Antibody against complement component c4d

Abstract

A specific immunogenic peptide for the production of rabbit antibody against human complement protein C4d. In transplants, rejection of the donor organ by the recipient may occur. Immunohistochemical examination can detect pathological changes in the transplant after its rejection. A part of the tissue is examined for the presence of the C4d component of complement as a marker of organ rejection. A unique immunogenic peptide was designed: CAQETGDNLYWGSVTGSQSNAVSP according the C4 complement protein sequence (P0C0L4 • CO4A_HUMAN), including the C4d fragment sequence. It is located in the interval of 1221 to 1243 amino acids in the original sequence and is part of the alpha chain of the complement C4 protein. The immunogenic peptide contains the selected linear epitope GSVTG for the antibody against C4d in the range of 1231 to 1235 amino acids. Anti-C4d antibody is applicable in clinical diagnosis of organ rejection after transplants.

Description

[0001] DESCRIPTION OF THE INVENTION

[0002] Antibody against complement component C4d. (Immunogenic peptide for Anti-C4d antibody)

[0003] A specific immunogenic peptide for the production of rabbit antibody against human complement protein C4d. Anti-C4d complement.

[0004] The field of technology

[0005] Antibodies (Ab) or immunoglobulins (Ig) are large proteins of the immune system of vertebrates, produced by cells of the immune system - effector B-lymphocytes, and serve to identify, mark and neutralize foreign elements in the body, such as pathogenic bacteria or viruses. Antibodies are created during the immune response, when the body's immune system reacts to the presence of foreign elements in the body. Naturally, the immune system serves to protect the body against pathogens. The ability of the immune system to create antibodies against foreign elements is purposefully used to create specific antibodies against any substances (primarily proteins and polysaccharides) that are capable of provoking an immune reaction. Such substances are referred to as antigens, or immunogen. The mentioned mechanism is e.g., the principle of inoculation (vaccination), when an antigen (part of a pathogen or the entire weakened or killed pathogen) is introduced into the body, which does not cause a disease, but stimulates the immune system to produce specific antibodies, which ensures full or partial protection of the organism against the given pathogen in the event of a real infections.

[0006] By targeting the immune system, it is possible to produce antibodies for use in science and research, medicine and diagnostics. An example of the use of antibodies in diagnostics are antigen rapid tests for the diagnosis of the disease COVID- 19. In the tests, specific antibodies are present that react with the proteins of the COVID-19 virus present in the positive sample, producing a colour reaction. Pregnancy tests work on a similar principle, using specific antibodies against human choriogonadotropin (hCG), which is found in the urine of pregnant women.

[0007] Specific antibodies are also used in other diagnostic investigation methods. One such method is immunohistochemistry. It is a combination of two scientific fields, histology and immunology. In histological preparations (micro-thin sections of tissue samples placed on a microscope slide), specific substances and molecular structures are searched for and highlighted using specific antibodies, ideally one specific substance using one specific antibody. The goal of this examination method is to prove the presence (visualization) of certain antigens in the examined tissue, directly in the tissue sections. The so-called indirect immunohistochemical staining, in which, in the first step, antibodies against specific structures are attached to the tissue section, the so-called primary antibodies. In the second step, antibodies are used that bind to the antibody from the first step (so- called secondary antibodies). Secondary antibodies are labelled with a label, e.g. peroxidase enzyme. In the final step, the tissue is incubated with the substrate, which is transformed into an insoluble coloured product by the action of an enzyme (present on the secondary antibody). This colour change of the substrate is visible under the microscope, which actually visualizes the presence and localization of the target structure.

[0008] Immunohistochemical staining is widely used in pathology in both routine and advanced diagnostics. During the routine examination of a number of tumours, this staining is used to determine, for example, the presence of the proliferative antigen Ki67, which is related to the biological behaviour of tumours. This examination allows oncologists to more accurately choose the optimal therapeutic strategy.

[0009] In transplants, rejection of the donor organ by the recipient's organism may occur despite thorough preoperative examinations. Immunohistochemical examination of the tissue of the transplanted organ is one of the examinations that detect pathological changes in the transplant after its rejection (rejection) by the recipient's body. After transplantation, part of the tissue is removed from the organ by biopsy, which is examined immunohistochemically after processing. With the help of antibodies, specific markers indicating rejection of the transplanted organ are searched for in tissue sections.

[0010] Current state of the art

[0011] Organ rejection is a serious post-transplant complication. The recipient's immune system reacts with different mechanisms to the presence of a foreign organ in the body. One of such mechanisms is called antibody-mediated acute organ rejection (acute antibody-mediated rejection, AMR), or acute humoral rejection (AHR) (1). Antibodies of the recipient bind to foreign antigens of the endothelial cells of the transplanted organ and trigger inflammatory processes with the aim of removing the foreign element. Among other things, the antibodies bound in this way on the labelled cells activate the complement system, which is a mixture of small proteins produced in the liver and circulating in the urinary tract as inactive precursors. Activation of complement via antibodies (the classic way of complement activation) triggers a set of cascading chemical reactions of a nonspecific immune response taking place on the surface of the marked cell with the aim of destroying the cell membrane by creating the so-called membrane of the attacking complex (microscopic holes in the cell membrane) and thereby cause lysis, i.e. rupture and death of the cell (2). During the ongoing chemical reactions, the individual precursors of the complement components are gradually activated and their disintegration to form various larger or smaller fragments, which become part of the emerging membrane-attacking complex or serve as triggers for further reactions of the complement system (3). One of these fragments is designated “C4d” and serves as a marker of antibody-mediated organ rejection (4). C4d binds covalently to the cell surface of the rejected organ and remains bound for several days. C4d deposition may precede histological evidence of AHR by 5-34 days (5). After a positive finding of C4d in biopsy samples of transplanted kidneys, clinical development of acute rejection occurred in 82% of cases (6).

[0012] Several monoclonal and polyclonal antibodies against the complement component C4d are currently available on the diagnostic antibody market. Their overview is shown in Table 1. The immunogenic peptides of the mentioned (polyclonal) antibodies are not identical to the unique peptide of DB Biotech a.s.

[0013] Table 1.

[0014] Literary sources:

[0015] 1. FARRIS III, ALTON B., LYNN D. CORNELL, and ROBERT B. COLVIN. "25 Pathology of Kidney." Kidney Transplantation-Principles and Practice E-Book (2019): 379.

[0016] 2. Janeway Jr, Charles A., et al. "The complement system and innate immunity." Immunobiology: The Immune System in Health and Disease. 5th edition. Garland Science, 3. Abbas, Abul K., Andrew H. Lichtman, and Shiv Pillai. Cellular and Molecular Immunology, - South Asia Edition-E-Book. Elsevier Health Sciences, 2021.

[0017] 4. Feucht, H. E., et al. "Vascular deposition of complement-split products in kidney allografts with cell-mediated rejection." Clinical & Experimental Immunology 86.3 (1991): 464-470.

[0018] 5. Haas, Mark, Lloyd E. Ratner, and Robert A. Montgomery. "C4d staining of perioperative renal transplant biopsies!" Transplantation 74.5 (2002): 711-717.

[0019] 6. Sund, Stale, et al. "Complement activation in early protocol kidney graft biopsies after living-donor transplantationl." Transplantation 75.8 (2003): 1204-1213.

[0020] The essence of the invention

[0021] Method for production of monospecific clonal antibodies at DB Biotech a.s.

[0022] 1. The process begins with a demanding structural analysis of the antigen (protein) molecule to identify possible linear epitopes, consisting of 18 to 22 steps. Possible linear epitopes are viewed in the context of the higher protein structure and then analyzed using special techniques and software ("Epitop Design and Analysis System" or "EDAS"). The EDAS epitope mapping process is unique and far exceeds traditional epitope mapping software and processes.

[0023] 2. By analyzing the protein, it is determined in what form the protein naturally occurs in nature, what is its structure, its hydrophobic and hydrophilic sites, intracellular and extracellular sites, possible interactions with other proteins within related cell signaling pathways, and possible interactions with nuclear acids (DNA / RNA). The final output of the analysis is three or four linear epitopes that will be accessible to antibody binding under any circumstances and this accessibility is not affected by conformational changes of the target protein (marker) itself.

[0024] 3. Target linear epitopes usually consist of 4 to 7 amino acids. By careful selection of adjacent amino acids at the beginning and end of the amino acid sequence of the epitope, an immunogenic peptide is selected. A rabbit is immunized with such an immunogenic peptide in order to stimulate its immune system to produce specific antibodies against the injected peptide. After approximately three months, blood is taken from the rabbit in order to obtain hyperimmune serum (antiserum) containing the desired antibody.

[0025] 4. Using proprietary antibody clone selection technology ("Epitop-Specific Entropic In-Vitro Antibody Capture System" or "EVAC"), the monospecific immunoglobulin is then separated from the crude anti-peptide polyclonal antiserum based on minimum entropy criteria against the respective originally designed epitope. This immunoglobulin is the product of one B-cell line. Traditional immunoaffinity purification is not used because it often results in a heterogeneous mixture of immunoglobulins. Purification results in a monospecific clonal antibody.

[0026] 5. 5. Using rabbits, it is possible to provide an antibody that is fully post-translationally modified and properly glycosylated and therefore more stable than antibodies produced by traditional cell systems. The use of rabbit to generate the native complete antibody structure avoids the use of a cellular system, which often results in an incorrectly or incompletely glycosylated antibody.

[0027] Methodology of DB Biotech a.s. overcomes the inherent limitations of traditional approaches to developing monoclonal antibodies because:

[0028] 1. Targeting specific epitopes using the EDAS system completely eliminates the use of hybridoma approaches and related technologies, including phage, ribosome and mRNA displays.

[0029] 2. The EDAS epitope mapping process allows us to dictate what antibody is produced for our specifically designed epitope. We tell nature exactly what antibody to make, instead of letting nature produce thousands of antibodies and then looking for the best one.

[0030] 3. Since proteins are known to be dynamic entities, steric epitopes often change their conformational parameters, causing lower affinity or complete disappearance of the physiological interaction between the antibody and the corresponding epitope. In practice, this means that traditional monoclonal antibodies may work very well in certain applications for which they are designed, but when the target protein is modified, the antibodies become ineffective.

[0031] 4. DB Biotech uses only rabbits for antibody production because they produce one type of immunoglobulin G, unlike mice, which have several subtractions of immunoglobulin G. In addition, rabbit immunoglobulin is optimally glycosylated, resulting in higher stability of the final immunoglobulin molecule.

[0032] 5. DB Biotech does not use traditional immunoaffinity purification techniques, but instead the EVAC system allows us to capture the monospecific homogeneous antibody product of a single B-lymphocyte lineage.

[0033] Immunogenic peptide for Anti - c4-d complement:

[0034] According to the above-mentioned epitope selection system for the subsequent production of a specific antibody, the amino acid sequence P0C0L4 ■ CO4A_HUMAN from the freely accessible database https: / / www.uniprot.org / was chosen for the structural analysis of the protein c4-d of the complement component. By careful analysis of the protein sequence, a unique epitope was selected using the EDAS method and a unique immunogenic peptide was designed:

[0035] CAQETGDNLYWGSVTGSQSNAVSP (Epitope: GSVTG)

[0036] The said immunogenic peptide is located in the interval of 1221 to 1243 amino acids in the original P0C0L4 sequence and is part of the alpha chain of the complement C4 protein. The immunogenic peptide contains a selected linear GSVTG epitope for the C4d antibody in the range of 1231 to 1235 amino acids. The indicated sequence of the immunogenic peptide must start with Cysteine (C) for conjugation to the carrier protein (KLH) for rabbit immunization purposes.

[0037] Overview of images on drawings

[0038] Figure 1.

[0039] Kidney rejection after transplantation. Histological section of kidney tissue after biopsy, fixed in formalin and embedded in paraffin, immunohistochemically stained using primary Anti C4d antibody against complement component C4d and secondary antibody labeled with horseradish peroxidase. Brown staining confirms the presence of the C4d component of complement in the peritubular capillaries, positively indicating kidney rejection.

[0040] Examples of implementation of the invention DB Biotech using its own comprehensive antibody design method designed a specific linear immunogenic peptide for antibody production against complement component C4d. The said antibody is used in the clinical diagnosis of rejection of a transplanted kidney, or other organs, using immunohistochemical laboratory methods.

[0041] Industrial applicability

[0042] From the previous points, it follows that the industrial applicability of the mentioned peptide is the production of a specific antibody against the complement component C4d, and this has application in the clinical diagnosis of organ rejection after transplantation.

[0043] DB Biotech, a.s.

Claims

Patent claimsAntibody against complement component C4d. (Immunogenic peptide for Anti-C4d antibody)1. Antibody specifically reactive with complement component C4d wherein the antibody reacts with a peptide comprising an epitope consisting of amino acid sequence CAQETGDNLYWGSVTGSQSNAVSP2. Antibody according to claim 1 , which is a monoclonal antibody.

3. Antibody according to claim 1 , which is a polyclonal antibody.

4. Antibody according to any one of claims 1 - 3, which is a mouse, rat or a rabbit antibody.

5. Method for generating an antibody according to any one of claims 1 - 4 wherein an animal is immunized with a peptide essentially consisting of the amino acid sequence CAQETGDNLYWGSVTGSQSNAVSP.

6. Antibody obtainable by a method according to claim 5.

7. In vitro method for the detection of complement component C4d in a sample wherein the sample is contacted with an antibody according to any one of claims 1 - 4 or 6 and wherein binding between the antibody and C4d is detected.

8. Method according to claim 7 wherein the sample is a blood, serum, plasma, synovial fluid or liquor sample.

9. Method according to claim 7 or 8 wherein the sample is heat-inactivated.

10. Method according to any one of claims 7 - 9, which is selected from the group consisting of ELISA, RIA, EIA, and immunohistochemistry.

11. In vitro method for the diagnosis or prognosis of a disease wherein an antibody according to any one of claims 1 - 4 or 6 is contacted with a sample from an individual and wherein it is concluded that the individual has an increased risk of having or developing said disease if the sample contains an elevated level of complement component C4d in comparison with a predetermined reference value.

12. In vitro method for monitoring disease activity in an individual wherein an antibody according to any one of claims 1 - 4 or 6 is contacted with a sample from the individual and wherein the level of C4d present in the sample is monitored over time.

13. In vitro method for determining the efficacy of the treatment of a disease in an individual, wherein an antibody according to any one of claims 1 - 4 or 6 is contacted with a sample taken from an individual before and after treatment and wherein it is concluded that the individual responds to the treatment if the sample taken after treatment has a decreased or lower level of C4d in comparison to the C4d level in the sample taken before treatment.

14. Kit for the detection of complement component C4d, comprising an antibody according to any one of claims 1 - 4 or 6 immobilized on a solid support and a reagent for the detection of immune complexes.

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