regions, AB and CD, and between them, the CDR which is to be replaced by a donor CDR. Primers A and B are used to amplify the framework region AB, and primers C and D used to amplify the framework region CD. However, the primers B and C each also contain, at their 5' ends, an additional sequence corresponding to all or at least part of the donor CDR sequence. Primers B and C overlap by a length sufficient to permit annealing of their 5' ends to each other under conditions which allow a PCR to be performed. Thus, the amplified regions AB and CD may undergo gene splicing by overlap extension to produce the humanized product in a single reaction.

Following the mutagenesis reactions to reshape the antibody, the mutagenised DNAs can be linked to an appropriate DNA encoding a light or heavy chain constant region, cloned into an expression vector, and transfected into host cells, preferably mammalian cells. These steps can be carried out in routine fashion. A reshaped antibody may therefore be prepared by a process comprising:

(a) preparing a first replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least a variable domain of an Ig heavy or light chain, the variable domain comprising framework regions from a human antibody and the CDRs required for the humanized antibody of the invention;

(b) preparing a second replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least the variable domain of a complementary Ig light or heavy chain respectively;

(c) transforming a cell line with the first or both prepared vectors; and

(d) culturing said transformed cell line to produce said altered antibody.

Preferably the DNA sequence in step (a) encodes both the variable domain and each constant domain of the human antibody chain. The humanized antibody can be prepared using any suitable recombinant expression system. The cell line which is transformed to produce the altered antibody may be a Chinese Hamster Ovary (CHO) cell line or an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line. The cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Barr virus. Most preferably, the immortalised cell line is a myeloma cell line or a derivative thereof.

The CHO cells used for expression of the antibodies may be dihydrofolate reductase (dhfr) deficient and so dependent on thymidine and hypoxanthine for growth (Urlaub et al, 1980). The parental dhfr ^" CHO cell line is transfected with the DNA encoding the antibody and dhfr gene which enables selection of CHO cell transformants of dhfr positive phenotype. Selection is carried out by culturing the colonies on media devoid of thymidine and hypoxanthine, the absence of which prevents untransformed cells from growing and transformed cells from resalvaging the folate pathway and thus bypassing the selection system. These transformants usually express low levels of the DNA of interest by virtue of co- integration of transfected DNA of interest and DNA encoding dhfr. The expression levels of the DNA encoding the antibody may be increased by amplification using methotrexate (MTX). This drug is a direct inhibitor of the enzyme dhfr and allows isolation of resistant colonies which amplify their dhfr gene copy number sufficiently to survive under these conditions. Since the DNA sequences encoding dhfr and the antibody are closely linked in the original transformants, there is usually concomitant amplification, and therefore increased expression of the desired antibody.

Another preferred expression system for use with CHO or myeloma cells is the glutamine synthetase (GS) amplification system described in WO 87/04462. This system involves the transfection of a cell with DNA encoding the enzyme GS and with DNA encoding the desired antibody. Cells are then selected which grow in glutamine free medium and can thus be assumed to have integrated the DNA encoding GS. These selected clones are then subjected to inhibition of the enzyme GS using methionine sulphoximine (Msx). The cells, in order to survive, will amplify the DNA encoding GS with concomitant amplification of the DNA encoding the antibody.

Although the cell line used to produce the humanized antibody is preferably a mammalian cell line, any other suitable cell line, such as a bacterial cell line or a yeast cell line, may alternatively be used. In particular, it is envisaged that E. co/i-derived bacterial strains could be used. The antibody obtained is checked for functionality. If functionality is lost, it is necessary to alter the framework of the antibody.

Once expressed, the whoje antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be recovered and purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (See, generally, Scopes, R., Protein Purification, Springer- Verlag, N.Y. (1982)). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, an antibody may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (See, generally, Immunological Methods, Vols. I and II, Lefkovits and Pernis, eds., Academic Press, New York, N.Y. (1979 and 1981)).

Antibodies with fully human variable regions against the CD52 glycoprotein can also be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Various subsequent manipulations can be performed to obtain either antibodies per se or analogs thereof (see, for example, US Patent No. 6,075,181). Methods of treatment .

The antibodies or fragments disclosed herein can be used to enhance an immune response in a subject. As used herein, the term "enhance" is intended to mean that the antibody or fragment causes the subject to exhibit a greater immune response to an antigen compared to the immune response that is exhibited against the same antigen in the absence of the antibody or fragment. The extent of the increase is not limited. It will be appreciated that the particular measurement of the immune response will depend on the method used to assess the immune response, and that the levels determined may vary depending on the experimental conditions used.

The level of a subject's immune response can be determined by any suitable means known in the art. For example, the level of an immune response may be determined by detecting the level of an activity of an immune cell or immune cell population from the subject.

In one example, the level of an immune response may be determined by determining the level of an activity of a T cell or a T cell population in a subject. T cells can be readily identified by the presence of any of one or more T cell markers known in the art. For example, the antibody or fragment may be capable of increasing T-cell proliferation in response to antigen challenge, and/or may be capable of increasing T-cell cytokine production (such as production of any one or more of IFN-γ, IL-2, IL-10, IL-17, G-CSF, TNF-a, and other cytokines known to be secreted by activated T-cells). For example, the glycoprotein may be capable of increasing IFN-γ production by T-cells.

The methods disclosed herein may further comprise administering any antigen to a subject, in addition to the antibody or fragment disclosed herein. The antigen may be any antigen known to be causative of or associated with any disease or condition, such as any of the diseases or conditions disclosed herein. For example, when the disease or condition is type 1 diabetes, the antigen may be GAD65. Thus, the methods may comprise a method of vaccinating a subject.

The methods disclosed herein may further comprise administering any therapeutic agent to a subject, in addition to the antibody or fragment disclosed herein. In one example, the therapeutic agent is a chemotherapeutic agent. Any known chemotherapeutic agent may be used.

The antibodies or fragments disclosed herein can be administered to a subject by any suitable means. For example, the antibodies or fragments disclosed herein may be administered in the form of a pharmaceutical composition.

The pharmaceutical composition may further comprise an antigen. Thus, the pharmaceutical composition may be provided as a vaccine.

Alternatively, the pharmaceutical composition may further comprise a therapeutic agent. Thus, the antibodies or fragments disclosed herein may be used as an adjuvant in therapy.

Thus, the present disclosure provides a pharmaceutical composition comprising an antibody or fragment thereof as disclosed herein, and a pharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier includes a veterinarily acceptable carrier. In one example, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

Therapeutic compositions can be prepared by mixing the desired compounds having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the form of lyophilized formulations, aqueous solutions or aqueous suspensions. Acceptable carriers, excipients, or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Additional examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, and cellulose-based substances.

A pharmaceutical composition as disclosed herein is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal), mucosal (e.g., oral, rectal, intranasal, buccal, vaginal, respiratory), enteral (e.g., orally, such as by tablets, capsules or drops, rectally) and transdermal (topical, e.g., epicutaneous, inhalational, intranasal, eyedrops, vaginal). Solutions or suspensions used for parenteral, intradermal, enteral or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions is brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compound (e.g., the antibody or fragment thereof) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound is incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions are also prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by mucosal or transdermal means. For mucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for mucosal administration, detergents, bile salts, and fusidic acid derivatives. Mucosal administration is accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

A pharmaceutically acceptable vehicle is understood to designate a compound or a combination of compounds entering into a pharmaceutical composition which does not cause side effects and which makes it possible, for example, to facilitate the administration of the active compound, to increase its life and/or its efficacy in the body, to increase its solubility in solution or alternatively to enhance its preservation. These pharmaceutically acceptable vehicles are well known and will be adapted by persons skilled in the art according to the nature and the mode of administration of the active compound chosen.

Pharmaceutical compositions to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The composition may be stored in lyophilized form or in solution if administered systemically. If in lyophilized form, it is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use. An example of a liquid formulation is a sterile, clear, colourless unpreserved solution filled in a single-dose vial for subcutaneous injection.

Pharmaceutical compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The compositions are preferably administered parenterally, for example, as intravenous injections or infusions or administered into a body cavity.

The methods of treatment may comprise administering a therapeutically effective amount of an antibody or fragment thereof to a subject in need thereof. The 'therapeutically effective amount' may be determined by a clinician and may vary from one patient to another, depending on factors such as age, weight, gender, and other factors. Diagnostic methods

Based on the inventors' finding that an antibody which specifically binds a CD52 glycoprotein (and in one example, a carbohydrate moiety on a CD52 glycoprotein) enhances a subject's immune response to a level that can be determined without the need to first expand a population of the subject's immune cells, the present disclosure also provides methods of determining a subject's immune response to an antigen.

The level of a subject's immune response can be detected by any suitable means known in the art. For example, the level of an immune response may be determined by detecting the level of an activity of an immune cell or immune cell population from the subject.

In one example, the level of an immune response may be determined by determining the level of an activity of a T cell or a T cell population in a subject. T cells can be readily identified by the presence of any of one or more T cell markers known in the art. For example, the level of T-cell proliferation in response to antigen challenge, and/or T-cell cytokine production (such as production of any one or more of IFN-γ, IL-2, IL-10, IL-17, G-CSF, TNF-a, and other cytokines known to be secreted by activated T-cells) may be determined by any suitable means. In one example, the level of IFN-γ production by T-cells may be determined by any suitable method, including the methods described in the examples herein. For example, the level of IFN-γ production by T-cells may be determined by ELISpot assay.

In one example, the diagnostic methods disclosed herein can be used to monitor patient response to therapy. Thus, the methods can be performed repeatedly over the course of a therapeutic treatment. The time period between performances of the diagnostic methods disclosed herein can vary as appropriate, according to the therapeutic treatment regime. For example, the methods can be performed hourly, daily, weekly, fortnightly or at longer time intervals. Such methods can be used to monitor a subject's response to any therapy, such as any cancer therapy (e.g., chemotherapy).

In another example, the diagnostic methods disclosed herein can be used to diagnose or predict a subject's response to a particular antigen. Thus, the methods may be used to diagnose a disease or condition or predict a subject's susceptibility to developing a disease or condition.

In one example, the disease or condition may be an autoimmune disease, allograft rejection, a graft-versus-host reaction, an infectious disease or an allergic disease. The term "autoimmune disease" refers to any disease in which the body produces an immunogenic (i.e., immune system) response to some constituent of its own tissue. Autoimmune diseases can be classified into those in which predominantly one organ is affected (eg, hemolytic anemia and anti-immune thyroiditis), and those in which the autoimmune disease process is diffused through many tissues (eg, systemic lupus erythematosus). The autoimmune disease may be (but is not limited to) any one or more of insulin-dependent diabetes mellitus (or type 1 diabetes), insulin autoimmune syndrome, rheumatoid arthritis, psoriatic arthritis, chronic lyme arthritis, lupus, multiple sclerosis, inflammatory bowel disease including Crohn's disease, ulcerative colitis, coeliac disease (or "celiac disease"), autoimmune thyroid disease, autoimmune myocarditis, autoimmune hepatitis, pemphigus, anti- tubular basement membrane disease (kidney), familial dilated cardiomyopathy, Goodpasture's syndrome, Sjogren's syndrome, myasthenia gravis, polyendocrine failure, vitiligo, peripheral neuropathy, autoimmnune polyglandular syndrome type 1, acute glomerulonephritis, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, Hashimoto's thyroiditis, Graves' disease, Addison's disease, chronic beryllium syndrome, ankylosing spondylitis, juvenile dermatomyositis, polychondritis, scleroderma, regional enteritis, distal ileitis, granulomatous enteritis, regional ileitis, and terminal ileitis, amyotrophic lateral sclerosis, ankylosing spondylitis, autoimmune aplastic anemia, autoimmune hemolytic anemia, Behcet's disease, chronic active hepatitis, CREST syndrome, dermatomyositis, dilated cardiomyopathy, eosinophilia- myalgia syndrome, epidermolisis bullosa acquisita (EBA), giant cell arteritis, Goodpasture's syndrome, Guillain-Barr syndrome, hemochromatosis, Henoch- Schonlein purpura, idiopathic IgA nephropathy, insulin autoimmune syndrome, juvenile rheumatoid arthritis, Lambert-Eaton syndrome, linear IgA dermatosis, myocarditis, narcolepsy, necrotizing vasculitis, neonatal lupus syndrome (NLE), nephrotic syndrome, pemphigoid, pemphigus, polymyositis, primary sclerosing cholangitis, psoriasis, rapidly-progressive glomerulonephritis (RPGN), Reiter's syndrome, stiff-man syndrome, inflammatory bowel disease, osteoarthritis, thyroiditis, and others. In one example, the autoimmune disease is type 1 diabetes. In another example, the autoimmune disease is rheumatoid arthritis. In another example, the autoimmune disease is coeliac disease. In another example, the condition is an allograft rejection or a graft- versus-host reaction. Thus, the methods disclosed herein may be carried out on a subject (or a sample taken from a subject) who is a transplant recipient or a potential transplant recipient.

Thus, the methods disclosed herein may comprise administering to a subject any antigen which is known to be causative of or associated with any of the diseases or conditions disclosed herein. For example, when the disease or condition is type 1 diabetes, the methods may comprise administering the GAD65 auto-antigen to a subject.

Alternatively, the methods disclosed herein may comprise administering to a subject an antigen which is not specific to any disease or condition. Such methods will allow a determination to be made of the subject's general level of immune response to an antigen. In some instances, an antigen such as tetanus toxin will be suitable for administration in order to allow a determination to be made of the subject's general level of immune response.

The greater the subject's immune response to an antigen associated with the disease or condition, the greater the risk of that subject developing the disease or condition. For example, the greater the subject's immune response to an auto antigen, the greater the risk of that subject developing an autoimmune disease. One example of such an auto antigen is the protein glutamic acid decarboxylase 65 (GAD65). Thus, in one example, the methods disclosed herein may be performed to determine the risk of a subject developing type 1 diabetes, comprising administering to the subject a GAD65 antigen and an antibody or fragment thereof which specifically binds a CD52 glycoprotein.

In any of the methods disclosed herein, the level of an immune response determined in a subject may be compared to a reference level. The reference level may be determined, for example, by administering the antigen and the antibody or fragment disclosed herein to one or more subjects, and calculating the average level of immune response from those subjects. The one or more subjects may constitute a population of subjects, which may be chosen based on any criteria. For example, the one or more subjects may be known to suffer from a particular disease or condition (e.g., type 1 diabetes or coeliac disease) or may be known to have a certain level of risk of developing a disease or condition (e.g., type 1 diabetes or coeliac disease).

Thus, the diagnostic methods disclosed herein, which comprise: i) contacting immune cells of the subject with an antigen in the presence of an antibody or fragment thereof which specifically binds a CD52 glycoprotein; and

ii) determining the level of an activity of the immune cells,

may further comprise a step of comparing the level of activity of the subject's immune cells with a reference level.

The reference level may be predetermined. Alternatively, the methods disclosed herein may further comprise a step of contacting the immune cells of one or more subjects with the antigen in the presence of an antibody or fragment thereof which specifically binds a CD52 glycoprotein, determining the level of activity of the immune cells of the one or more subjects, and determining a reference level of activity. The reference level may be, for example, an average level of activity. Other calculations may be performed in order to determine the reference level, as appropriate. Again, any number of subjects may be assayed in order to determine the reference level.

The methods disclosed herein may additionally comprise a step of comparing the level of an activity of the subject's immune cells with the reference level. If the level of activity of a subject is greater than the reference level, this may be taken as an indication that the subject has an increased likelihood of developing the disease or condition associated with the antigen used in the diagnostic methods. For example, when the antigen is GAD65, a subject who is shown to have a level of activity equal to or greater than a reference level determined from administering GAD65 to one or more (or a population of) subjects known to have, or to be susceptible of developing type 1 diabetes, may be considered to have an increased likelihood of developing type 1 diabetes. Conversely, if the level of activity of a subject is lower than the reference level, this may be taken as an indication that the subject has a decreased likelihood of developing the disease or condition associated with the antigen used in the diagnostic methods.

In another example, the diagnostic methods disclosed herein can be used to diagnose or predict a subject's response to a particular antigen. The particular antigen may be a peptide fragment. Thus, the methods disclosed herein can be used to determine an antigenic epitope.

Accordingly, the present disclosure provides a method of identifying an antigenic epitope, the method comprising:

i) contacting immune cells of a subject with one or more potentially

antigenic epitopes and an antibody or fragment thereof which specifically binds a CD52 glycoprotein; and ii) determining the level of an activity of the immune cells,

wherein an increased level of an activity of the immune cells in the presence of a potentially antigenic epitope identifies that epitope as an antigenic epitope.

In one example, the methods comprise contacting immune cells of a subject with a plurality of peptide fragments derived from a polypeptide. The plurality of peptide fragments may comprise partially overlapping amino acid sequences. For example, peptide fragments sequentially scanning the length of a given polypeptide can be used to identify an antigenic epitope within the full length epitope. The peptide fragments may be of any length. For example the peptide fragments may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids.

The methods of identifying an antigenic epitope may also comprise a step of comparing the level of activity of the immune cells to a reference level as described herein. For example, the reference level may be determined as the average level of immune cell activity in response to a known antigenic epitope, or a peptide fragment which is known not to be an antigenic epitope.

The diagnostic methods disclosed herein may be performed on a subject in situ, or on a sample taken from a subject. Preferably, the methods are performed on a sample taken from the subject.

The sample may comprise any bodily fluid. Preferably, the sample is a blood sample. More preferably, the sample comprises peripheral blood mononuclear cells (PBMCs). In another preferred embodiment, the sample comprises T cells or a T cell population.

The invention will now be further described with reference to the following, non-limiting examples.

EXAMPLES

EXPERIMENTAL PROCEDURES

Blood donors

Venous blood drawn into sodium heparin tubes was obtained with informed consent and Human Research Ethics Committee approval from 5 healthy young adults (3 males, 2 females) and a young adult male at risk for type 1 diabetes, all known to have blood T-cell responses to GAD65. All donors had been vaccinated to tetanus toxoid. Peripheral blood mononuclear cells (PBMCs) were isolated on Ficoll/Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden), washed twice in human tonicity phosphate buffered saline (PBS) and resuspended in Iscove's modified Dulbecco's medium (Gibco, Melbourne, Australia) containing 5% pooled, heat-inactivated human serum, lOOmM non-essential amino acids, 2mM glutamine and 5xlO-5M 2- mercaptoethanol (complete Iscove's modified Dulbecco's medium [IMDM]). Antibodies and other reagents

Reagents and suppliers were as follows: fluorescent-labelled mouse monoclonal antibody to human CD52 (clone CF1D12) (Caltag); mouse IgG3 (Caltag); mouse monoclonal antibodies to human IFN-γ (Mabtech, Sydney, NSW, Australia); carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, USA); 3H-thymidine (ICN, Sydney, Australia); PNGase F (New England Biolabs, Ipswich, MA, USA). Tetanus toxoid (TT) was provided by CSL (Parkville, Victoria, Australia). Recombinant GAD65 produced in Baculovirus and purified as described (Bach et al., 1997) was purchased from Dr Peter Van Endert, Hopital Necker, Paris. The endotoxin concentration of the GAD65 stock solution, measured by Limulus lysate assay (BioWhittaker, Walkerville, MD, USA), was 1.2 EU/mg/ml. TT and GAD65 were used at concentrations of 10 Lyons flocculating units (LFU)/ml and 5μg/ml, respectively, unless otherwise stated.

Statistical analysis

Replicates were expressed as mean ± sem. Significance between groups was determined by unpaired (2-tail) Student t test, using GraphPad Prism version 3.0cx for Macintosh (GraphPad Software Inc., San Diego, CA).

EXAMPLE 1 : Monoclonal antibody CF1D12 abrogates ^' CD52 ^h, CD4 ⁺ T cell suppression of TT-stimulated CD52 ^l0 CD4 ⁺ T cells.

Methods

PBMCs stained with carboxyfluorescein succinimidyl ester (CFSE) were cultured in IMDM in 96-well round-bottom plates, without or with GAD65 or TT, at 2 x 10 ⁵ in 200μ1 in replicates of six. After 7 days, replicates were pooled, washed in 0.1% BSA- PBS and stained on ice with anti-human CD4-PE, -PECy7 or -APC and CD52-PE (clone CF1D12) antibodies. Viable (propidium-iodide negative) CFSE ^dim CD4 ⁺ cells that had undergone division in response to GAD65 were sorted in a FACSAria (BD Biosciences, North Ryde, NSW, Australia) into fractions with the highest to the lowest CD52 expression, and single cells cloned as described (Dromey et al., 2011). Subsequently, in response to GAD65 or TT, CD52 ^hl and CD52 ¹⁰ populations corresponding, respectively, to the upper 10% and lower 10% of CD52 expression on undivided CD4 ⁺ cells were sorted for further study.

CD52 ^hi and CD52 ¹⁰ CD4 ⁺ T cells were incubated alone or together (5xl0 ³ each/200ml) in triplicate in IFN-γ ELI Spot plate wells with irradiated PBMCs (4xl0 ⁴ ) ± TT and either l(^g/ml anti-CD52 (clone CF1D12) or isotype control (IgG ₃ ) monoclonal antibody. After 24 h, cells were removed by washing and spots developed by incubation with biotinylated second anti-IFN-γ antibody, followed by streptavidin- alkaline phosphatase and BCIP/NBT colour reagent.

Results

Results are representative of three independent experiments. As shown in Figure 1, TT-activated CD52 ^hl CD4 ⁺ T cells displaced only a slight increase in IFN-γ production upon exposure to TT, whereas TT-activated CD52 ¹⁰ CD4 ⁺ T cells showed a marked increase. The combination of CD52 ¹ " and CD52 ¹⁰ CD4 ⁺ T cells resulted in the suppression of CD52 ¹⁰ CD4 ⁺ T cell IFN-γ production by CD52 ^hi CD4 ⁺ T cells. · However, this suppressive effect was abrogated by the addition of the monoclonal antibody CF1D12. The suppressive effect of CD52 ^h 'CD4 ⁺ T cells was not observed if these cells were first incubated with CF1D12 antibody for an hour at 37°C then washed with fresh medium before incubation with CD52 ^lo CD4 ⁺ T cells, irradiated PBMCs and TT. This indicates that blockade of suppression by the antibody is due to its interaction with soluble, extracellular rather than cell-bound CD52.

EXAMPLE 2: CF1D12 enhances responses of T cells in PBMCs to antigens. Methods

PBMCs (2xl0 ⁶ /well) in 0.4 ml of IMDM containing either IgG3 isotype control or CF1D12 antibody (20 μg/ml each) in triplicate were incubated in duplicate 48-weIl plates for 1 h at 37°C. Non-adherent cells were removed from each well, gently washed in IMDM at 37°C, re-constituted to 0.4 ml in IMDM and added back to their original wells in the plates. In one plate, isotype control or CF1D12 antibody (20 μg ml each) was added again to triplicate wells containing cells initially exposed to these antibodies (filled histograms); no antibodies were added to cells in the other plate ('Antibody pre- bound only'; open histograms). No antigen ('Nil'), TT (2 LFU/ml) or GAD65 (6 μg/ml) was added to each of the triplicate wells. After 1-2 h at 37°C, 0.1 ml of cell suspension from each well was dispensed in triplicate into wells of an IFN-γ ELISpot plate. After 24 h at 37°C, the wells were washed free of cells and developed for IFN-γ spots. Endotoxin in the assays was < lEU/ml.

Results

As shown in Figure 2, CF1D12 enhanced IFN-γ production in T cells exposed to TT and GAD65, compared to an isotype control antibody. However, this effect was not observed in cells which were pre-incubated with CF1D12 for an hour at 37°C before exposure to medium containing antigen but lacking CF1D12 ('Antibody pre-bound only'). Thus, CF1D12 was required to be present in the medium in order to provide an effect. This indicates that CF1D12 exerts its effect by interacting with soluble, rather than cell-bound CD52. EXAMPLE 3: CF1D12 exhibits a dose-dependent effect in enhancing responses of T cells in PBMCs to antigens.

Methods

PBMCs (5xl0 ⁵ /well) were incubated with CF1D12 antibody for 1 h at 37°C as described in Example 2, but with increasing concentrations (0, 0.5, 5 and 50 μg/ml) of antibody. After washing, the same CF1D12 antibody concentrations were added to cells in one plate (filled histograms); no antibodies were added to cells in the other plate ('Antibody pre-bound only'; open histograms). TT was added to all wells. After 1-2 h at 37°C, 0.1 ml of cell suspension from each well was dispensed in triplicate into wells of an IFN-γ ELISpot plate. After 24 h at 37°C, the wells were washed free of cells and developed for IFN-γ spots.

Results

As shown in Figure 3, CF1D12 was shown to have a dose-dependent effect in enhancing T cell IFN-γ production. Again, CF1D12 was required to be present in the medium in order to provide an effect, indicating that CF1D12 exerts its effect by interacting with soluble, rather than, cell-bound CD52.

EXAMPLE 4: CF1D12 enhances T cell response to antigens in people at risk for type / diabetes. Methods

PBMCs (2xl0 ⁶ /well) were incubated in 48- well plates in 0.4 ml of IMDM containing either isotype control or CF1D12 antibody (10 mg/ml each) in triplicate. No antigen, TT (2 LFU/ml) or GAD65 (6 mg/ml) were added to each of the triplicate wells. After 1-2 h at 37°C, 0.1 ml of cell suspension from each well was dispensed in triplicate into wells of an IFN-γ ELISpot plate. After 24 h at 37°C, the wells were washed free of cells and developed for IFN-γ spots. Endotoxin in the assays was < 1 EU/ml. '

Results

As shown in Figure 4, CF1D12 significantly enhanced T cell IFN-γ production in response to challenge with both TT and GAD65, demonstrating that a patient's likely immune response to a particular antigen can be unmasked using an antibody which specifically binds a carbohydrate moiety of soluble CD52. Such an antibody can therefore be used to indicate a patient's susceptibility to developing a disease such as an autoimmune disease. The results in Figure 4 also demonstrate that the enhancing effect of an antibody which specifically binds a carbohydrate moiety of soluble CD52 is not limited to any particular antigen.

EXAMPLE 5: The carbohydrate moiety is necessary for soluble CD52 effector function as replicated with CD52-Fc.

Methods

To further explore the role of the carbohydrate moiety in effecting soluble CD52 immunosuppressor function, mature cell surface CD52 was cloned in a lentivirus vector as a fusion protein in-line with the Fc fragment of immunoglobulin G and a C-terminal Strep-tag sequence for purification. An Fc only construct was cloned as a control. Constructs were expressed stably in Daudi cells or transiently in HEK 293T cells and soluble recombinant proteins purified from medium by elution with desthiobiotin from Streptactin resin.

The scheme for constructing DNAs encoding fusion proteins is shown in Figure 5. A mutated human IgGl Fc fragment (Armour et ai, 2003) joined to the signal peptide (SigP) sequence of CD52 was generated by PGR. This included a flexible GGSGG linker and two cleavage sites for Precission and Xa proteases between the SigP and Fc fragment, and a Strep-tag II sequence for purification (Schmidt and Skerra, 2007) at the terminus of the Fc fragment. Primers, as designated in Figure 5, used to generate and clone Fc constructs, were:

1F1 : GAAGTTCTGTTCCAGGGGCCCATCGAAGGTCGTGGTG (SEQ ID NO: 58); 1R1 : TCATTTTTCGAACTGCGGGTGGCTCCAGGCGCTTTTACCCGGAGACAG (SEQ ID NO: 59);

1F2: GGGGGTTCCGGGGGACTGGAAGTTCTGTTC (SEQ ID NO: 60);

1R2: CTTGATATCGAATTCTCATTTTTCGAACTG (SEQ ID NO: 61);

2F: CGCTGTTACGGATCCCCACCATGAAGCGCTTCCTC (SEQ ID NO: 62); 2R1 : TCCACCGCTACCTCCTGAGGGGCTGCTGGT (SEQ ID NO: 63);

2R2: TCCACCGCTACCTCCTGAGAGTCCAGTTTG (SEQ ID NO: 64).

A CD52-Fc construct comprising the CD52 SigP and extracellular domain (ECD) joined to the Fc fragment was generated by PCR. Primers used were: 2F, 2R1, 1F2 and 1R2. PCR products were digested with BamHl/EcoKL and ligaied into the FTGW lentivirus vector (Herold MJ et al., 2008). Clones were also verified by sequencing. Lentivirus particles were produced by CaP04-mediated transfection of HEK293T cells seeded in 6 cm dishes with 10 ug of vector DNA together with three helper plasmids (pMDLRRE, pRSV-REV, and pVSV-g). Virus-containing cell culture medium was collected 48 hrs after transfection and passed through a 0.45 μπι filter. One milliliter was used to transducer lxl 0 ⁶ Daudi cells grown in DME media supplemented with 10% FCS, lOOmM non-essential amino acids, 2mM glutamine and 5x10 ^"5 M 2- mercaptoethanol. Cells were screened for the highest expression of protein by intracellular staining and flow cytometry. CD52-Fc or Fc control proteins were purified from medium by single-step affinity chromatography on Streptactin resin and elution with 2.5mM desthiobiotin in lOOmM Tris-HCl, 150mM NaCl, lmM EDTA, pH 8.0, as per the manufacturer's instructions. After dialysis, SDS-PAGE revealed single Coomassie blue-stained bands of predicted size whose specificity was confirmed by Western blotting.

Assays for effects of recombinant Fc fusion proteins

PBMCs (2xl0 ⁵ cells/well) or purified CD4 ⁺ T cells (5xl0 ⁴ cells/well) in complete IMDM medium-5% heat-inactivated pooled human serum were incubated in round- bottomed 96- well plates with or without 10 Lfu/ml TT and different concentrations of CD52-Fc or Fc proteins, in a total volume of 200 μΐ, at 37°C in 5% C0 ² -air for up to 7 days. ³ H-thymidine (l μΟΛνεΙΙ) was added and after a further 18 h cells were harvested and radioactivity incorporated into DNA was measured by scintillation counting. Medium was sampled for assay of cytokines after 48hr incubation. Dendritic cells (DCs) were isolated from PBMCs as described (Mittag et al, 201 1). In brief, PBMCs were first enriched for DCs by magnetic bead depletion of cells labelled with antibodies to lineage markers (CD3, CD 19, CD56). Cells were then stained with fluorescent antibodies to HLA-DR, CDl lc, CDlb/c, CD304 and CD14 and flow sorted to purify CDlb/c+HLA-DR+CDl lc+ conventional DC, CD304+HLA-DR+CDl lc- plasmacytoid DC and CD14+CD16-CDl lc+ monocytes. Purified DCs were pre- incubated with CD52-Fc or Fc protein at 3.3μΜ for 30min at 37°C and washed twice. They were then serially diluted from 6000 cells/well in a 96-round bottom well plate and incubated with CFSE-labelled CD4+ T cells (5xl0 ⁴ /well) isolated from a different donor. After 6 days, the allogeneic T cell response was measured as frequency of dividing CFSE ¹⁰ ceils determined by flow cytometry. As described above, PBMCs (200,000) were cultured with TT for 7 days and purified CD4 ⁺ T cells (20,000) with anti-CD3 (100 ng/ml) and anti-CD28 (200 ng/ml) antibody for 48 hr, with 4 times the number of irradiated PBMCs in 200μ1 round bottom wells, in the presence of recombinant CD52-Fc or Fc protein control protein at the indicated concentrations. ³ H-thymidine uptake was measured over the final 16 hr of incubation. Results (mean ± sem of triplicates) are representative of six independent experiments.

Media from PBMCs activated with TT ± 3.3 μΜ CD52-Fc or Fc proteins were sampled after 48 hr incubation and assayed for cytokines by multiplex bead array. CD52-Fc (0.66 μΜ) was incubated with PNGase F (10 ³ units) or reaction buffer only in 20 ml PBS overnight at 37°C, as recommended by the supplier, and the reaction terminated by heating at 75°C for 10 min. The decrease in size of CD52-Fc after treatment was determined by SDS-PAGE and Coomassie staining. PBMCs were incubated with TT and either PNGase F-treated or untreated CD52-Fc (final 0.33 μΜ), in triplicate, for 7 days at 37°C, before measurement of ³ H-thymidine uptake as above.

Results

With PBMCs, the proliferative response of T cells to TT was suppressed by CD52-Fc in a dose-dependent manner (Figure 6A), and CD52-Fc suppressed the secretion of cytokines typifying different T-cell lineages (Figure 6C). The effect of CD52-Fc on T cell function was direct because it suppressed proliferation of purified CD4+ T cells in response to T cell receptor cross-linking with anti-CD3 antibody and co-stimulation with anti-CD28 antibody (Figure 6B). Evidence that CD52-Fc did not require antigen- presenting cells for T cell suppression was obtained by showing that exposure of purified dendritic cells to CD52-Fc did not affect their ability to elicit an allogeneic T cell response (Figure 7).

The ability of the CF1D12 antibody to block suppression by native CD52 implied that suppression may be mediated by the carbohydrate moiety of CD52. To examine its role in recombinant CD52-Fc the N-linked carbohydrate was cleaved with the endoglycosidase PNGase F, which cleaves asparagine-linked oligosaccharides between two N-acetylglucosamine subunits immediately adjacent to the asparagine residue to generate a truncated carbohydrate with one N-acetylglucosamine residue remaining on the asparagine. Treatment reduced the molecular weight of CD52-Fc from -48 to -30 kDa, as predicted from loss of the carbohydrate. As shown in Figure 6D, treatment with PNGase F reduced the suppressive effect of CD52-Fc, confirming the role of the carbohydrate moiety in mediating the suppressive effect of soluble CD52.

EXAMPLE 6: CD52 antibody enhances host immune response to antigens. Methods

To demonstrate further the effect of an anti-CD52 antibody in enhancing a subject's immune response to antigen, peripheral blood mononuclear cells (PBMCs) were incubated together with a known causative antigen of coeliac disease (gliadin or a gluten peptide) in the presence or absence of the monoclonal antibody CF1D12.

Participants and wheat challenge

It has previously been shown that 3-day oral wheat challenge induces T cells specific for gluten and gluten peptides in peripheral blood of people with coeliac disease in remission, but not in healthy FILA-matched controls (Tye-Din et al., 2010; Anderson et al., 2005). Here, eight HLA DQ2.5-positive subjects with biopsy-proven coeliac disease (as per ESPGHAN criteria [Walker-Smith et al., 1990] in remission on a gluten-free diet and two HLA DQ2.5-positive non-coeliac healthy control subjects were studied. Subjects with coeliac disease undertook a three-day oral wheat challenge (four slices of wheat bread, approximately 13 gram of gluten daily) as previously described (Tye-Din et al., 2010; Anderson et al, 2005), whilst the healthy controls continued on a normal gluten-containing diet. ELISpot assay

Overnight interferon-gamma (IFN-γ) ELISpot is a robust and sensitive tool to enumerate antigen-specific T cells. In coeliac disease subjects, blood was collected immediately before and 6 days after commencing wheat challenge and assessed for the presence of gliadin- or gluten peptide-specific peripheral blood T cells using IFN-γ ELISpot. In healthy controls, blood was collected at a single time point for the same assay. PBMCs were isolated and overnight IFN-γ ELISpot assays (Mabtech; Stockholm, Sweden) performed using 96-well plates (MSIP-S45-10; Millipore, Bedford, MA) (Tye-Din et al, 2010; Anderson et al, 2005). Gliadin (#0210177810, MP Biomedicals, Santa Ana, CA) was incubated for 4 hours, 37° C in 10-fold excess with chymotrypsin (Sigma C3142) in ammonium bicarbonate pH 8, and finally boiled for 15min. Deamidation of chymotrypsin-digested gliadin was performed with guinea pig liver tissue transglutaminase (tTG) (Sigma T5398) (Anderson et ah, 2000), in order to produce an antigen closely resembling that which is causative of coeliac disease in human subjects (tTG activity is increased in the inflamed intestinal mucosa). Protein concentration was determined by BCA method (Pierce, USA).

Deamidated chymotrypsin-digested gliadin and two wheat gliadin peptides ("Peptide 1" and "Peptide 2") were assessed in duplicate at concentrations of 1-320 μg/ml and 0.1- 32 μg ml, respectively. "Peptide 1" was a 15mer containing the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 65), which encompassed the immunodominant T-cell epitopes DQ2-a-l/2. Peptide 2 was a 14mer containing the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 66), which encompassed DQ2-glia-co-l/2 (Tye- Din et al, 2010). Both had acetyl- and amide-groups at the N- and C-termini, respectively. Positive controls were tetanus toxoid (10 LFU/ml) and CEF (a peptide pool containing CD8+ T-cell epitopes from cytomegalovirus, Epstein Barr virus, and influenza virus, 1 μg ml, Mabtech, Sweden). Spot forming units (SFU) in individual ELISpot wells were counted using an automated reader (AID ELISPOT Reader System, AID Autoimmun Diagnostika GmbH; Strassberg, Germany). Criteria for a positive response to chymotrypsin-digested gliadin and Peptide 1/2 were previously established to be >10 SFU/10 ⁶ PBMCs, and also to be at least twice the background (medium alone) SFU value. Results were expressed as mean± SEM SFU per million PBMCs plated with mean Nil (no antigen) SFU value subtracted. In the ELISpot assay, deamidated chymotrypsin-digested gliadin, Peptide 1 and Peptide 2 were incubated with PBMCs in medium alone or with CF1D12 antibody or IgG3 isotype control (final concentration of each 20 g/ml). In 3 coeliac disease subjects and 1 healthy control, tetanus toxoid and CEF were also tested together with CF1D12 or isotype control.

Results

IFN-γ ELISpot responses of PBMCs from two representative HLA DQ2.5-positive coeliac disease subjects 6 days after commencing wheat challenge, and in 2 HLA DQ2.5-positive healthy subjects at baseline are shown in Figures 8 and 9, respectively. Positive controls were positive in all individuals (data not shown). In coeliac disease subjects, IFN-γ responses to deamidated gliadin and Peptides 1 and 2 were substantially increased (up to 10-fold) by the addition of CF1D12 compared to no antibody or isotype control (Figure 9). This was observed in all (8/8) coeliac disease subjects in response to Peptide 1 and Peptide 2, and in 7/8 coeliac disease subjects in response to deamidated gliadin. IFN-γ responses to positive control antigens were modestly increased in the presence of CF1D12. In healthy controls, IF -γ responses to deamidated gliadin and Peptides 1 and 2 were also increased by the addition of CF1D12 compared to no antibody or isotype control (Figure 9). Consistent with previous findings, the IFN-γ responses to Peptides 1 or 2 alone were not significant (Anderson et al, 2005).

Thus, the anti-CD 2 antibody CF1D12 increases IFN-γ responses to coeliac disease- specific antigens in a short-term functional T cell assay in both disease and HLA- matched healthy control subjects. This demonstrates, for the first time, the utility of anti-CD52 antibodies in enhancing a host immune response and in allowing an improved determination of a host's immune response to an antigen. For example, the IFN-γ ELISpot responses of PBMCs from coeliac disease subjects shown in Figure 8 are significantly greater than the 10 SFU/10 ⁶ PBMCs cut-off value previously regarded as a criterion for defining a positive host response, when the PBMCs were incubated with the anti-CD52 antibody CF1D12. Accordingly, anti-CD52 antibodies such as CF1D12 can be used to reveal a host's immune response to a particular antigen without the need to perform costly and lengthy intermediary steps of expanding a subject's T cell population to an amount which is large enough to producer detectable result. The results also represent the first time effector T cell responses to coeliac disease- specific gluten peptides have been demonstrated in healthy individuals who do not have clinical coeliac disease. This raises the possibility that the gluten-specific T cells are present in healthy individuals with coeliac disease susceptibility HLA haplotypes, but that CD52-based regulation keeps them in check. Accordingly, anti-CD52 antibodies such as CF1D12 can be used to reveal the likelihood of a host responding to coeliac disease-specific gluten peptides and hence, such antibodies can be used to determine a subject's susceptibility to developing coeliac disease. In a broader context, the present disclosure demonstrates that an anti-CD52 antibody such as CF1D12 can be used to reveal a subject's immune response to any antigen and hence, to determine a subject's susceptibility to disease.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed and/or referenced herein are incorporated herein in their entirety. ^"

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention asi it existed before the priority date of each claim of this application.

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