CN119490588A - A VHH antibody against human FcRn and its application - Google Patents

Abstract

The present invention belongs to the biological field, and discloses an anti-human FcRn VHH antibody and its application. The complementary determining regions CDR1 to CDR3 of the VHH antibody are shown in SEQ ID NO.1 to 9. The anti-human FcRn VHH antibodies of some examples of the present invention can specifically bind to human FcRn; can replace traditional antibodies and be used in the treatment and diagnosis of autoimmune diseases; the fused Fc fragment targets and binds to the FcRn receptor, effectively reducing the half-life of IgG in the blood while enhancing the targeting of the antibody. The anti-human FcRn VHH antibodies of some examples of the present invention can increase their affinity with the FcRn receptor by fusing the Fc fragment, effectively reducing the half-life of IgG in autoimmune diseases, and reducing the effects of ADCC and CDC.

Description

VHH antibody for resisting human FcRn and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a VHH antibody for resisting human FcRn and application thereof.

Background

The molecular structure of neonatal Fc receptor (neonatal Fc receptor, abbreviated as FcRn) is similar to MHC-I molecule, and is formed by non-covalent combination of a heavy chain (containing alpha 1-alpha 2-alpha 3 structural domain and transmembrane region) and beta 2-MG. FcRn is an atypical fcγr that acts only on IgG and not on other immunoglobulins. Wherein α1 is responsible for binding IgG and α2- α3 is responsible for binding albumin, and the two binding sites are independent of each other and do not interfere with each other. FcRn is expressed in a variety of cells and organs in the body, including epidermal cells, endothelial cells, APC, hematopoietic stem cells, small intestine, kidneys, lungs, central nervous system, etc., and is a multifunctional receptor that can perform diverse functions. Under acidic conditions (pH 6.0-6.5), fcRn has high affinity with Fc segment, igG molecules are swallowed into cells by pinocytosis, and fuse with acidic granules to form inner nuclear bodies with acidic internal environment, and unbound IgG and other proteins enter lysosomes after separation and are degraded. At physiological pH (7.4), the two binding capacities are weak, and the non-binding IgG is released outside the cell again through exocytosis, so that the IgG is protected by cyclic reciprocation, and the half-life period of the IgG is maintained.

An antibody, also called an immunoglobulin (Ig), is a protein that specifically binds to an antigen. The antibody consists of 4 peptide chains, two heavy chains of larger molecular weight (H chain, 50 kD) and two light chains of smaller molecular weight (L chain, 23 kD), the H chain and L chain being linked by disulfide bonds. The hydroxyl end of the polypeptide chain, which contains relatively stable amino acids with no significant number difference or sequence change, is called the constant region or stabilizing region, i.e., the C region, which accounts for 1/2 of the L chain and 3/4 of the H chain. The amino acid sequence of the different species at the amino terminus is changed, and the region is called a variable region, namely a V region, and the characteristic of the variable region that the variable region is highly changed determines the diversity of the antibody and also determines that the antibody only binds to a specific antigen. Humans have five different classes of antibodies, including IgA (including subclasses IgA1 and IgA 2), igD, igE, igG (including subclasses IgG1, igG2, igG3, and IgG 4), and IgM. IgG is the predominant immunoglobulin class in humans and is commonly used in therapy. In autoimmune diseases, the immune system of the body erroneously recognizes an own tissue or organ as a foreign antigen, initiating an immune response. After abnormal activation, B lymphocytes proliferate and differentiate into plasma cells, producing an excess of immunoglobulins, including IgG. The excess IgG may bind to antigen in the self tissue or organ to form an immune complex. These immune complexes deposit in tissues, activate the complement system, and cause inflammatory reactions, resulting in tissue damage. IgG can also directly kill autologous tissue cells by antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), exacerbating tissue damage. Therefore effective reduction of IgG levels in autoimmune diseases is of paramount importance.

Unlike conventional antibody molecules having 4 polypeptide chains, heavy and light chain, single domain antibodies (Single domain antibody, sdabs) refer to a class of antibodies that lack the light chain of an antibody and only the heavy chain variable region, known as VHHs (Variable domain of HEAVY CHAIN of HEAVY CHAIN antibodies), which are also known as nanobodies (nanobodies) due to their small molecular weight. Single domain antibodies, while simple in structure, can still achieve an affinity for specific antigen that is comparable to or even higher than conventional antibodies. Compared with the traditional antibody, the single-domain antibody has the advantages of small molecular weight, strong stability, easy recombinant expression and the like.

"Autoimmune disease" encompasses a disease that occurs when the immune system of the body attacks its own normal tissues, organs or other in vivo components due to an immune system abnormality for which a cause cannot be found. These autoimmune diseases are systemic diseases that can occur in almost all parts of the body, including the nervous system, the gastrointestinal system, the endocrine system, the skin, the skeletal system and vascular tissue. Autoimmune diseases are known to affect about 5-8% of the world population, but reported prevalence of autoimmune diseases is lower than practical due to understanding autoimmune diseases and limitations in the methods of diagnosing these diseases.

The etiology of autoimmune diseases has been studied for a long time in terms of genetic, environmental and immune factors, but has not yet been clearly identified. Many recent studies have revealed that various autoimmune diseases are caused by IgG-type autoantibodies. Indeed, according to studies on the treatment of diseases and autoimmune diseases, the relationship between the presence or absence of disease-specific autoantibodies and the treatment of autoimmune diseases has been widely identified. Thus, the presence of disease-specific autoantibodies in a large number of autoimmune diseases and their pathological roles have been identified, and when the autoantibody of interest is removed from the blood, the effect of rapidly treating the disease can be obtained.

Autoimmune diseases and alloimmune diseases are mediated by pathogenic antibodies, and common examples thereof include immune neutropenia, guillain-Barre syndrome, epilepsy, autoimmune encephalitis, isaac syndrome, nevi syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Grave's disease, goodpasture's syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic Thrombocytopenic Purpura (ITP), lupus nephritis, or membranous nephropathy, or others.

The use of antibodies with novel mechanisms for the treatment of autoimmune diseases by clearing pathogenic autoantibodies is expected to have therapeutic effects against pathogenic IgG-mediated autoimmune diseases (such as pemphigus vulgaris, neuromyelitis optica and myasthenia gravis), and immune complex-mediated glomerular diseases (such as lupus nephritis or membranous nephropathy).

Methods of treating autoimmune diseases by intravenous bulk administration of IgG (IVIG) have been widely used (Arnson Auto immunity 42:553, 2009). The effects of IVIG are explained by a variety of mechanisms, and in addition by mechanisms that compete for FcRn with endogenous IgG to increase pathogenic antibody clearance. Intravenous administration of human immunoglobulin (IVIG) in large amounts has been shown to increase platelet count in infants suffering from immune ITP, and IVIG has been shown to be beneficial as a therapy for several other autoimmune disorders. Many studies have investigated the mechanism by which IVIG is effective in the treatment of autoimmune diseases. For ITP, early studies concluded that IVIG effects were primarily due to blocking of Fc receptors on platelets responsible for phagocytic antibody opsonization. Subsequent studies showed that Fc-depleted IVIG formulations caused elevated platelet counts in some ITP patients, and that IVIG effects were recently reported to be due to stimulation of fcyriib expression on macrophage cells, resulting in inhibition of the platelet phagocytosis process.

Treatment of autoimmune diseases with inhibitors that competitively inhibit IgG binding to FcRn is a promising treatment. However, due to the high affinity of endogenous IgG for FcRn and the high concentration of endogenous IgG in the blood, competitive inhibition of FcRn will likely require extremely high doses and thus have the same limitations as existing IVIG treatments.

Disclosure of Invention

The present invention aims to overcome at least one of the disadvantages of the prior art and to provide a VHH antibody against human FcRn and its use.

The technical scheme adopted by the invention is as follows:

In a first aspect of the invention, there is provided:

A VHH antibody against human FcRn comprising framework region FR and complementarity determining region CDR, the amino acid sequence of the VHH antibody complementarity determining regions CDR1 to CDR3 being selected from any one of the following combinations:

The amino acid sequences of the CDR1 to the CDR3 of the combination 1 are respectively shown as SEQ ID NO.1 to SEQ ID NO. 3;

The amino acid sequences of CDR 1-CDR 3 of the combination 2 are respectively shown as SEQ ID NO. 4-SEQ ID NO. 6;

the amino acid sequences of the CDR1 to the CDR3 of the combination 3 are respectively shown as SEQ ID NO.7 to SEQ ID NO. 9.

In some examples of VHH antibodies, the FR of the VHH antibodies is selected from any one of the following combinations:

the amino acid sequences of FR 1-FR 4 of the combination 1 are respectively shown as SEQ ID NO. 10-SEQ ID NO. 13;

The amino acid sequences of FR 1-FR 4 of the combination 2 are respectively shown as SEQ ID NO. 14-SEQ ID NO. 17;

The amino acid sequences of FR 1-FR 4 of the combination 3 are respectively shown as SEQ ID NO. 18-SEQ ID NO. 21.

In some examples of VHH antibodies, the VHH antibodies are selected from one shown by the amino acid sequence:

the amino acid sequences of CDR 1-CDR 3 of the VHH-1 are respectively shown in SEQ ID NO. 1-SEQ ID NO.3, and the amino acid sequences of FR 1-FR 4 of the VHH-1 are respectively shown in SEQ ID NO. 10-SEQ ID NO. 13;

The amino acid sequences of CDR 1-CDR 3 of VHH-2 are respectively shown as SEQ ID NO. 4-SEQ ID NO.6, and the amino acid sequences of FR 1-FR 4 are respectively shown as SEQ ID NO. 14-SEQ ID NO. 17;

The amino acid sequences of the CDR1 to the CDR3 of the VHH-3 are respectively shown as SEQ ID NO.7 to SEQ ID NO.9, and the amino acid sequences of the FR1 to the FR4 of the VHH-3 are respectively shown as SEQ ID NO.18 to SEQ ID NO. 21.

In some examples of VHH antibodies, they are chimeric antibodies, humanized antibodies, nanobodies fused to Fc fragments, bivalent or multivalent nanobodies.

In some examples of VHH antibodies, the Fc fragment is selected from the group consisting of an Fc fragment of human IgG1, igG2, igG3, or IgG4, or a variant, and variant thereof.

In some examples of VHH antibodies, the amino acid sequence of the Fc fragment is ：EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMITRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTPLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVGSCSVMHEALHAHYTQKSLSLSPGK.

In a second aspect of the invention, there is provided:

a nucleic acid molecule encoding a VHH antibody according to the first aspect of the invention.

In a third aspect of the invention, there is provided:

an expression vector comprising a nucleic acid molecule according to the second aspect of the invention.

In a fourth aspect of the invention, there is provided:

The use of a VHH antibody according to the first aspect of the invention, the use comprising:

preparing an autoimmune disease diagnostic reagent;

Preparing an FcRn adsorbent;

preparing a formulation for the treatment of autoimmune disease;

preparing an FcRn detection reagent;

Preparing a drug delivery vehicle;

Preparing a biosensor;

And (3) preparing a reagent for researching protein structure and function.

VHH antibodies have a high specificity and are capable of recognizing and binding to specific antigens associated with autoimmune diseases. In autoimmune diseases, the immune system of the body erroneously attacks self tissues and organs, producing antibodies against self antigens. Meanwhile, when VHH antibodies bind to the antigen of interest, a detectable signal can be generated in a variety of ways.

In some examples of use, the autoimmune disease is one selected from the group consisting of immune neutropenia, guillain-Barre syndrome, epilepsy, autoimmune encephalitis, isaac syndrome, nevus syndrome, pemphigus vulgaris, pemphigus largehead, bullous pemphigoid, acquired epidermolysis bullosa, pregnant pemphigoid, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Grave disease, goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura, lupus nephritis, and membranous nephropathy.

The beneficial effects of the invention are as follows:

VHH antibodies of some examples of the invention against human FcRn may specifically bind to human FcRn.

The VHH antibodies against human FcRn of some examples of the invention may be used in place of conventional antibodies in the therapeutic diagnosis of autoimmune diseases.

In some examples of the invention, the fusion Fc fragment targets binding to FcRn receptor, while enhancing antibody targeting, effectively reducing IgG half-life in blood.

VHH antibodies against human FcRn of some embodiments of the invention, by fusing the Fc fragment, can increase their affinity for FcRn receptor, effectively reducing the half-life of IgG in autoimmune diseases, while mitigating ADCC and CDC effects.

Drawings

FIG. 1 is an electrophoretogram (45 kD) of a purified VHH-Fc antibody against human FcRn.

FIG. 2 is a schematic representation of the results of a VHH-Fc antibody binding activity assay against human FcRn.

Detailed Description

The technical scheme of the invention is further described below by combining examples and experiments.

Example 1 construction of VHH-Fc antibodies specifically targeting human FcRn Using a Natural alpaca library

FcRn protein expressed by CHO cells is utilized to immunize the Bactrian camel. After 4 immunizations, lymphocytes from 100ml peripheral blood of camels were extracted and total RNA was extracted, and the extracted RNA was reverse transcribed into cDNA. A phage display library of VHH-Fc antibodies against human FcRn was constructed by nested PCR amplification of the nucleic acid fragment encoding the variable region of the heavy chain antibody and the Fc fragment (the amino acid sequence of which is shown in SEQ ID NO. 22). The size of the reservoir is 1.33 multiplied by 10 ¹¹, and the insertion rate reaches 100 percent.

The amino acid sequence of the Fc fragment is as follows:

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMITRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTPLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVGSCSVMHEALHAHYT QKSLSLSPGK（SEQ ID NO.：22）.

Example 2 screening of monoclonal antibodies against VHH-Fc targeting human FcRn

100NM biotinylated human FcRn was bound to the beads for 30min at room temperature, the antigen not bound to the beads was discarded, and the blocked phage display library was placed into the beads and bound for 1h at room temperature. Unbound phage were washed away with sodium acetate solution, phage bound to antigen was eluted with Tris buffer saline at ph7.4, and the resulting phage was infected with e.coli TG1 grown in log phase, and phage were generated and purified for the next round of screening. Positive clones were enriched after 2-3 rounds of the same screening procedure.

Monoclonal colonies were picked from screening the enriched clones for phage ELISA testing. ELISA plates were coated with 2. Mu.g/mL FcRn protein, left overnight at 4℃and PBST washed 3 times. Blocking with 2% skimmed milk at room temperature for 1h, washing with PBST for 3 times, adding phage supernatant diluted with blocking solution, reacting at room temperature for 1h, and washing with PBST for 6 times. anti-M13 secondary antibody was added and reacted at room temperature for 1h, and PBST was washed 3 times. 100. Mu.L of TMB chromogenic substrate was added and the reaction was stopped with 100. Mu.L of 1M sulfuric acid and the absorbance was read at 450nm using an ELISA reader. Clones with OD450 values greater than 0.5 in the ELISA binding assay were sequenced. Finally, 3 VHH-Fc antibodies with the best effect are screened, and are respectively marked as NO. 1-3, wherein:

The VHH amino acid sequence of NO.1 is ：QVQLVESGGGLVQPGGSLRLSCAASGFTESGSGFQYHAWFRQAPGKERERVALSWSGSRIRETISRDNSKNTVYLQMNSLRAEDTAVYYCAATADRMLGYPQGHEDDYGTLVTVSS（SEQ ID NO.23）.

The VHH amino acid sequence of NO.2 is ：QVQLVESGGGLVQPGGSLRLSCTGSGRGFTDFGIGWFRQAPGKERKFVAGISWSGHSTWYGDSVKGRFTISRDNAKNVVYLQMNDLQPEDTGVYYCGVIGLHLWGQGTEVTVSS（SEQ ID NO.24）.

The VHH amino acid sequence of NO.3 is ：AVQLVDSGGGLVQAGGSLRLSCEASGFTFDDYEIGWFRQAPGKEREGVSWIIPKYGDTYYADPVKGRFTISRGNAKSTVSLQMNSLKPEDTAVYYCAADVRTTEWGAPLRYWGQGTQVTVSS（SEQ ID NO.25）.

The 3 VHHs were further analyzed to determine their CDR and FR regions, and the analysis results are shown in Table 1.

TABLE 1 Structure analysis results of different VHHs

Example 3 in vitro expression and purification of VHH-Fc antibodies targeting human FcRn

The gene sequence of the VHH-Fc antibody of No.1 anti-FcRn was transferred into PET28 plasmid through cleavage site and expressed in E.coli BL 21. After the expansion culture with LB medium containing 70. Mu.g/mL kanamycin, the cells were collected, sonicated (5 s on, 10s off, 20min on) and centrifuged at 10000rpm for 10min, and the supernatant was collected.

Purification of the antibody of interest was performed using protein a packing, eluting the antibody of interest with 0.1M acetic acid. The target antibody was analyzed by SDS-PAGE, and the expression level of the VHH-Fc antibody was determined to be 38.4 mg/L.

FIG. 1 is an electrophoretogram of a purified anti-human FcRn VHH-Fc antibody having a molecular weight of about 45 kD.

Example 4 VHH-Fc antibody affinity assay against FcRn

The antibodies prepared in example 3 were subjected to affinity assays using an octet@red96 intermolecular interaction detection system. Ka (association rate constant, binding rate constant), which refers to the rate at which an antibody binds to an antigen to form an immune complex per unit time, higher Ka values mean that the antibody and antigen can bind rapidly. Kd (dissociation rate constant ), which refers to the rate at which an antibody in an immune complex dissociates from an antigen per unit time, is lower, indicating that an antibody does not dissociate readily after binding to an antigen. KD is the ratio of Ka to KD, i.e. kd=kd/Ka. The KD value reflects the magnitude of the affinity of an antibody for antigen binding, with smaller KD values indicating higher affinity of the antibody for antigen. Human FcRn protein was diluted to 10. Mu.g/mL, the antibody obtained in example 3 was diluted to 50. Mu.g/mL, PBS+0.1% Tween 20+0.1% BSA was used as the diluent, and the affinity detection results are shown in Table 2.

TABLE 2 VHH-Fc antibody affinity assay for FcRn

The results in Table 2 show that the 3 VHH-Fc antibodies obtained by screening all have better affinity for FcRn protein, wherein the affinity of NO.1 for FcRn is highest.

Example 5 detection of FcRn protein binding Activity of VHH-Fc antibodies against FcRn

FcRn protein was diluted at 100. Mu.L/well (1000 ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.63 ng/mL) and coated overnight at 4 ℃. PBST plates were washed 3 times, 300. Mu.L of 5% BSA was added and blocked at 37℃for 2h. PBST plates were washed 3 times, 100. Mu.L of diluted VHH-Fc antibody was added and incubated for 1h at 37 ℃. Plates were washed 6 times with PBST, 100. Mu.L of HRP-labeled M13 secondary antibody was added, and incubated at 37℃for 1h. PBST plates were washed 6 times, 100. Mu.L of TMB substrate solution was added, developed for 10min, and the absorbance was read at a wavelength of 450nm, and the results are shown in FIG. 2. As can be seen from FIG. 2, the VHH-Fc antibody has binding activity to FcRn protein, and the highest binding activity is NO.1.

The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.

Claims (10)

1. A VHH antibody against human FcRn comprising framework region FR and complementarity determining region CDR, characterized in that the amino acid sequence of the VHH antibody complementarity determining regions CDR1 to CDR3 is selected from any one of the following combinations:

The amino acid sequences of the CDR1 to the CDR3 of the combination 1 are respectively shown as SEQ ID NO.1 to SEQ ID NO. 3;

The amino acid sequences of CDR 1-CDR 3 of the combination 2 are respectively shown as SEQ ID NO. 4-SEQ ID NO. 6;

the amino acid sequences of the CDR1 to the CDR3 of the combination 3 are respectively shown as SEQ ID NO.7 to SEQ ID NO. 9.

2. The VHH antibody according to claim 1, characterized in that the FR of the VHH antibody is selected from any one of the following combinations:

the amino acid sequences of FR 1-FR 4 of the combination 1 are respectively shown as SEQ ID NO. 10-SEQ ID NO. 13;

The amino acid sequences of FR 1-FR 4 of the combination 2 are respectively shown as SEQ ID NO. 14-SEQ ID NO. 17;

The amino acid sequences of FR 1-FR 4 of the combination 3 are respectively shown as SEQ ID NO. 18-SEQ ID NO. 21.

3. The VHH antibody according to claim 1, characterized in that it is selected from one of the following amino acid sequences:

the amino acid sequences of CDR 1-CDR 3 of the VHH-1 are respectively shown in SEQ ID NO. 1-SEQ ID NO.3, and the amino acid sequences of FR 1-FR 4 of the VHH-1 are respectively shown in SEQ ID NO. 10-SEQ ID NO. 13;

The amino acid sequences of CDR 1-CDR 3 of VHH-2 are respectively shown as SEQ ID NO. 4-SEQ ID NO.6, and the amino acid sequences of FR 1-FR 4 are respectively shown as SEQ ID NO. 14-SEQ ID NO. 17;

The amino acid sequences of the CDR1 to the CDR3 of the VHH-3 are respectively shown as SEQ ID NO.7 to SEQ ID NO.9, and the amino acid sequences of the FR1 to the FR4 of the VHH-3 are respectively shown as SEQ ID NO.18 to SEQ ID NO. 21.

4. The VHH antibody according to any one of claims 1-3, which is a chimeric antibody, a humanized antibody, a nanobody fused to an Fc fragment, a bivalent or multivalent nanobody.

5. The VHH antibody according to claim 4, characterized in that the Fc fragment is selected from the group consisting of the Fc fragment of human IgG1, igG2, igG3 or IgG4 or variants, variants thereof.

6. The VHH antibody according to claim 4, characterized in that the amino acid sequence of the Fc fragment is ：EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMITRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTPLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVGSCSVMHEALHAHYTQKSLSLSPGK.

7. A nucleic acid molecule encoding a VHH antibody according to any one of claims 1-6.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. The use of a VHH antibody according to claims 1-6, characterized in that the use comprises:

preparing an autoimmune disease diagnostic reagent;

Preparing an FcRn adsorbent;

preparing a formulation for the treatment of autoimmune disease;

preparing an FcRn detection reagent;

Preparing a drug delivery vehicle;

Preparing a biosensor;

And (3) preparing a reagent for researching protein structure and function.

10. The use according to claim 9, wherein the autoimmune disease is one selected from the group consisting of immune neutropenia, guillain-Barre syndrome, epilepsy, autoimmune encephalitis, isaac syndrome, nevi syndrome, pemphigus vulgaris, pemphigus largehead, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anaemia, autoimmune Grave disease, goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura, lupus nephritis and membranous nephropathy.