HK1222554B - Methods of use of soluble cd24 for therapy of rheumatoid arthritis - Google Patents

Description

Method for treating rheumatoid arthritis using soluble CD24

This case is a divisional case of application number 201180021423.6, application date 2011-4-28, and title “Method for treating rheumatoid arthritis using soluble CD24”.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 61/329,078, filed April 28, 2010, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to compositions and methods for treating rheumatoid arthritis.

Background Art

This section provides background information that is not necessarily prior art and is a general summary of the disclosure, which is not a comprehensive disclosure of its full scope or all of its features.

CD24 is known as a heat-resistant antigen (1). It is expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule (2) and has a wide distribution in different lineages (3). Since CD24 tends to be expressed on immature cells, it has also been used as a partial stem cell marker and for lymphocyte differentiation. The first function associated with CD24 is its co-stimulatory activity for antigen-specific T cell responses (4-6). In vivo experiments have shown that CD24 is redundant as a co-stimulatory factor for T cell activation in lymphoid organs, but becomes necessary in the absence of CD28 (7,8). This does not apply to the case of local target organs that are not "co-stimulatory factor-rich". Consistent with this concept, we have demonstrated that mice with a targeted mutation of CD24 are completely resistant to the induction of experimental autoimmune encephalomyelitis (EAE) (9)(10).

Polymorphisms in human CD24 have been associated with the risk and progression of several autoimmune diseases (11-15), including multiple sclerosis and rheumatoid arthritis (RA). In the case of multiple sclerosis, we have reported that soluble CD24 composed of the extracellular portion of murine CD24 and human IgG1 Fc improves clinical symptoms in a murine model of the experimental autoimmune disease multiple sclerosis (9). Recent studies conducted by one of us have shown that CD24 interacts with and inhibits host responses to danger-associated molecular patterns (DAMPs) (16).

RA affects 0.5-1% of the population. Although many disease-modifying antirheumatic drugs (DMARDs) are currently available, even the gold standard of biological DMARDs, therapy targeting tumor necrosis factor alpha, results in a 50% improvement (ACR50) in less than 50% of patients receiving treatment according to the American College of Rheumatology improvement criteria (17). A cure for RA cannot be achieved. Therefore, there is a need to test additional RA treatments. RA is assumed to be an autoimmune disease in the joints, although the cause of the disease remains largely unknown. Many studies have implicated T cells in the pathogenesis of rheumatoid arthritis (18). Recently, it has been shown that antibody transfer can trigger the development of joint inflammation in mice (19-21). The lesion pathology is similar to that of human rheumatoid arthritis.

One of the most interesting concepts established from studies of RA via passive transfer of antibodies is that tissue-specific autoimmune disease can be observed even when the antibodies are specific for ubiquitously expressed proteins (19-21). This concept is important because it suggests that autoimmune diseases of different organs/tissues may require different treatments despite a common pathogenesis. Supporting this concept is the fact that interferon beta, which is widely used to treat multiple sclerosis, has little effect on the treatment of RA (22).

Animal models relevant to human RA have played an important role in the advancement of therapeutic development with DMARDs. For example, collagen-induced arthropathy in mice and rats has been crucial for the development of RA therapeutics (23). Recently, it has been demonstrated that adaptive transfer of anti-collagen antibodies induces robust RA-like lesions in mice (19). Because autoantibodies are increased in RA patients before disease onset (24,25), the passive transfer of collagen-specific antibodies is a relevant model for human RA.

Because the pathogenesis of RA involves host responses to DAMPs (26,27) and because the CD24 molecule negatively regulates host responses to DAMPs (16), we investigated the possibility of using soluble CD24 to treat RA. The passive transfer model of RA was chosen because of its relevance to human disease and the simplicity of the experimental design.

Summary of the Invention

Provided herein are methods for treating rheumatoid arthritis by administering a CD24 protein to a mammal in need thereof. The CD24 protein may comprise a sequence of mature mouse CD24 or mature human CD24, or a variant thereof. Mature human CD24 may consist of SEQ ID NO: 1 or 2. Mature mouse CD24 may consist of the sequence of SEQ ID NO: 3. The CD24 protein may further comprise an extracellular domain of mouse or human CD24, which may be fused to the N-terminus of mature CD24. The extracellular domain of CD24 may consist of the sequence of SEQ ID NO: 4. The CD24 protein may further comprise a portion of a mammalian immunoglobulin (Ig), which may be fused to the N-terminus or C-terminus of mature CD24. The Ig portion may be the Fc portion of a human Ig protein, which may be IgG1, IgG2, IgG3, IgG4, or IgA. The Fc portion may consist of the hinge region and CH2 and CH3 domains of a human Ig protein. The Fc portion may also be a combination of the hinge region and CH3 and CH4 domains of human IgM.

The CD24 protein can be soluble and glycosylated. The CD24 protein can be produced using a eukaryotic protein expression system. The expression system can include a vector contained in a Chinese hamster ovary cell line or a replication-deficient retroviral vector. The replication-deficient retroviral vector can stably integrate into the genome of eukaryotic cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Amino acid composition of the CD24 fusion protein CD24IgG1Fc (SEQ ID NO: 5). The underlined 26 amino acids are the CD24 signal peptide (SEQ ID NO: 4). The boxed, bold portion of the sequence is the mature CD24 protein used in the fusion protein (SEQ ID NO: 1). The final amino acids (A or V) typically present in the mature CD24 protein have been deleted from the construct to prevent immunogenicity. The non-underlined, non-bold letters are the IgG1 Fc sequence (SEQ ID NO: 6), which includes the hinge region and the CH1 and CH2 domains.

Figure 2. Methods for purification and processing of CD24 IgG1 Fc expressed from mammalian cell lines.

Figure 3. Amino acid sequence changes between the mature CD24 protein from mouse (SEQ ID NO: 3) and the mature CD24 protein from human (SEQ ID NO: 2). Potential glycosylation sites are bolded, with N-glycosylation sites in red.

Figure 4. The therapeutic effect of CD24Ig on RA. 8-10 week old male BALB/c mice were intravenously immunized with 2 mg/mouse of the ArthritoMab arthritis-inducing antibody cocktail (MDbioproducts, St Paul, MN). Two days later, the mice were intraperitoneally injected with 9 μg of LPS dissolved in PBS. Disease progression was monitored daily using the following scoring system: 0, no reaction, normal; 1, mild but definite redness and swelling of the ankle/wrist or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2, moderate to severe redness and swelling of the ankle/wrist; 3, redness and swelling of the entire paw; 4, maximal inflammation of all four limbs, including the digits, involving multiple joints. Data shown are composite scores from all four limbs (mean + SE). *P < 0.05, **P < 0.01, ***P < 0.001. The differences between the two groups were also significant based on the Fisher's PLSD test.

Figure 5. WinNonlin compartmental simulation analysis of the pharmacokinetics of CD24 IgG1. Open circles represent the average of three mice, and the lines are the expected pharmacokinetic curves. a. 1 mg CD24 IgG1 injected intravenously. b. 1 mg CD24 IgG1 injected subcutaneously. c. Comparison of total antibody content as measured by area under the curve (AUC), half-life, and maximum blood concentration. Note that the overall AUC and Cmax for the subcutaneous injection are approximately 80% of those for the intravenous injection, although this difference is not statistically significant.

Figure 6. CD24-Siglec G(10) interaction identification between PAMPs and DAMPs. A. Host responses to PAMPs are not affected by CD24-Siglec G(10) interaction. B. CD24-Siglec G(10) interaction inhibits host responses to DAMPs, likely through Siglec G/10-bound SHP-1.

Figure 7. A. A single injection of CD24Fc reduces the clinical score of CAIA. a. Experimental diagram. BALB/c mice (8 weeks old) received mAbs in combination with vector or fusion protein on day 1. Mice were injected with LPS on day 3 and observed daily for 3 consecutive weeks. b. CD24Fc reduces the clinical score of CAIA. Fusion protein (1 mg/mouse) or vector was injected once on day 1. Clinical scores were determined in a double-blind manner. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The effect of CD24 was reproduced in 6 independent experiments, including a total of 52 mice in the PBS group and a total of 54 mice in the CD24Fc group.

Figure 8. CD24Fc reduces the levels of inflammatory cytokines and CAIA in joints. CAIA was initiated and treated as illustrated in Figure 7a. Inflammatory cytokines were measured by cytokine bead microarray from BD pharmingen. a. Representative FACS curves, b. Summary of reduced cytokines measured in joint homogenates (mean + SE).

Figure 9. CD24Fc reduces inflammation and cartilage destruction in joints. On day 7, front and hind paws from CD24Fc-treated and control mice were excised and fixed in 4% paraformaldehyde for 24 hours, followed by decalcification with 5% formic acid. The paws were then embedded in paraffin and longitudinal sections were stained with H&E and Safranin O red (Sigma-Aldrich).

Figure 10. Therapeutic effect of CD24Fc administered on day 5 of CAIA induction. CAIA-induced mice were randomly divided into two groups to receive vehicle (PBS) or CD24Fc. Mice were scored in a double-blind manner. Representative results of three independent experiments are shown.

Figure 11. Low-dose CD24Fc prevents the development of CAIA. a. Experimental image, b. Clinical score of arthritis, scored in a double-blind manner.

Figure 12. Siglecg is essential for the therapeutic effect of CD24Fc. WT (a) and Siglecg-/- mice (b) received vehicle control or CD24Fc combined with a cocktail of anti-collagen mAbs. Clinical scores were recorded daily in a double-blind manner.

DETAILED DESCRIPTION

The present inventors have discovered that a soluble form of CD24 is highly effective in treating rheumatoid arthritis.

1. Definition

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

With respect to the description of numerical ranges herein, each intervening number with the same degree of precision is expressly contemplated. For example, for the range 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

A "peptide" or "polypeptide" is a linked sequence of amino acids and may be natural, synthetic, or a modification or combination of natural or synthetic.

"Substantially identical" may mean that the first amino acid sequence and the second amino acid sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 amino acids that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical over a region of 1, 2, 3, 4, 5, 6, 7, 8, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 amino acids.

"Treatment" or "treating," when referring to protecting an animal from a disease, means preventing, suppressing, inhibiting, or completely eliminating the disease. Preventing a disease includes administering a composition of the present invention to an animal prior to the onset of the disease. Suppressing a disease includes administering a composition of the present invention to an animal prior to the onset of the disease but prior to its clinical manifestation. Inhibiting a disease includes administering a composition of the present invention to an animal after clinical manifestation of the disease.

"Variant" may refer to a peptide or polypeptide whose amino acid sequence differs due to insertion, deletion, or conservative substitution of amino acids, but which retains at least one biological activity. Typical examples of "biological activity" include the ability to bind to a Toll-like receptor and to be bound by a specific antibody. A variant may also refer to a protein whose amino acid sequence is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are generally recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydropathicity index of the amino acids, which is well known in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathicity index of an amino acid is based on considerations of its hydrophobicity and charge. It is well known in the art that amino acids with similar hydropathic indices can be substituted while still retaining protein function. In one aspect, amino acids with a hydropathicity index of ±2 are substituted. The hydrophilicity of amino acids can also be used to indicate substitutions that will result in a protein that retains biological function. Considering the hydrophilicity of amino acids in the case of peptides makes it possible to calculate the maximum local average hydrophilicity of the peptides, which is a useful measure that has been reported to be very relevant to antigenicity and immunogenicity. U.S. Patent number 4,554,101, which is incorporated herein by reference in its entirety. The replacement of amino acids with similar hydrophilicity values can produce peptides that retain biological activity (e.g., immunogenicity), as is well known in the art. Replacement can be performed using amino acids with hydrophilicity values within ± 2 of each other. Amino acidic hydrophobicity index and hydrophilicity values are all affected by the specific side chains of the amino acid. Consistent with this observation, it should be understood that amino acid replacements compatible with biological functions depend on the relative proximity of the amino acids, particularly on the side chains of those amino acids, as disclosed by hydrophobicity, hydrophilicity, charge, size and other properties.

2.CD24

Provided herein are CD24 proteins having the amino acid sequence of mature human CD24, which may be SETTTGTSSNSSQSTSNSGLAPNPTNATTK (SEQ ID NO: 1) or SETTTGTSSNSSQSTSNSGLAPNPTNATTK (V/A) (SEQ ID NO: 2), or the amino acid sequence of mouse CD24, which may be NQTSVAPFPGNQNISASPNPTNATTRG (SEQ ID NO: 3), or variants thereof. The CD24 may be soluble. The CD24 may also include an N-terminal signal peptide, which may have the amino acid sequence MGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID NO: 4). The CD24 may also have the amino acid sequence described in Figures 1 or 3. The CD24 may exist in one of two allelic forms, such that the C-terminal amino acid of mature human CD24 may be valine or alanine. C-valine or alanine may be immunogenic and may be omitted from CD24 to reduce its immunogenicity. The difference between the two alleles can affect the risk of autoimmune diseases, including multiple sclerosis and RA. However, because both alleles affect the expression levels of the membrane-bound form, the variation should not affect CD24 function.

Despite considerable sequence variation in the amino acid sequences of mature CD24 proteins from mice and humans, they are functionally equivalent in their interactions with danger-associated molecular patterns (DAMPs). Because host responses to DAMPs are thought to be important in the pathogenesis of RA, mouse and human CD24 are functionally equivalent in treating RA. As a result of sequence conservation between mouse and human CD24, primarily at the C-terminus and at many glycosylation sites, significant changes in the mature CD24 protein may be tolerated in the treatment of RA using CD24, particularly if these changes do not affect conserved residues at the C-terminus or glycosylation sites from mouse or human CD24.

a. Fusion

CD24 can be fused at its N-terminus or C-terminus to a portion of a mammalian Ig protein (which can be human or mouse). The Fc region can include the hinge region and CH2 and CH3 domains of the Ig protein. The Ig protein can be human IgG1, IgG2, IgG3, IgG4, IgM, or IgA. The Fc portion can include SEQ ID NO: 6. The Ig protein can also be IgM, and the Fc portion can include the hinge region and CH3 and CH4 domains of IgM. The CD24 can also be fused at its N-terminus or C-terminus to a protein tag, which can be GST, His, or FLAG. Methods for preparing and purifying fusion proteins are well known in the art.

b. Production

CD24 can be heavily glycosylated and can participate in the functions of CD24, such as costimulation and interaction with danger-associated molecular patterns. The CD24 is prepared using a eukaryotic expression system. The expression system may require expression from a vector in mammalian cells, such as Chinese hamster ovary (CHO) cells. The system can also be a viral vector, such as a replication-deficient retroviral vector, which can be used to infect eukaryotic cells. CD24 can also be produced from a stable cell line that expresses CD24 from a vector or a portion of a vector that has been integrated into the cell genome. The stable cell line can express CD24 from an integrated replication-deficient retroviral vector. The expression system can be GPEx(TM).

3. Treatment Methods

The CD24 can be used to treat rheumatoid arthritis. The CD24 can be administered to a subject in need thereof. The subject can be a mammal such as a human.

a. Combined CD24 therapy

The CD24 can be combined with another drug, such as a disease-modifying antirheumatic drug (DMARD). The drug can be a nonsteroidal anti-inflammatory drug (NSAID), which can be a propionic acid derivative, an acetic acid derivative, an enolic acid derivative, a fenamic acid derivative or a selective Cox2 inhibitor. The drug can also be a corticosteroid or methotrexate. The drug can be biological, which can be a TNF-α antagonist, such as an anti-TNF-α antibody or a fusion protein (Enbrel) bound to TNF-α, an anti-CD20 mAb, an antagonist of the co-stimulatory molecules CD80 and CD86, such as a monoclonal antibody or fusion protein (CTLA4Ig) bound to these two molecules, or an antagonist of IL-1 or IL-6 receptors. CD24 and another drug can be administered together or sequentially.

b. Pharmaceutical Compositions

CD24 can be included in a pharmaceutical composition that can also include a solvent that can stabilize CD24 for a long period of time. The solvent can be PBS, which can stabilize CD24 at -20°C for at least 36 months. The solvent can also be suitable for combining CD24 with another drug.

c. Dosage

The dosage for human use will ultimately be determined by clinical trials to determine the dose with acceptable toxicity and efficacy. The initial clinical dosage for human use can be estimated by pharmacokinetic and toxicity studies in rodents and non-human primates. The dosage of CD24 can be from 0.01 mg/kg to 1000 mg/kg, and can be from 1 to 500 mg/kg, depending on the severity of the disease being treated and the route of administration.

d. Application

The route of administration of the pharmaceutical composition can be parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, intraarticular, and direct injection into the affected joint. For veterinary use, the drug can be administered as an appropriate acceptable formulation in accordance with normal veterinary practice. Veterinarians can easily determine the dosage regimen and route of administration that is most suitable for a particular animal. The pharmaceutical composition can be administered to human patients, cats, dogs, large animals, or birds.

CD24 can be administered simultaneously or metronomically with other treatments. As used herein, the term "simultaneous" or "simultaneously" means that CD24 and the other treatment can be administered within 48 hours, preferably within 24 hours, more preferably within 12 hours, still more preferably within 6 hours, and most preferably within 3 hours or less of each other. As used herein, the term "metronomically" means that the agent is administered at a different time than the other treatment and at a certain frequency associated with repeated administration.

CD24 can be administered at any time prior to another treatment, including about 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 hours, 98 hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours, 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes and 1 minute. CD24 can be administered at any time prior to the second CD24 treatment, including about 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 hours, 98 hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours, 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, and 1 minute

CD24 can be administered at any time after another treatment, including about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours. , 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours and 120 hours. CD24 can be administered at any time after a prior CD24 treatment, including about 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 hours, 98 hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours, 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes and 1 minute.

The following examples are provided to illustrate the method of the present invention and are in no way intended to limit the application of the method.

Example

Example 1

Soluble CD24 protein

The extracellular domain of CD24 is fused to IgG1 Fc. The amino acid composition of the CD24 fusion protein is provided in Figure 1. A replication-deficient retrovirus is then produced that promotes the expression of the CD24Ig fusion protein. The GPEx™ (acronym for gene product expression) system provides several important advantages, the most important of which is that on average >1000 insertions/cell are achieved, but each insertion produces only 1 copy. In addition, because the retrovirus preferably inserts into a transcriptionally active locus, GPEx™ produces high levels of target protein expression. A stable cell line is produced that produces a high yield of CD24Ig. In addition, 45 g of GLP-grade product and ~100 g of cGMP-grade product are produced. The method for downstream processing of the culture medium harvested from the bioreactor is summarized in the flow chart below (Figure 2).

Harvest clarification

The biotin culture medium was clarified using a Cuno 60M02 Maximizer depth filter followed by a Millipore Opticap 0.22 μm filter. The filtrate was collected into a sterile collection bag. The CD24-Fc yield of the samples was measured by ELISA.

Protein A capture

The clarified medium was passed through a Protein A resin column (GE Healthcare MabSelect) at a concentration not exceeding 16 g/L resin (based on ELISA) with a contact time of 4 minutes. The column was washed with equilibration buffer (50 mM Tris + 0.15 M NaCl pH 7.5) and then with 10 mM sodium citrate/citric acid pH 6.0 for 5 column volumes (cv). Bound CD24Ig was eluted from the column using 10 mM sodium citrate/citric acid pH 3.5.

Virus inactivation

The Protein A elution fraction was immediately brought to pH 3.0 by adding 2 M hydrochloric acid and maintained at this pH for 30 minutes at ambient temperature. It was then brought to pH 5.0 by adding 1 M Tris base and filtered through a 0.65 μm glass fiber filter (Sartorius Sartopure GF2) until clear and through a 0.2 μm filter (Sartorius Sartopore 2) into a sterile collection bag.

SP-Sepharose chromatography

The virus-inactivated material was applied to an SP-Sepharose column (GE Healthcare) at a concentration not exceeding 25 g/L resin (based on A280 nm, 1.22 = 1 mg/mL) and a linear flow rate of 250 cm/hr. The column was washed with equilibration buffer (10 mM sodium citrate/citric acid pH 5.0) and bound CD24Ig was eluted from the column using 10 mM sodium citrate/citric acid + 0.2 M NaCl pH 5.0. The flow-through was collected in a sterile collection bag.

Mustang Q chromatography

The SP-Sepharose eluate was adjusted to pH 7.5 by adding 1 M Tris base and diluted with WFI to reduce conductivity. The diluted material was applied to a Mustang Q filter (Pall) at a concentration not exceeding 0.5 g/L resin (based on A280 nm, 1.22 = 1 mg/mL) at a flow rate of 5 column volumes/minute. The filter was washed with equilibration buffer (10 mM Tris pH 7.5), and CD24-Fc was contained in the flow-through and collected in a sterile collection bag.

Virus filtration

The Mustang Q was then filtered through a 0.2 mM filter and a Millipore NFP virus filter (nominal pore size 20 nm) at a constant pressure of 30 psi and collected into a sterile collection bag.

Concentration and final formulation

The product was concentrated and filtered into 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2, using a 10 kDa ultrafiltration membrane (Millipore Prep/Scale) at a final concentration of approximately 10 mg/mL (as measured by absorbance at 280 nm). Analytical samples were drawn from the batch while it was in the biosafety kitchen. Labeling was performed and the samples were sent to QC for testing while aliquots of the batch were stored at 2-8°C pending release.

Viral clearance study

Viral clearance validation was performed on samples prepared at CHM at Cardinal Health, NC. Qualified scientists from GalaBiotech performed the chromatography and filtration steps at the Cardinal Health Viral Validation Laboratory with the assistance of Cardinal Health staff. Scaling down the steps was carried out from a 200 L scale process. Two viruses were selected for this study. The first was heterotropic murine leukemia virus (XMuLv), an enveloped RNA virus from the Retroviridae family with a size of 80-130 nm. The second was porcine parvovirus (PPV), a non-enveloped DNA virus with a size of 18-26 nm. This is considered a hardy virus and was expected to show much lower viral reduction through the purification protocol than XMuLv.

Example 2

Using CD24Fc to treat RA

This example demonstrates that CD24 can be used to treat RA. Because anti-collagen antibodies are present in RA patients before the onset of a primary attack, and because they can induce RA-like pathology in mice, the efficacy of soluble CD24 was tested using an established passive transfer model of RA. The fusion protein was dissolved in a PBS vehicle at 10 mg/ml. As shown in FIG4 , the combination of the four anti-collagen antibodies caused severe clinical symptoms in all four limbs, which peaked on day 7 in both the vehicle- and CD24Fc-treated groups. The disease is characterized by redness and swelling of the entire paw in all four limbs. Some limbs were most inflamed, including the toes and multiple joints. Therefore, the soluble protein did not affect the onset of the disease. Surprisingly, the CD24Fc-treated group showed a much more rapid recovery. The decrease in clinical scores was very significant starting on day 9 and ultimately persisted throughout the 24-day observation period. Therefore, CD24Fc provides an effective treatment for RA. Interestingly, because this effect was observed after the peak of the disease, it is likely that blocking CD24 affects the chronic disease process after inflammation has begun.

Example 3

CD24 pharmacokinetics

1 mg of CD24IgG1 was injected into naive C57BL/6 mice, and blood samples were collected at different time points (5 minutes, 1 hour, 4 hours, 24 hours, 48 hours, day 7, day 14, and day 21), with 3 mice at each time point. The serum was diluted 1:100, and the levels of CD24Ig were detected using a sandwich enzyme-linked immunosorbent assay using purified anti-human CD24 (3.3 μg/ml) as the capture antibody and peroxidase-conjugated goat anti-human IgG Fc (5 μg/ml) as the detection antibody. As shown in Figure 5a, the decay curve of CD24Ig shows a typical biphasic decay of the protein. The first bioturbation phase has a half-life of 12.4 mice. The second phase follows a first-order elimination model from the central compartment. The half-life of the second phase is 9.54 days, which is similar to the in vivo half-life of the antibody. These data indicate that the fusion protein is very stable in the bloodstream. In another study where the fusion protein was injected subcutaneously, an almost identical half-life of 9.52 days was observed (Figure 5b). More importantly, although it takes about 48 hours for CD24Ig to reach peak levels in the blood, the total amount of fusion protein in the blood (as measured by AUC) is essentially the same for either route of injection. Therefore, from a therapeutic perspective, different routes of injection should not affect the therapeutic effect of the drug. This observation greatly simplifies the experimental design of primate toxicity and clinical trials.

Example 4

CD24 for the treatment of RA

For decades, it was assumed that RA was primarily a T-cell mediated autoimmune disease. In the last two decades, there has been a reawakening of the role that antibodies and B lymphocytes may play in the pathogenesis of RA. Thus, in addition to rheumatoid factor, a host of autoreactive antibodies have been found in RA patients, although they have not yet been conclusively elucidated and identified in humans. However, several lines of evidence have demonstrated that antibodies specific for ubiquitous or tissue-specific antigens are sufficient to induce RA symptoms in mouse models. For example, antibodies from K/BxN TCR transgenic mice were found to be fully capable of transferring an RA-like disease in a new host. Similarly, a cocktail (mixture) of four anti-collagen antibodies is now widely used to induce RA in mice. This model is now known as CAIA, i.e. collagen antibody-induced arthritis.

Genetic analysis of the CAIA model has revealed a key role for complement. Although other possibilities exist, these findings suggest the potential involvement of antibody-mediated tissue damage in the pathogenesis of RA. The link between tissue damage and inflammation is a long-standing observation in immunology. Almost two decades ago, Matzinger proposed what is commonly known as the danger theory. Essentially, she stated that the immune system turns on when it senses danger in the host. Although the nature of the danger was not fully defined at the time, it was established that necrosis is associated with the release of intracellular components such as HMGB1 and heat shock proteins, which are known as DAMPs, or danger-associated molecular patterns. DAMPs have been found to promote the production of inflammatory cytokines and autoimmune diseases. Inhibitors of HMGB1 and HSP90 have been found to ameliorate RA in animal models. The involvement of DAMPs has raised the prospect that negative regulation of the host response to DAMPs could be used for RA treatment. CD24-Siglec 10 interacts in the host response to tissue damage.

Using paracetamol-induced hepatic necrosis and inflammation, we observed that, through interaction, Siglec G and CD24 provide negative regulation of the host response to tissue damage. CD24 is a GPI-anchored molecule that is widely expressed in hematopoietic cells and other tissue hepatocytes. Genetic analysis of various autoimmune diseases in humans, including multiple sclerosis, systemic lupus erythematosus, RA, and giant cell arthritis, has shown a significant association between CD24 polymorphisms and the risk of autoimmune disease. Siglec G is a member of the I-lectin family, which is defined by its recognition of sialic acid-containing structures. Siglec G recognizes sialic acid-containing structures on CD24 and negatively regulates the production of inflammatory cytokines by dendritic cells (16). Human Siglec 10 and mouse Siglec G are functionally equivalent in terms of their ability to interact with CD24. However, it is unclear whether there is a one-to-one correspondence between the mouse and human homologs. Although the mechanism remains to be fully elucidated, it is plausible that Siglec G-related SHP1 may be involved in negative regulation. These data, recently reported in Science, lead to a new model in which the CD24-Siglec G/10 interaction may play a key role in the recognition of pathogen-associated molecular patterns (PAMPs) from DAMPs (Fig. 6).

At least two overlapping mechanisms may explain the function of CD24. First, by binding to a variety of DAMPs, CD24 can capture inflammatory stimuli, preventing them from interacting with TLRs or RAGE. This concept is supported by the observation that CD24 associates with several DAMP molecules, including HSP70, 90, HMGB1, and nucleolin. Second, CD24 may stimulate signaling through Siglec G, possibly after association with DAMPs. The two mechanisms may work in concert, as mice with targeted mutations in either gene mount a much stronger inflammatory response. Indeed, DCs cultured from the bone marrow of CD24-/- or Siglec G-/- mice produce much higher levels of inflammatory cytokines when stimulated with HMGB1, HSP70, or HSP90. In contrast, no effect was found in their response to PAMPs (such as LPS and PolyLC). These data not only provide a mechanism by which the innate immune system distinguishes pathogens from tissue damage, but also suggest that CD24 and Siglec G may serve as potential therapeutic targets for diseases associated with tissue damage.

The therapeutic effect of CD24Fc on collagen-antibody-induced arthritis

Given the suspected role of innate immunity to tissue damage in the pathogenesis of RA, and the role of the CD24-Siglec G/10 pathway in negatively regulating this response, the potential for stimulating this pathway to treat RA has been investigated. The pathogenesis of virtually all autoimmune diseases involves the induction of an immune response to self-antigens and autoimmune destruction. The autoimmune destruction phase is centrally based on the novel function of the CD24-Siglec G interaction. Therefore, for initial analysis, collagen antibody-induced arthritis was used to evaluate potential therapeutic effects.

As shown in Figure 7a, CAIA was induced in 8-week-old BALB/c mice by intravenous injection of four anti-collagen mAbs (MD Biosciences, St. Paul, MN) at 2 mg/mouse on day 1 and intraperitoneal injection of 100 μg/mouse LPS (MD Bioscience) on day 3. Mice were treated with 1 mg of CD24Fc or an equal volume of 1xPBS vehicle as a negative control on day 1. As shown in Figure 7b, CD24Fc provided a highly significant therapeutic effect compared to the vehicle control.

To understand the mechanism by which CD24Fc reduces arthritis in this model, cytokines were measured in homogenized joints from CD24Fc-treated mice or PBS controls, as well as in the supernatant of 200 μg of tissue homogenate by cytokine bead microarray. A typical example is shown in FIG8a, while summary data are shown in FIG8b. These data demonstrate that systemic administration of CD24 reduces the levels of multiple inflammatory cytokines, including TNF-α, IL-6, MCP-1 (CCL2), and IL-1β.

The effect of CD24Fc was confirmed by histological analysis of the synovial joints of CAIA mice, as presented in Figure 9. On day 7, after arthritis induction, H&E staining confirmed that the joint synovium in the PBS group was severely invaded by inflammatory cells including neutrophils, macrophages and lymphocytes (Figure 9, upper left panel). This was much lower in CD24Fc-treated mice (Figure 9, lower left panel). In addition, severe joint damage was exposed by the loss of Safranin O red staining in PBS-treated (Figure 9, upper right panel) mice, but did not occur in the CD24Fc-treated group (Figure 9, lower right panel).

To determine whether mouse CD24Fc has a therapeutic effect on ongoing RA, treatment was initiated 5 or 7 days after RA induction. As shown in Figure 10, a significant reduction in RA scores was observed two days after QD CD24Fc treatment. Even without additional treatment, the therapeutic effect persisted throughout the remaining observation period. These data further strengthen the therapeutic potential of CD24Fc for ongoing disease.

To evaluate the therapeutic dose of CD24Fc in humans, CD24Fc was titrated through doses that resulted in a significant response. As shown in Figure 11, as little as 2 micrograms per mouse was sufficient to have a statistically significant therapeutic effect.

Siglecg-dependent therapeutic effects of CD24Fc

To determine whether CD24Fc protects mice by interacting with Siglec G, we determined whether the therapeutic effect depends on the Siglecg gene. Because Siglecg-deficient mice were generated using ES cells from C57BL/6 mice, we used WT C57BL/6 mice as a control. As shown in Figure 12, because B mice are known to be less sensitive to CAIA, the total disease score is lower than that observed in BALB/c mice. However, a single injection of CD24Fc essentially eliminated the clinical symptoms in WT mice. Importantly, even though the disease was less severe in Siglecg-deficient mice, CD24Fc had no therapeutic effect. Therefore, the therapeutic effect of CD24Fc is strictly dependent on the Siglecg gene.

In summary, the data presented here demonstrate that CD24Fc has a high therapeutic effect on CAIA. Given our extensive safety and stability data and our successful production of CD24Fc, all of this suggests that this fusion protein has great potential as a therapeutic agent for RA.

Example 5

toxicity

Extensive toxicity studies in rodents and non-human primates have shown no drug-related toxicity at doses of 12.5 to 125 mg/kg in mice and non-human primates.

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Claims (9)

1. A fused CD24 protein comprising a mature human CD24 polypeptide and an Fc region of a human IgG protein, wherein the amino acid sequence of the mature human CD24 polypeptide is SEQ ID NO:1, wherein the CD24 protein does not include an alanine or valine immediately following the C-terminus of SEQ ID NO:1, and wherein the Fc region is fused to the C-terminus of the mature human CD24 polypeptide. 2. The fused CD24 protein of claim 1, wherein the Fc region comprises a hinge region of IgG2, IgG3 or IgG4 and CH2 and CH3 domains. 3. The fused CD24 protein of claim 2, wherein the Fc region comprises a hinge region of IgG2, IgG3 or IgG4 and CH2 and CH3 domains. 4. The fused CD24 protein of claim 1, wherein the CD24 protein is soluble. 5. The fused CD24 protein of claim 4, wherein the CD24 protein is glycosylated. 6. The fused CD24 protein of claim 5, wherein the CD24 protein is produced using a eukaryotic protein expression system. 7. Use of the fused CD24 protein as described in any one of claims 1-6 in the preparation of a medicament for the treatment of multiple sclerosis. 8. The use as claimed in claim 7, wherein the Fc region of the fused CD24 protein is composed of the hinge region of IgG1 and CH2 and CH3 domains. 9. Use of the fused CD24 protein as described in any one of claims 1-6 in the preparation of a medicament for treating rheumatoid arthritis.