JP7791605B2 - Diagnostic markers - Google Patents

Description

The present invention relates to diagnostic markers, diagnostic compositions, kits, methods, etc. for identifying specific groups of patients with cutaneous polyarteritis nodosa. The present invention also relates to pharmaceutical compositions, methods, etc. for treating specific groups of patients with cutaneous polyarteritis nodosa.

Polyarteritis nodosa is a disease characterized by inflammation and necrosis of the vascular walls, primarily affecting small and medium-sized arteries (muscular-walled arteries that lead to organs and their branches) throughout the body. Polyarteritis nodosa presents with a variety of symptoms, including fever, kidney damage, and muscle pain. Approximately 25-60% of cases are accompanied by skin symptoms, such as subcutaneous nodules, livedopathies, ulcers, and gangrene. The cause remains unknown in many cases, and effective treatments have yet to be established. Cutaneous polyarteritis nodosa has been considered a form of polyarteritis nodosa that is limited to the skin. However, since the Chapel-Hill Criteria 2012, it has been referred to as "cutaneous arteritis," and it is unclear whether it is limited to a specific symptom of polyarteritis nodosa or an independent disease.

Currently, corticosteroids and immunosuppressants are used to treat cutaneous polyarteritis nodosa, but response to treatment varies from case to case (Non-Patent Document 1). In particular, severe cases involving peripheral neuropathy require intensive treatment and may require hospitalization (Non-Patent Document 2). Therefore, it is desirable to be able to accurately diagnose cutaneous polyarteritis nodosa, especially the severity of cutaneous polyarteritis nodosa.

However, there are no specific serum diagnostic markers for cutaneous polyarteritis nodosa, and diagnosis must rely on skin biopsy. However, depending on the biopsy site, insufficient findings may be obtained, necessitating repeated skin biopsies, which places a significant burden on both patients and medical professionals. There was a need for a marker to properly diagnose cutaneous polyarteritis nodosa.

K. M. Matsuda et al. , J. Dermatol. , vol. 48, no. 7, pp. 1021-1026, 2021 T. Kawakami et al. , J. Dermatol. , vol. 40, no. 12, pp. 955-961, 2013 E. Fukuda et al. , Genes Cells. , vol. 26, no. 3, pp. 180-189, 2021 C. Mukhtyar et al. , Ann Rheum Dis. , vol. 68, no. 3, pp. 310-317, 2009 E. V. Quadros et al. Blood. , vol. 113, no. 1,,pp. 186-192, 2009

As a result of intensive research to solve the above problems, the inventors discovered that autoantibodies against CD320 (sometimes called "transcobalamin receptor") are detected in some patients with cutaneous polyarteritis nodosa, and that patients with cutaneous polyarteritis nodosa in whom autoantibodies against CD320 are detected have mild (low severity) cutaneous polyarteritis nodosa or do not have peripheral neuropathy. This led to the conception of the present invention, based on the belief that anti-CD320 antibodies could be an indicator of low severity of cutaneous polyarteritis nodosa.

Furthermore, in the present invention, it was discovered that the symptoms of cutaneous polyarteritis nodosa are suppressed by administering a CD320 ligand to mildly severe cutaneous polyarteritis nodosa, and a pharmaceutical composition for treating cutaneous polyarteritis nodosa was devised.

The present invention includes, but is not limited to, the following aspects.
[Aspect 1]
Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A diagnostic marker for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii),
The diagnostic marker as described above, which comprises an anti-CD320 antibody.
[Aspect 2]
The marker according to Aspect 1, wherein the cutaneous polyarteritis nodosa is of low severity when the Birmingham Vasculitis Activity Score is 6 or less.
[Aspect 3]
contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a substance that identifies an anti-CD320 antibody; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
a method of using an anti-CD320 antibody as a marker for distinguishing, among patients with cutaneous polyarteritis nodosa, a group that satisfies any of the requirements (i) to (iii) above from a group that does not satisfy any of the requirements (i) to (iii), comprising:
[Aspect 4]
Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A diagnostic composition for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not having any of the requirements (i) to (iii), comprising:
The diagnostic composition as defined above, comprising a substance that identifies an anti-CD320 antibody.
[Aspect 5]
Aspect 5. The diagnostic composition according to aspect 4, wherein the cutaneous polyarteritis nodosa is of low severity when the Birmingham Vasculitis Activity Score is 6 or less.
[Aspect 6]
A diagnostic composition according to Aspect 4 or 5, wherein the substance that identifies the anti-CD320 antibody is CD320 or a soluble form of CD320.
[Aspect 7]
When an anti-CD320 antibody is identified in a biological sample from a patient with cutaneous polyarteritis nodosa, any one of the following requirements is met:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) the patient is identified as belonging to a group comprising: no peripheral neuropathy; and cutaneous polyarteritis nodosa of low severity.
[Aspect 8]
Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A kit for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii), comprising:
The kit further comprises a substance that identifies an anti-CD320 antibody.
[Aspect 9]
The kit according to Aspect 8, wherein the cutaneous polyarteritis nodosa is of low severity when the Birmingham Vasculitis Activity Score is 6 or less.
[Aspect 10]
A kit according to aspect 8 or 9, wherein the substance that identifies the anti-CD320 antibody is CD320 or a soluble form of CD320.
[Aspect 11]
contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a composition containing an anti-CD320 antibody identifying substance; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
A method for distinguishing, among patients with cutaneous polyarteritis nodosa, a group that satisfies any of the requirements (i) to (iii) from a group that does not satisfy any of the requirements (i) to (iii), comprising:
[Aspect 12]
The method according to Aspect 11, wherein the cutaneous polyarteritis nodosa is of low severity when the Birmingham Vasculitis Activity Score is 6 or less.
[Aspect 13]
Aspect 13. The method of aspect 11 or 12, wherein the substance that identifies the anti-CD320 antibody is CD320 or a soluble form of CD320.
[Aspect 14]
Aspect 14. The method of any one of aspects 11-13, wherein the biological sample is serum or plasma.
[Aspect 15]
Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A pharmaceutical composition for distinguishing and treating a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements of (i) to (iii), comprising:
The pharmaceutical composition comprising a CD320 ligand.
[Aspect 16]
16. The pharmaceutical composition of aspect 15, wherein said CD320 ligand is vitamin B12.
[Aspect 17]
17. The pharmaceutical composition of claim 16, wherein the vitamin B12 is methylcobalamin, cyanocobalamin, hydroxocobalamin, or adenosylcobalamin.

The present invention provides diagnostic markers, diagnostic compositions, kits, methods, etc. that enable the identification of specific groups of patients with cutaneous polyarteritis nodosa, which was previously impossible. The present invention enables testing to identify groups of patients with mild cutaneous polyarteritis nodosa, with only the relatively minor invasiveness of blood sampling. Identifying a previously unknown patient population, the anti-CD320 antibody-positive group, makes it possible to predict the severity of cutaneous arteritis, which can contribute to the selection of appropriate treatments and the reduction of treatment-related adverse events.

Figure 1 is a schematic diagram of one embodiment of a method for detecting autoantibodies characteristic of patients with cutaneous arteritis: STEP 1: whole proteomic-scale human protein synthesis; STEP 2: comprehensive antibody screening from pooled patient sera; STEP 3: antibody concentration measurement using sera from individual patients. Figure 2 shows the serum concentrations of anti-CD320 antibodies in each group: cutaneous arteritis (CA) patient group (28 patients), IgA vasculitis patient group (5 patients), ANCA-associated vasculitis patient group (31 patients), and control group (healthy individuals) (20 patients). Figure 3 shows the specific steps and results of immunoblotting to detect anti-sCD320 antibodies in human serum. The specific steps are as explained on the left side of Figure 3. The right side of Figure 3 shows the results of immunoblotting. The positive control on the right side of Figure 3 shows the results for rabbit anti-sCD320 antibodies. The three lanes labeled "Anti-CD320 AB(+)CA" show the results using sera collected from three anti-CD320 antibody-positive cutaneous arteritis patients. The three lanes labeled "Anti-CD320 AB(-)CA" show the results using sera collected from three anti-CD320 antibody-negative cutaneous arteritis patients. Figure 4 shows a summary of the relationship between the clinical features of CA patients and the presence or absence of anti-CD320 antibodies, as determined by examining the presence or absence of anti-CD320 antibodies (immunoblot analysis) and peripheral neuropathy. BVAS and the presence or absence of peripheral neuropathy are described in detail in Example 2. The presence or absence of ulcers was examined by visual inspection by a dermatologist. In Figure 4, P values for continuous variables were calculated using Wilcoxon's rank-sum test, and for non-continuous variables using Fisher's exact test. WBC: white blood cell count in blood; CRP: C-reactive protein concentration in serum; ESR: erythrocyte sedimentation rate Figure 5 shows micrographs (magnification ×200) of CD320 expression in human skin stained by immunohistochemistry. From left to right, they show the subcutaneous tissue (lesion), subcutaneous tissue (non-lesion), and dermis. The upper row shows the results for anti-CD320, which is a rabbit anti-sCD320 antibody, and the lower row shows the results for Isotype control, which is a control IgG. 6 shows immunofluorescent micrographs of CD320 and CD31 expression in human skin. Magnification: ×200; H&E: hematoxylin and eosin staining; CD31: CD31 staining only; CD320: CD320 staining only; DAPI: cell nuclei stained with DAPI (4',6-diamidino-2-phenylindole); Merge: a composite of CD320, CD31, and DAPI results. Figure 7 shows the results of a single administration of anti-CD320 antibody to mice. In Figure 7, "Anti-CD320" indicates the results for the side administered with mouse anti-CD320 antibody, and "Ctrl IgG" indicates the results for the side administered with control IgG. The left image in Figure 7 is a photograph taken with the mouse in a prone position after administration of Evans blue and antibody. The middle image in Figure 7 is a micrograph at a magnification of ×100, and the right image is a micrograph at a magnification of ×200. Figure 8 shows the skin weight, thickness of the dermis and subcutaneous tissue, and the concentration of Evans blue eluted from the collected skin sections (staining intensity) following a single administration of anti-CD320 antibody to mice. In Figure 9, "Anti-CD320" indicates the results for mice administered with mouse anti-CD320 antibody, and "Ctrl IgG" indicates the results for mice administered with control IgG. "MetCbl-" indicates the results for mice not administered with methylcobalamin, and "MetCbl+" indicates the results for mice administered with methylcobalamin. NS P>0.05, P<0.05 by Wilcoxon's signed rank test. Figure 9 shows the results of examining the expression levels of each inflammatory cytokine and adhesion molecule gene (ratio to the expression level of the endogenous control, Gapdh) following a single administration of anti-CD320 antibody to mice. "MetCbl-" indicates the results when methylcobalamin was not administered, and "MetCbl+" indicates the results when methylcobalamin was administered. NS P>0.05, P<0.05 by Wilcoxon's signed rank test. Figure 10 shows the results of flow cytometry analysis of cell surface expression of CD320 in cultured human vascular endothelial cells. "Ctrl" indicates the results for a sample labeled with fluorescein-conjugated control IgG; "MetCbl-" indicates the results for a sample labeled with fluorescein-conjugated anti-CD320 antibody; and "MetCbl+" indicates the results for a sample labeled with fluorescein-conjugated anti-CD320 antibody after methylcobalamin administration. The left panel of Figure 10 shows a histogram of fluorescein fluorescence intensity for a representative sample. The right panel of Figure 10 shows the mean and standard error of the mean fluorescein fluorescence intensity observed for each sample. NS P>0.05, P<0.05 by Wilcoxon's rank-sum test. Figure 11 shows the results of multiple administrations of anti-CD320 antibody to mice. "Anti-CD320" is a photograph of an individual administered with mouse anti-CD320 antibody, and "Ctrl IgG" is a photograph of an individual administered with control IgG (an antibody that does not react with a specific antigen). Figure 12 shows the results of examining the expression levels (ratio to the expression level of the endogenous control, Gapdh) of each inflammatory cytokine and adhesion factor gene when anti-CD320 antibody was administered multiple times to mice. "Anti-CD320" shows the results for mice administered with mouse anti-CD320 antibody, and "Ctrl" shows the results for mice administered with control IgG. Figure 13 shows the results of examining the expression levels (ratio to the expression level of the endogenous control, Gapdh) of various intercellular adhesion factors, coagulation activation factors, and inflammatory cytokines when anti-CD320 antibody was added to cultured human vascular endothelial cells. "Anti-CD320" shows the results for individuals administered with mouse anti-CD320 antibody, and "Ctrl IgG" shows the results for individuals administered with control IgG. NS P>0.05, P<0.05 by Wilcoxon's rank-sum test.

The present invention includes, but is not limited to, the following embodiments. Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The substances, materials, and examples disclosed herein are merely illustrative and are not intended to be limiting. When used herein, the phrase "in one embodiment" means that the embodiment is not limited, i.e., is non-limiting.

1. Diagnostic Markers In one aspect, the present invention relates to diagnostic markers.
The diagnostic marker is
Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A diagnostic marker for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii),
Includes anti-CD320 antibodies.

Polyarteritis nodosa is a disease that causes inflammation and necrosis in the walls of blood vessels distributed throughout the body, primarily in small and medium-sized arteries (muscular-walled arteries that lead to organs and their branches). Polyarteritis nodosa presents with a variety of symptoms, including fever, kidney damage, and muscle pain, and in approximately 25-60% of cases, it is accompanied by skin symptoms such as subcutaneous nodules, livedopathic symptoms, ulcers, and gangrene. The cause is unknown in many cases, and effective treatment methods have not yet been established. Furthermore, there are no highly specific diagnostic markers.

"Cutaneous polyarteritis nodosa" has been considered to be a form of "polyarteritis nodosa" limited to the skin. However, since the Chapel-Hill Criteria 2012, it has been referred to as "cutaneous arteritis," and it is not clearly distinguished whether it is limited to a partial symptom of polyarteritis nodosa or whether it is an independent disease. In this specification, "cutaneous polyarteritis nodosa" refers to vasculitis in the broad sense, and unless otherwise specified, it is used to include both "polyarteritis nodosa" present on the skin and "cutaneous arteritis" in the narrow sense as an independent disease. Furthermore, in this specification, when the terms "cutaneous arteritis" and "CA" are used, they are synonymous with "cutaneous polyarteritis nodosa," the target of diagnosis using the above-mentioned markers, unless otherwise specified.

Peripheral nerves refer to nerves other than the central nervous system (brain and spinal cord). They are spread throughout the body and are responsible for moving muscles and receiving sensations. "Peripheral neuropathy" is a general term for conditions in which damage to peripheral nerves causes difficulty moving the limbs and autonomic nervous system disorders. Damage to motor nerves can cause difficulty moving the limbs. Damage to sensory nerves can cause numbness and loss of sensation. Damage to autonomic nerves can cause urinary and bowel problems.

In one embodiment, "cutaneous polyarteritis nodosa" diagnosed using the marker does not have peripheral neuropathy. The presence or absence of peripheral neuropathy can be confirmed by known methods. For example, it can be determined by a neurological examination by a neurologist or a nerve conduction velocity test.

In one embodiment, the "cutaneous polyarteritis nodosa" diagnosed by the marker is of low severity. Without limitation, "low severity" means that, when the severity of patients with "cutaneous polyarteritis nodosa" is ranked in order, the severity falls within the range of mild to half, preferably the range of mild to one-third, or the range of mild to one-fifth.

In one aspect, cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score (BVAS) is 6 or less. In one aspect, cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 5 or less.

The "Birmingham Vasculitis Activity Score" is a score assigned to each of the various symptoms of vasculitis in nine organ systems, as listed in Table 1 below. The higher the total score, the more active the vasculitis and the worse the prognosis. A score of 0 is obtained when all symptoms have disappeared. Details are described, for example, in Mukhtyar C et al., "Modification and validation of the Birmingham Vasculitis Activity Score (version 3)." Ann Rheum Dis. 2009 68(12):1827-32 (Non-Patent Document 4).

The Birmingham Vasculitis Activity Score (BVS) is a score of 9. Peripheral neuropathy is given a score of 6. A BVS score of 6 or less is consistent with the absence of peripheral neuropathy.
The diagnostic marker can identify cutaneous polyarteritis nodosa,
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A group having either no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa.

The inventors discovered that autoantibodies against CD320 are detected in some patients with cutaneous polyarteritis nodosa, and that patients with cutaneous polyarteritis nodosa in whom autoantibodies against CD320 are detected have mild (low severity) cutaneous polyarteritis nodosa or do not have peripheral neuropathy. They then came up with the present invention based on the belief that anti-CD320 antibodies could be an indicator of low severity of cutaneous polyarteritis nodosa.

Therefore, the diagnostic marker includes an anti-CD320 antibody. CD320 is a membrane protein belonging to the LDL receptor family and has the function of endocytosing the transcobalamin-cobalamin complex into cells. It is known that soluble CD320 (sCD320) consisting only of the extracellular domain exists in CD320 (Non-Patent Document 5, E.V. Quadros et al., Blood., vol. 113, no. 1, pp. 186-192, 2009). The soluble CD320 described in Non-Patent Document 5 consists of amino acids ^Ser36 - ^Tyr229 of the amino acid sequence of human CD320 (SEQ ID NO: 2).

The anti-CD320 antibody is not particularly limited as long as it can recognize all or part of CD320. It may be a monoclonal or polyclonal antibody.

The diagnostic marker is
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
A group comprising (iii) no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa is distinguished from a group comprising none of the requirements (i) to (iii). In one embodiment, preferably, a group comprising "(iii) no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa" is distinguished from a group comprising none of the requirements (i) to (iii).

The present invention, in one aspect, comprises:
Patients with cutaneous polyarteritis nodosa treated with anti-CD320 antibodies who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) Use as a diagnostic marker for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii).

In one aspect, the present invention relates to a patient with cutaneous polyarteritis nodosa who meets any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) An anti-CD320 antibody used as a diagnostic marker for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii).

The present invention relates to a method of using an anti-CD320 antibody as a marker for distinguishing a certain group of cutaneous polyarteritis nodosa from a group that does not meet any of the requirements (i) to (iii) above.
contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a substance that identifies an anti-CD320 antibody; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
and a method of using an anti-CD320 antibody as a marker for distinguishing, among patients with cutaneous polyarteritis nodosa, a group that satisfies any of the above requirements (i) to (iii) from a group that does not satisfy any of the above requirements (i) to (iii), comprising:

Methods for distinguishing certain groups of cutaneous polyarteritis nodosa from groups that do not meet any of the requirements (i)-(iii) above, and each term, are described in detail in separate sections.

2. Diagnostic Compositions In one aspect, the present invention relates to diagnostic compositions.
The diagnostic composition comprises:
Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A diagnostic composition for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not having any of the requirements (i) to (iii), comprising:
Contains substances that identify anti-CD320 antibodies.
"Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii) above" is as explained in "1. Diagnostic markers."

In one aspect, cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 6 or less. In one aspect, cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 5 or less. The "Birmingham Vasculitis Activity Score" is as described in "1. Diagnostic Markers."

The substance that identifies an anti-CD320 antibody is not particularly limited as long as it is capable of directly or indirectly identifying the anti-CD320 antibody. In one embodiment, the substance that identifies an anti-CD320 antibody is a substance that can bind directly or indirectly to the anti-CD320 antibody. In one embodiment, the substance that identifies an anti-CD320 antibody is CD320 or soluble CD320. Soluble CD320 is a soluble portion of CD320 that can be bound by an anti-CD320 antibody. For example, it refers to a portion containing amino acids Leu ³¹ -Tyr ²²⁹ of the amino acid sequence of human CD320 (SEQ ID NO: 2), more preferably a portion containing amino acids Ser ³⁶ -Tyr ²²⁹ .

When anti-CD320 antibodies are identified in a biological sample from a patient with cutaneous polyarteritis nodosa, any of the following requirements must be met:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) identified as belonging to a group with no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa;

Without limitation, the biological sample is serum or plasma.

Methods for determining the presence (identification) of anti-CD320 antibodies in a biological sample can be any known method, including, but not limited to, ELISA and protein arrays. Contact of the biological sample with a substance that identifies anti-CD320 antibodies is preferably carried out in vitro or ex vivo.

The diagnostic composition may include, but is not limited to, a blocking agent (e.g., skim milk, bovine serum albumin (BSA), etc.) in addition to a substance that identifies the anti-CD320 antibody.

The present invention, in one aspect, comprises:
For identifying anti-CD320 antibodies, patients with cutaneous polyarteritis nodosa must meet one of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) Use of the composition as a diagnostic composition for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii).

In one aspect, the present invention relates to a patient with cutaneous polyarteritis nodosa who meets any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A substance identifying an anti-CD320 antibody, which is used as a diagnostic composition for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not having any of the requirements (i) to (iii).

The present invention relates to a method of using a substance that identifies an anti-CD320 antibody as a diagnostic composition for distinguishing certain groups of cutaneous polyarteritis nodosa from groups that do not meet any of the requirements (i)-(iii) above.

In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising:
contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a composition containing an anti-CD320 antibody identifying substance; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
and a method for using a substance that identifies an anti-CD320 antibody as a diagnostic composition for distinguishing, among patients with cutaneous polyarteritis nodosa, a group that meets any of the requirements (i) to (iii) above from a group that meets none of the requirements (i) to (iii), the method comprising:

3. Kits In one aspect, the present invention relates to kits.
The kit is for use in patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A kit for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii), comprising:
Contains substances that identify anti-CD320 antibodies.

"Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) distinguish a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not having any of the requirements (i) to (iii) above.
The terms are as explained in "1. Diagnostic markers."

In one aspect, cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 6 or less. In one aspect, cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 5 or less. The "Birmingham Vasculitis Activity Score" is as described in "1. Diagnostic Markers."

The "substance for identifying anti-CD320 antibodies" is as described in "2. Diagnostic compositions."

The kit may include, but is not limited to, a container for containing a substance that identifies the anti-CD320 antibody and/or other components, a blocking agent (e.g., skim milk, BSA, etc.), a positive control containing the anti-CD320 antibody, or a negative control that does not contain the anti-CD320 antibody.

The present invention, in one aspect, comprises:
For identifying anti-CD320 antibodies, patients with cutaneous polyarteritis nodosa must meet one of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) Use of the method as a kit for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii).

In one aspect, the present invention relates to a patient with cutaneous polyarteritis nodosa who meets any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A substance for identifying an anti-CD320 antibody, which is used as a kit for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not having any of the requirements (i) to (iii).

The present invention relates to a method of using a substance that identifies an anti-CD320 antibody as a kit for distinguishing a certain group of cutaneous polyarteritis nodosa from a group that does not meet any of the requirements (i)-(iii) above.

In one embodiment, the method comprises:
contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a kit containing a substance that identifies an anti-CD320 antibody; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
and a method for using a substance that identifies an anti-CD320 antibody as a kit for distinguishing, among patients with cutaneous polyarteritis nodosa, a group that meets any of the requirements (i) to (iii) above from a group that meets none of the requirements (i) to (iii), the method comprising:

4. Methods of Identification and Diagnosis In one aspect, the present invention relates to a method for distinguishing a certain group of patients with cutaneous polyarteritis nodosa from a group that does not meet any of the requirements (i) to (iii) above.

In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising:
contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a composition containing an anti-CD320 antibody identifying substance; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
The present invention relates to a method for distinguishing, among patients with cutaneous polyarteritis nodosa, a group that satisfies any of the requirements (i) to (iii) above from a group that does not satisfy any of the requirements (i) to (iii), comprising:

In one aspect, the present invention relates to a method for diagnosing a certain group of patients with cutaneous polyarteritis nodosa.

In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising:
contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a composition containing an anti-CD320 antibody identifying substance; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
The present invention relates to a method for diagnosing cutaneous polyarteritis nodosa by distinguishing a group of patients with cutaneous polyarteritis nodosa that meets any of the requirements (i) to (iii) from a group that meets none of the requirements (i) to (iii), the method comprising:

"Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) distinguish a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not having any of the requirements (i) to (iii) above.
The terms are as explained in "1. Diagnostic markers."

In one aspect, cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 6 or less. In one aspect, cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 5 or less. The "Birmingham Vasculitis Activity Score" is as described in "1. Diagnostic Markers."

The "substance for identifying anti-CD320 antibodies" is as described in "2. Diagnostic compositions."

Without limitation, the biological sample is serum or plasma.

The method for determining the presence or absence (identification) of an anti-CD320 antibody in a biological sample is not particularly limited. Contacting the biological sample with a substance that identifies an anti-CD320 antibody is preferably performed in vitro or ex vivo.

The method for identifying a specific group of cutaneous polyarteritis nodosa patients can be performed not only by medical professionals (doctors, etc.), but also by non-medical professionals, for example, at testing companies, research institutions, etc. In one embodiment, the identification method is performed by a non-medical professional. Even when the identification method is performed by a non-medical professional, the results can be communicated to medical professionals, for example, to aid in the medical professional's diagnosis and treatment decisions.

5. Pharmaceutical Composition In the Examples herein, studies using individual mice and cultured human cells demonstrated that vitamin B12 suppresses the pathogenicity of anti-CD320 antibodies.

CD320 is a membrane protein belonging to the LDL receptor family and functions as a receptor for the internalization of vitamin B12 into cells. Example 6 of the present specification demonstrated that, for example, when vitamin B12 binds to CD320 on the surface of vascular endothelial cells, the vitamin B12-CD320 complex is internalized into the cell, resulting in attenuation of CD320 cell surface expression in vascular endothelial cells. Vitamin B12, a ligand for CD320, reduces CD320 on the cell surface, preventing it from being recognized by anti-CD320 antibodies. This means that vitamin B12 can induce the internalization of CD320 into cells and indirectly inhibit the binding of anti-CD320 antibodies to CD320. Vitamin B12, a ligand for CD320, can function as a direct or indirect inhibitor of anti-CD320 antibodies.

Therefore, CD320 ligand containing vitamin B12 is effective as a pharmaceutical composition for treating patients with mild cutaneous polyarteritis nodosa.

In one aspect, the present invention relates to a pharmaceutical composition.

The pharmaceutical composition is administered to patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A pharmaceutical composition for distinguishing and treating a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements of (i) to (iii), comprising:
Contains CD320 ligand.

In the Examples herein, administration of anti-CD320 antibodies to healthy mice resulted in symptoms similar to those of cutaneous polyarteritis nodosa, particularly mild cutaneous polyarteritis nodosa, including the development of subcutaneous edema, periarterial neutrophil infiltration, and expression of various inflammatory cytokines and adhesion molecules. Administration of methylcobalamin, a form of vitamin B12, to these mice clearly suppressed cutaneous polyarteritis nodosa.

The "CD320 ligand" is not particularly limited as long as it binds to CD320 and preferably competes with an anti-CD320 antibody. A non-limiting example of a competitive inhibitor of an anti-CD320 antibody is selected from the group consisting of vitamin B12 and soluble CD320. A non-limiting example of a CD320 ligand is vitamin B12.

In one embodiment, vitamin B12 is methylcobalamin, cyanocobalamin, hydroxocobalamin or adenosylcobalamin.

In this specification, vitamin B12 refers to a group of physiologically active substances that share a common skeletal structure and are collectively referred to as "cobalamins." Cyanocobalamin, along with hydroxocobalamin, is a representative example of vitamin B12 and is classified as a water-soluble vitamin. It is used in the metabolism of amino acids and fatty acids, but does not have coenzyme activity itself and is converted in the body to its coenzyme form, methylcobalamin or adenosylcobalamin. Methylcobalamin is a form of vitamin B12 that differs from cyanocobalamin in that the cyanide is replaced by a methyl group. It is used in the initial treatment of peripheral neuropathy, diabetic neuropathy, and other conditions.

Hydroxocobalamin is a natural form of vitamin B12 and, like other forms of vitamin B12, is bright red in color. Hydroxocobalamin is not found in the human body, but it can be easily converted into useful vitamin B12 coenzymes in the human body. Adenosylcobalamin, also known as cobamamide or dibencozide, is a type of activated vitamin B12.

In one embodiment, the vitamin B12 is methylcobalamin.

In this specification, the administration form of the pharmaceutical composition is not particularly limited and may be oral or parenteral. Examples of parenteral administration include injections such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal). In one embodiment, the pharmaceutical composition is administered by subcutaneous injection.

The pharmaceutical composition may contain the active ingredient as is, or may be formulated with the addition of pharmaceutically acceptable carriers, excipients, additives, etc. Dosage forms include, for example, liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices. Formulations can be carried out using conventional methods, using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc., as appropriate.

Examples of ingredients used in formulations include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol, and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin.

If the pharmaceutical composition is in the form of a pill or tablet, it may be coated with a sugar coating, gastric or enteric coating material.

When the pharmaceutical composition is an injection, it may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizers, preservatives, etc. may also be added.

The pharmaceutical composition may be intended for use not only in humans, but also in non-human mammals or birds. Examples of non-human mammals include non-human primates (monkeys, chimpanzees, gorillas, etc.), livestock animals (pigs, cows, horses, sheep, etc.), dogs, cats, rats, mice, guinea pigs, rabbits, etc.

The dosage, particularly when administered to humans, varies depending on the symptoms, the patient's age, sex, weight, and sensitivity, the administration method, administration interval, type of active ingredient, and type of formulation. Non-limiting examples include 100 μg to 50,000 μg administered once or multiple times (e.g., 2 to 6 times) per day. Non-limiting examples include 1,500 μg administered three times per day. In the case of injection, 500 μg can be administered intramuscularly or intravenously once a day, three times a week.

In one aspect, the present invention relates to a method for treating cutaneous polyarteritis nodosa in patients with CD320 ligand who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) Use of the composition as a pharmaceutical composition for distinguishing and treating a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii).

In one aspect, the present invention relates to a patient with cutaneous polyarteritis nodosa who meets any of the following requirements:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A CD320 ligand used as a pharmaceutical composition for distinguishing and treating a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii).
In one aspect, the present invention relates to a patient with cutaneous polyarteritis nodosa who meets any of the following requirements:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A method for treating a group having any of (i) to (iii) above, which comprises administering a CD320 ligand to a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa.

The treatment method of the present invention is a method for treating cutaneous polyarteritis nodosa in which, before administering CD320 ligand, any of the following conditions is satisfied in a patient:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii).

The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. Those skilled in the art will be able to easily modify and alter the present invention based on the description in this specification, and such modifications and alterations will fall within the technical scope of the present invention.

Example 1: Detection of Autoantibodies Characteristic of Patients with Cutaneous Arteritis In this example, autoantibodies were detected in the serum of patients with cutaneous arteritis. Specifically, the antibody was detected according to the protein array (human comprehensive wet protein array; CWPA) method described in Non-Patent Document 3, following the steps of STEP-1 to STEP-3 outlined in Figure 1. CWPA comprehensively synthesizes human proteins in a form close to their physiological state, and then mounts them on an array substrate to enable analysis.

First, 12,946 human proteins were expressed in a wheat germ cell-free expression system (STEP 1). Taking advantage of the affinity of the GST-tag attached to the N-terminus of each expressed human protein and the glutathione modified on the array substrate, CWPAs were created in which the human proteins were bound to the substrate surface (STEP 1).

Next, two groups of pooled sera were prepared by mixing sera from seven patients with cutaneous arteritis. Each group was diluted with antibody diluent and added to CWPA, allowing the mixture to react at room temperature for one hour. After discarding the reaction solution and washing the CWPA, a secondary antibody (Goat anti-Human IgG (H+L) Alexa Flour® 647 conjugate, ThermoFisher, #A-21445) diluted with antibody diluent was added to the CWPA and allowed to react at room temperature for one hour (STEP 2). After discarding the reaction solution and washing the CWPA, fluorescent images were captured using a fluorescent imager. Proteins bound by the secondary antibody were analyzed from the fluorescent images, and the antigens of the antibodies contained in the serum were identified (STEP 2).

Experiments using CWPA narrowed down the group of autoantibody antigens found in patient serum, and focused protein arrays loaded with these antigens were prepared (STEP 3). Sera from 28 patients with cutaneous arteritis (CA), as well as five patients with related IgA vasculitis, 31 patients with ANCA-associated vasculitis, and 20 healthy controls were diluted with antibody diluent and individually added to the focused array (STEP 3). IgA vasculitis is an Oligocene small-vessel vasculitis characterized by palpable purpura on the skin and sometimes accompanied by arthritis, gastrointestinal symptoms such as abdominal pain and bloody stool, and renal damage. IgA vasculitis is classified as an immune complex vasculitis, in which inflammation occurs due to the deposition of immune complexes containing IgA antibodies in small blood vessels. ANCA-associated vasculitis is a syndrome characterized by autoantibody-induced inflammation of blood vessels (arterioles and capillaries) throughout the body. Although both IgA vasculitis and ANCA-associated vasculitis are related diseases to CA, they are different diseases that should be diagnosed separately.

After reacting for 1 hour at room temperature, the solution was discarded, and after washing, a secondary antibody (Goat anti-Human IgG (H+L) Alexa Flour® 647 conjugate, ThermoFisher, #A-21445) diluted with antibody diluent was added to the focus array and reacted for 1 hour at room temperature. After discarding the solution and washing the focus array and air-drying it, a fluorescent image was captured using a fluorescent imager. The proteins bound by the secondary antibody were analyzed from the fluorescent image, and the antigens of the antibodies contained in the serum were identified (STEP 3).

As a result, CD320 was identified as one of the antigens for autoantibodies specifically positive in patients with cutaneous arteritis (Figure 2). The threshold was set as the mean (Mean) of the control group plus three times the standard deviation (SD) of the control group, i.e., Mean + 3SD. As shown in Figure 2, autoantibodies against CD320 were detected in the serum of some CD patients. It is estimated that anti-CD320 antibodies in the serum of CD patients are likely to be polyclonal, but not limited to these.

Example 2 Detection of anti-sCD320 antibodies in human serum by immunoblotting Anti-sCD320 antibodies in human serum were detected by immunoblotting.

First, 1 μg/5 μL of sCD320 recombinant protein (Aviscera Bioscience, Santa Clara, CA, USA, #S00209-01-100) was mixed with 1.4 μL of 6x SDS-PAGE Sample Buffer (Tokyo Chemical Industry, Tokyo, Japan, #B6105) and 0.6 μL of β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA, #516732), and the mixture was loaded onto NuPAGE 4-12%, Bis-Tris, 1.0-1.5 mm, Mini Protein Gels (Thermo Fisher Scientific). The sCD320 recombinant protein used in this example is a 40-50 kDa recombinant protein corresponding to the extracellular domain of CD320.

After transfer to an Immobilon-P membrane, PVDF, 0.45 μm (Merck Millipore, Burlington, MA, USA, #IPVH00010), the cells were buffered in Tris-buffered saline (Tween-20) containing 5% skim milk (Wako Pure Chemical, Osaka, Japan, #190-12865) and 1% bovine serum albumin (Iwai Chemicals, Tokyo, Japan, #A001) (Cell Signaling). The cells were blocked with 1:250 diluted patient serum or 1:1000 diluted rabbit anti-sCD320 antibody (Aviscera Bioscience, #A00209-01-100) at 4°C overnight. Subsequently, the cells were reacted with 1:100 diluted horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (Cappel Research Reagents, Cochranville, PA, USA, #55226) or 1:2000 diluted HRP-conjugated goat anti-rabbit IgG antibody (Cell Signaling Technology, #7074S) at room temperature for 1 hour. Signals were detected using Chemi-Lumi One Ultra (Nacalai Tesque, Kyoto, Japan, #11644).

The results are shown in Figure 3. Figure 3 reveals that anti-CD320 antibodies in the serum of patients with cutaneous arteritis (CA) recognize sCD320, i.e., the extracellular domain of CD320.

The serum of 26 patients with cutaneous arteritis (CA) was examined for the presence or absence of anti-CD320 antibodies (immunoblot analysis) and peripheral neuropathy, and the relationship between the clinical features of CA patients and the presence or absence of anti-CD320 antibodies was clarified. The results are summarized in Figure 4. Of the 26 CA patients, 7 were positive for anti-CD320 antibodies and 19 were negative. A positive result was determined when a band was detected at the same molecular weight as the positive control, and a negative result when no band was detected. The results showed that anti-CD320 antibody-positive CA patients had a mean Birmingham Vasculitis Activity Score (BVAS) of 4.1, while anti-CD320 antibody-negative CA patients had a mean BVAS of 8.6, demonstrating that anti-CD320 antibody-positive CA patients had a lower BVAS than negative CA patients.

In addition, all patients were examined for the presence or absence of peripheral neuropathy by neurological examination or nerve conduction velocity testing. None of the seven CA patients who were anti-CD320 antibody positive had peripheral neuropathy, whereas 10 of the 19 CA patients who were anti-CD320 antibody negative had peripheral neuropathy. There were no differences in WBC and ESR between patients who were anti-CD320 antibody positive and negative. A low BVAS and the absence of peripheral neuropathy indicate that the patient's cutaneous arteritis is mild. The results of this example support the idea that the presence or absence of anti-CD320 antibodies in CA patient serum is associated with the severity of CA, and that positive anti-CD320 antibodies indicate mild CA.

Example 3 Evaluation of CD320 Expression in Human Skin by Immunohistochemistry In this example, CD320 expression in human skin was evaluated by immunohistochemistry.

Skin samples from CA patients were prepared from subcutaneous tissue (lesional area), subcutaneous tissue (non-lesional area), and dermis. Formalin-fixed, paraffin-embedded skin samples from CA patients were thinly sliced and placed on glass slides, then deparaffinized. Antigen retrieval was then performed for 10 minutes at 95°C using Antigen Unmasking Solution (Vector Laboratories, Gdynia, Poland, #H-3300).

After blocking with 20-fold diluted goat serum, the cells were reacted overnight at 4°C with 200-fold diluted rabbit anti-sCD320 antibody (Aviscera Bioscience, #A00209-01-100) or control IgG (Cell Signaling Technologies) (an antibody that does not react with a specific antigen).

After quenching with hydrogen peroxide (Wako Pure Chemical, #086-07445), signals were detected using the Vectastain ABC Kit (Vector Laboratories, #PK-4001). Nuclear counterstaining was performed with Mayer's hematoxylin. After dehydration and clearing, the specimens were mounted in Marinol solution and then observed.

The results are shown in Figure 5. In the micrographs in Figure 5, the upper rows of the subcutaneous tissue (lesioned area) and subcutaneous tissue (non-lesioned area) are strongly stained, indicating that CD320 is more strongly expressed in the arterioles of the subcutaneous tissue than in the blood vessels of the dermis. CD320 was strongly expressed in the subcutaneous tissue not only in the lesioned area but also in the non-lesioned area.

Example 4: Expression of CD320 and CD31 in Human Skin by Fluorescent Immunostaining In this example, the expression of CD320 and CD31 in human skin was evaluated by fluorescent immunostaining. CD31 (platelet endothelial cell adhesion molecule-1) is a cell surface receptor belonging to the immunoglobulin superfamily (IgSF). In this example, fluorescent immunostaining was performed on CD320 as a marker for vascular endothelium.

Formalin-fixed, paraffin-embedded patient skin specimens (subcutaneous tissue (lesion area)) were thinly sliced and placed on glass slides. After deparaffinization, antigen retrieval was performed using Antigen Unmasking Solution (Vector Laboratories, #H-3300) at 95°C for 10 minutes.

After blocking with 20x diluted goat serum, the sections were incubated overnight at 4°C with 200x diluted rabbit anti-sCD320 antibody (Aviscera Bioscience, #A00209-01-100) and 2000x diluted mouse anti-CD31 antibody (Abcam, Cambridge, UK, #AB9498). Subsequently, the sections were incubated with 1000x diluted Alexa Fluor 594-labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific, #A-11037) and Alexa Fluor 488-labeled goat anti-mouse IgG antibody (Thermo Fisher Scientific, #A-11001).

Finally, the sections were embedded in Vectashield Mounting Medium with DAPI (Vector Laboratories, #H-1200) and images were taken using a BioZero BZ-X800 (Keyence, Osaka, Japan). The results are shown in Figure 6. The "Merge" image in Figure 6 shows the overlay of the results of fluorescent immunostaining for CD320 and CD31. It is clear that CD320 is localized with the vascular endothelial marker (CD31).

Example 5 Experiment on Single Administration of Anti-CD320 Antibody to Mice In this example, a single administration of anti-CD320 antibody to mice was performed, and the results were examined.

Female wild-type C57BL/6 mice were purchased from Jackson Laboratory and shaved at 6 weeks of age. Mouse anti-CD320 antibody (Sigma-Aldrich, #SAB1409408) or control IgG (Bio X Cell, Lebanon, NH, USA, #BE0085) was diluted to 2 mg/mL in phosphate buffered saline (PBS) and subcutaneously injected at 30 μL into the left or right dorsal region of each mouse using a 29-gauge needle. Simultaneously, 200 μL of Evans blue (Sigma-Aldrich, #E2129) diluted to 500 mg/mL in PBS, either dissolved at 3 mg/mL or undissolved, was intravenously administered. Methylcobalamin is a form of vitamin B12 that differs from cyanocobalamin in that the cyanide is replaced by a methyl group.

Six hours later, the mice were sacrificed, and skin samples from the subcutaneous injection site were collected using a 4 mm punch. Histological examination of formalin-fixed, paraffin-embedded skin samples was performed by hematoxylin-eosin staining or toluidine blue staining. The results are shown in Figures 7 and 8. In Figures 7 and 8, "Anti-CD320" indicates the results for the site where mouse anti-CD320 antibody was administered, and "Ctrl IgG" indicates the results for the site where control IgG was administered. The middle image in Figure 7 is an optical microscope photograph at a magnification of 100x, and the right image is at a magnification of 200x. As can be seen from the right image in Figure 7, when anti-CD320 antibody was administered, strong purple staining was observed around the arteries, indicating that neutrophil infiltration around the arteries was induced.

In Figure 8, "Skin Weight" was measured by placing a skin section taken with a 6 mm punch on an electronic balance. The weight is relative to the weight of the area ("Ctrl IgG") administered to the same individual where control IgG was administered, defined as 1.0. The thickness of the "Dermis" and "Subcutaneous Tissue" was measured by microscopic observation of formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained skin specimens. The thickness is relative to the weight of the area ("Ctrl IgG") administered to the same individual where control IgG was administered, defined as 1.0. The results for "Skin Weight" in Figure 8 show that anti-CD320 antibody administration increases skin weight (approximately 1.5-fold), and the results for "Dermis" and "Subcutaneous Tissue" show that anti-CD320 antibody administration increases subcutaneous tissue thickness (approximately 1.8-fold), resulting in edema. Both the increase in skin weight and edema were inhibited by administration of methylcobalamin.

Evans blue is a blue pigment with a peak absorbance at a wavelength of 620 nm. In the blood, it binds strongly to albumin. Therefore, vascular permeability can be assessed by immersing a skin section from an individual after intravenous administration in formamide and measuring the concentration of eluted Evans blue using an absorbance meter. The "Evans blue" results in Figure 8 show that administration of anti-CD320 antibodies increases vascular permeability (approximately 1.9-fold), and that administration of methylcobalamin prevents this increase in vascular permeability.

The results in Figures 7 and 8 show that administration of anti-CD320 antibody induces edema in the subcutaneous tissue of mice and neutrophil infiltration around arteries, and that these phenomena are suppressed by administration of methylcobalamin, a type of vitamin D.

In addition to histological examination, gene expression levels were also examined. Specifically, total RNA was extracted from skin samples (subcutaneous tissue) preserved in RNA Later (Invitrogen, #AM7021) using RNeasy Mini Kit (Qiagen, Crawley, UK, #74106), and reverse-transcribed to cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo, #FSQ-201). Real-time PCR was performed using SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan, #QPK-201) on an ABI Prism 7000 sequence detector (Applied Biosystems, Waltham, MA, USA). Gene expression levels were evaluated by the △△ _CT method using Gapdh as an endogenous control.

The results are shown in Figure 9.
IL6: Interleukin 6
Tnfa: tumor necrosis factor-alpha
IL1b: Interleukin 1β
Icam1: intercellular adhesion factor 1
Icam2: intercellular adhesion factor 2
Vcam1: Vascular cell adhesion factor 1

Il6, Tnfa, and Il1b are inflammatory cytokines. A single dose of anti-CD320 antibody increased the expression levels of these inflammatory cytokines and various adhesion cell factor genes by more than 1.5-fold. The expression levels of these genes were suppressed by the administration of methylcobalamin.

Figures 7-9 show that administering anti-CD320 antibodies to wild-type mice can artificially induce symptoms similar to mild cutaneous polyarteritis nodosa, and that these symptoms of mild cutaneous polyarteritis nodosa can be suppressed by administering methylcobalamin, a type of vitamin D12.

Example 6: Effect of Methylcobalamin on Cell Surface Expression of CD320 in Cultured Human Dermal Vascular Endothelial Cells Human dermal vascular endothelial cells (HDBEC) were purchased (PromoCell, Heidelberg, Germany, #C-12225) and cultured in Endothelial Cell Growth Medium (PromoCell, #C-22110) in a 37°C _CO2 incubator. After the cell density reached 70-80%, the cells were starved for 24 hours and then stimulated for 6 hours with methylcobalamin adjusted to a concentration of 100 ng/mL in Endothelial Cell Basal Medium (PromoCell, #C-22210). Alternatively, the cells were cultured in Endothelial Cell Basal Medium without methylcobalamin for 6 hours.

The cells were then detached using a cell scraper and transferred to a FACS tube. After rinsing once with PBS, the cells were mixed with rabbit anti-sCD320 antibody (Aviscera Bioscience, #A00209-01-100) or control IgG (Cell Signaling Technologies) conjugated to fluorescein using the Fluorescein Labeling Kit-NH2 (Dojindo, Kumamoto, Japan, #347-90911) and left to stand at room temperature for 30 minutes. After rinsing once with PBS, the cells were suspended in BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (BD Biosciences, Franklin Lakes, New Jersey, #554722) and incubated at room temperature for 15 minutes. Fluorescence intensity was then measured using a FACSVerse (BD Biosciences). Analysis was performed using a Kaluza (Beckman Coulter, Brea, California).

The results are shown in Figure 10. It was revealed that administration of methylcobalamin reduced the cell surface expression of CD320 in cultured human dermal vascular endothelial cells.

Example 7 Experiment on Multiple Administration of Anti-CD320 Antibody to Mice In this example, anti-CD320 antibody was administered multiple times to mice, and the results were examined.

Female wild-type C57BL/6 mice were purchased from Jackson Laboratory and had their backs shaved at 6 weeks of age. Mouse anti-CD320 antibody (Sigma-Aldrich, #SAB1409408) or control IgG (Bio X Cell, #BE0085) was diluted to 2 mg/mL in PBS and subcutaneously administered daily in 30 μL doses using a 29-gauge needle into the dorsum. Seven days later, Evans blue (Sigma-Aldrich, #E2129) diluted to 500 mg/mL in PBS was intravenously administered. 30 minutes later, the mice were sacrificed and skin samples from the subcutaneous injection site were collected using a 6 mm punch. For histological analysis, formalin-fixed, paraffin-embedded skin samples were stained with hematoxylin and eosin.

The results are shown in Figure 11. In Figure 11, "Anti-CD320" is a photograph of an individual administered a mouse anti-CD320 antibody, "Ctrl IgG" is a photograph of an individual administered a control IgG (an antibody that does not react with a specific antigen), and "PBS" is a photograph of an individual administered saline only. In the "Anti-CD320" group, vasculitis was observed, vascular permeability was increased, and Evans blue was observed to leak out.

In addition to histological examination, gene expression levels were also examined. As in Example 6, total RNA was extracted from skin samples (subcutaneous tissue) preserved in RNA Later (Invitrogen, #AM7021) using the RNeasy Mini Kit (Qiagen, #74106) and reverse-transcribed to cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo, #FSQ-201). Real-time PCR was performed using SYBR Green Realtime PCR Master Mix (Toyobo, #QPK-201) on an ABI Prism 7000 sequence detector (Applied Biosystems). The gene expression level was evaluated by the _ΔΔCT method using Gapdh as an endogenous control.

The results are shown in Figure 12. Multiple administrations of anti-CD320 antibody increased the expression levels of the inflammatory cytokines Il6, Tnfa, and Il1b, as well as the genes for various adhesion cell factors, by more than two-fold.

Figures 11 and 12 show that administration of anti-CD320 antibodies to wild-type mice can artificially induce symptoms similar to mild cutaneous polyarteritis nodosa.

Example 8 Stimulation of Vascular Endothelial Cells with Anti-CD320 Antibody In this example, anti-CD320 antibody was added to cultured vascular endothelial cells, and the effect on the expression levels of various genes was examined.

Specifically, cultured human dermal vascular endothelial cells (HDBEC) were purchased (PromoCell, Heidelberg, Germany, #C-12225) and cultured in Endothelial Cell Growth Medium (PromoCell, #C-22110) at 37°C in a _CO2 incubator. After the cell density reached 70-80%, the cells were starved for 24 hours, and then stimulated for 6 hours with anti-CD320 antibody (Sigma-Aldrich, #SAB1409408) or control IgG (Bio X Cell, #BE0085) adjusted to a final concentration of 1 μg/mL in Endothelial Cell Basal Medium (PromoCell, #C-22210).

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, #74106) and reverse-transcribed to cDNA using the ReverTra Ace qPCR RT Master Mix (Toyobo, #FSQ-201). Real-time PCR was performed using the SYBR Green Realtime _PCR Master Mix (Toyobo, #QPK-201) on an ABI Prism 7000 sequence detector (Applied Biosystems). Gene expression was assessed using the ΔΔCT method with GAPDH as an endogenous control.

The results are shown in Figure 13.
Icam2: intercellular adhesion factor 2
PAI1: Plasminogen activator inhibitor 1
IL6: Interleukin 6
IL8: Interleukin 8

PAI1 is a coagulation activation factor. In vitro administration of anti-CD320 antibody increased the expression levels of the genes encoding intercellular adhesion factors, coagulation activation factors, and inflammatory cytokines by more than 1.4 times. It was revealed that stimulation of vascular endothelial cells with anti-CD320 antibody enhanced the expression of intercellular adhesion factors, coagulation activation factors, and inflammatory cytokines.

Figure 13 shows that symptoms similar to mild cutaneous polyarteritis nodosa can be artificially induced by stimulating cultured vascular endothelial cells with anti-CD320 antibody.

The present invention has revealed that patients with cutaneous arteritis in whom anti-CD320 antibodies are detected in their serum do not suffer from peripheral neuropathy and have relatively mild symptoms. The clinical significance of this invention lies in the fact that it makes it possible to identify a previously unknown unique subgroup of vasculitis patients (a group of patients with cutaneous polyarteritis nodosa who are of low severity and/or do not have peripheral neuropathy) using a minimally invasive, highly reproducible blood test. Utilizing this invention, testing is possible with only the relatively minimal invasiveness of blood sampling. Furthermore, because the measurement target is blood circulating throughout the body, it is highly reproducible.

By identifying a previously unknown patient population, the anti-CD320 antibody-positive group, the present invention enables prediction of the severity of cutaneous arteritis, contributing to appropriate treatment selection and reduction of treatment-related adverse events. Without being bound by theory, the patient population that can now be selected for the first time by the present invention differs from other vasculitides in that anti-CD320 antibodies in peripheral blood may directly contribute to the pathology. Indeed, studies using individual mice and cultured human cells have shown that vitamin B12, a CD320 ligand, suppresses the pathogenicity of anti-CD320 antibodies. The pharmaceutical composition of the present invention, containing a CD320 ligand, provides a pathology-specific treatment with minimal side effects.
[Sequence Listing]

Claims (17)

Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A diagnostic marker for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii),
The diagnostic marker as described above, which comprises an anti-CD320 antibody. The marker described in claim 1, wherein the cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 6 or less. contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a substance that identifies an anti-CD320 antibody; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
A method of using an anti-CD320 antibody as a marker for distinguishing a group of cutaneous polyarteritis nodosa patients who meet any of the requirements (i) to (iii) above from a group who do not meet any of the requirements (i) to (iii), comprising :
The method is performed by a non-medical professional . Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A diagnostic composition for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not having any of the requirements (i) to (iii), comprising:
The diagnostic composition as defined above, comprising a substance that identifies an anti-CD320 antibody. The diagnostic composition according to claim 4, wherein the cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 6 or less. The diagnostic composition according to claim 4 or 5, wherein the substance that identifies the anti-CD320 antibody is CD320 or soluble CD320. When an anti-CD320 antibody is identified in a biological sample from a patient with cutaneous polyarteritis nodosa, any one of the following requirements is met:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
The diagnostic composition according to claim 4 or 5, wherein the patient is identified as belonging to a group comprising (iii) no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa. Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A kit for distinguishing a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements (i) to (iii), comprising:
The kit further comprises a substance that identifies an anti-CD320 antibody. The kit according to claim 8, wherein the cutaneous polyarteritis nodosa is considered to be of low severity when the Birmingham Vasculitis Activity Score is 6 or less. The kit described in claim 8 or 9, wherein the substance that identifies the anti-CD320 antibody is CD320 or soluble CD320. contacting a biological sample from a patient with cutaneous polyarteritis nodosa with a composition containing an anti-CD320 antibody identifying substance; and
When an anti-CD320 antibody is identified in a biological sample from the patient with cutaneous polyarteritis nodosa, the patient with cutaneous polyarteritis nodosa is diagnosed with any of the following:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) The patient is judged to have no peripheral neuropathy and to have low severity of cutaneous polyarteritis nodosa.
A method for distinguishing a group of cutaneous polyarteritis nodosa patients who meet any of the requirements (i) to (iii) from a group who do not meet any of the requirements (i) to (iii), comprising :
The identification method is performed by a non-medical professional . The method of claim 11, wherein the cutaneous polyarteritis nodosa is of low severity when the Birmingham Vasculitis Activity Score is 6 or less. The method of claim 11 or 12, wherein the substance that identifies the anti-CD320 antibody is CD320 or soluble CD320. The method of any one of claims 11 or 12, wherein the biological sample is serum or plasma. Patients with cutaneous polyarteritis nodosa who meet any of the following criteria:
(i) no peripheral neuropathy;
(ii) cutaneous polyarteritis nodosa is of low severity; or
(iii) A pharmaceutical composition for distinguishing and treating a group having no peripheral neuropathy and low severity of cutaneous polyarteritis nodosa from a group not satisfying any of the requirements of (i) to (iii), comprising:
The pharmaceutical composition comprising a CD320 ligand. The pharmaceutical composition of claim 15, wherein the CD320 ligand is vitamin B12. The pharmaceutical composition of claim 16, wherein the vitamin B12 is methylcobalamin, cyanocobalamin, hydroxocobalamin, or adenosylcobalamin.