Antibodies against porphyromonas gingivalis and methods of use

Abstract

The present invention provides monoclonal antibodies and methods of use thereof for the diagnosis and / or treatment of Porphyromonas gingivalis infection and / or chronic diseases caused by or associated with such infection. The invention also identifies a specific antigen-antibody reaction region on the outer membrane protein of the porphyromonas gingivalis, and is used for developing vaccines for prevention and treatment. The monoclonal antibody provided by the invention has a cross reaction with different antigen regions of different porphyromonas gingivalis outer membrane proteins. The invention also provides an animal model and a diagnostic kit for serological evaluation, prognosis and therapeutic effect evaluation after porphyromonas gingivalis infection.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and biological agents, and specifically, to multi-target monoclonal antibodies against Porphyromonas gingivalis infection. Background Art

[0002] Periodontal disease is one of the most common oral infectious diseases globally. The annual global cost of treating oral diseases is approximately $442 billion, of which periodontal disease accounts for 10.5 - 12%. In the United States, 17 million people seek medical treatment for periodontal disease each year, with costs exceeding $6 billion. The UK National Health Service (NHS) estimates that more than £500 million is spent on periodontal care in England and Wales each year. These costs only apply to routine acute periodontal disease treatment by NHS dentists and do not include specialist treatment, follow-up, hospital services, and private dentist consultations. 80 - 97% of adults in China have varying degrees of periodontal problems.

[0003] Porphyromonas gingivalis is considered in the research field to be the cause of "adult" periodontal disease. Published literature shows that periodontal disease caused by Porphyromonas gingivalis infection in children is not as severe as in adults. The survival rate of dental implants is closely related to the health of oral periodontal tissues. Porphyromonas gingivalis is the main pathogen causing peri-implantitis. In fact, the course of most periodontal disease patients is chronic, progressive, and recurrent, which means that bacterial infection has never been eradicated.

[0004] Many clinical and statistical studies have shown that Porphyromonas gingivalis not only causes oral and periodontal lesions, but is also associated with cardiovascular diseases, Alzheimer's disease, diabetes, and tumors of the digestive and respiratory systems. Porphyromonas gingivalis infection is also closely related to an increased incidence of low birth weight infants and premature infants, and is closely related to many chronic systemic diseases. So far, the pathogenic mechanisms linking local infection and systemic diseases are still unclear, and sometimes the experimental results conflict with the proposed research directions.

[0005] Porphyromonas gingivalis has many different subtypes, and these subtypes have different bacterial virulence. When discussing bacterial pathogenic mechanisms, bacterial virulence and abnormal immune responses have always been the focus of debate in the scientific community.

[0006] Currently, the main methods for clinically treating periodontal disease are using broad-spectrum antibiotics, physical / mechanical methods to remove local lesions, and surgical treatment if necessary. In clinical practice, the problems that need to be comprehensively addressed include how to prevent, treat, and control Porphyromonas gingivalis infection, reduce local and systemic damage caused by bacterial infection, how to select biological products with immunoprotective effects, and how to use animal experimental models to evaluate the functions of new drugs. Summary of the Invention

[0007] The present invention provides a safe, stable and effective monoclonal antibody drug for preventing and treating diseases caused by Porphyromonas gingivalis infection, including but not limited to periodontal diseases.

[0008] The technical problems to be solved by the present invention are to provide evidence that antibodies against the outer membrane protein of Porphyromonas gingivalis are involved in local tissue and systemic damage of infected individuals. Another technical problem to be solved by the present invention is to provide the heterophilic antigen characteristics of the outer membrane protein of Porphyromonas gingivalis and the pathogenesis of local and systemic damage caused by Porphyromonas gingivalis infection. Another technical problem to be solved by the present invention is to provide a new animal model for evaluating the protective function of products.

[0009] In some embodiments, the present invention discloses the pathogenic mechanism of Porphyromonas gingivalis, specific monoclonal antibodies and their preparation methods. The present invention also discloses specific antigen targets corresponding to the monoclonal antibodies and methods for identifying these antigen targets. In some embodiments, novel tagless RagB and Cra4S1 recombinant plasmids and their nucleotide and amino acid sequences (see SEQ ID NO: 1-10) are also disclosed. The present invention also provides a method from plasmid to production of recombinant proteins, including the design of artificial synthetic genes, expression cassettes and recombinant vectors or cells. In some embodiments, the specific antigen target sequences of the above-mentioned recombinant proteins are shown as SEQ ID NO: 11-126.

[0010] The monoclonal antibodies described in the present invention are directed against a variety of antigen targets disclosed in the present invention. In some embodiments, the nucleotide and amino acid sequences of the monoclonal antibodies corresponding to the antigen targets are shown as SEQ ID NO: 128-167. In some embodiments of the present invention, bispecific antibodies and their preparation methods are also provided. In some embodiments, the nucleotide and amino acid sequences of the bispecific antibodies are shown as SEQ ID NO: 168-174, and the present invention also provides methods for preparing these bispecific antibodies.

[0011] Furthermore, the present invention provides a method for preventing and treating periodontal diseases / peri-implantitis and other diseases caused by chronic Porphyromonas gingivalis infection using one or more monoclonal antibodies disclosed in the present invention. In some embodiments, the mass ratio of the RagB-4-1B11-4-7-7 monoclonal antibody to the RagB-4-1C3-7-8 monoclonal antibody in the combined antibody formulation is 1:0.5-5. The present invention also provides the application of expression cassettes, recombinant vectors and eukaryotic cells for preparing monoclonal and / or bispecific antibody drugs for preventing or treating periodontal diseases / peri-implantitis and diseases caused by chronic Porphyromonas gingivalis infection. The monoclonal and / or bispecific antibodies in the present invention are used for preventing primary diseases, recurrent diseases, and patients with chronic infectious diseases and non-oral / dental diseases caused by Porphyromonas gingivalis infection.

[0012] The present invention also provides a prophylactic and / or therapeutic vaccine for preventing and / or treating periodontal diseases and / or peri-implantitis or diseases associated with Porphyromonas gingivalis infection. In some embodiments, the amino acid sequence of such a vaccine is shown in SEQ ID NO. 127. The vaccines of the present invention are used for preventing primary diseases, recurrence of diseases in patients with periodontal diseases / peri-implantitis, and patients with chronic infectious diseases and non-oral / dental diseases caused by Porphyromonas gingivalis infection.

[0013] The present invention also provides a novel animal model. In some embodiments, the animals are infected with Porphyromonas gingivalis and used to evaluate the monoclonal antibodies and / or antibody-antigen reactions or cross-reactions involved in the present invention and their effects on animal reproduction.

[0014] The present invention also provides a serological diagnostic kit that uses specific diagnostic reagents to diagnose and evaluate Porphyromonas gingivalis infection and the treatment effects and prognosis of related diseases. In some embodiments, the amino acid sequences of the diagnostic reagents are shown in SEQ ID NOs. 2, 4, 6, 8, and 10. The diagnostic kit also provides instructions for operating the reagents to detect specific antibodies in biological samples, which include but are not limited to body fluids such as blood, gingival crevicular fluid, urine, saliva, cerebrospinal fluid, pleural and ascitic fluids, and amniotic fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Various aspects of the present disclosure can be better understood by reference to the drawings. The content in the drawings is not necessarily drawn to scale, and the emphasis is on clearly illustrating the principles of the present invention. Further, the parts corresponding to the reference numerals in the drawings have multiple meanings. The drawings (hereinafter referred to as "FIGS.") apply the principles of the invention and, in conjunction with the detailed description in the embodiments, can better understand the features and advantages disclosed by the present invention:

[0016] Figure 1A-1B Shows the RagB-1 nucleic acid and amino acid sequences of Porphyromonas gingivalis. Figure 1A Shows SEQ ID NO: 1, with a full length of 1461 bases and an average C+G content of 55.10%. Figure 1B Shows SEQ ID NO: 2, with a full length of 482 amino acids. For details, see Example 1.

[0017] Figure 2A-2B Shows the RagB-2 nucleic acid and amino acid sequences of Porphyromonas gingivalis. Figure 2A Shows SEQ ID NO: 3, with a full length of 1449 bases and an average C+G content of 51.62%. Figure 2BDisclosed is SEQ ID NO:4, with a full length of 480 amino acids. For details, see Example 1.

[0018] Figure 3A-3B Disclosed are the RagB-3 nucleic acid and amino acid sequences of Porphyromonas gingivalis. Figure 3A Disclosed is SEQ ID NO:5, with a full length of 1470 bases and an average C+G content of 51.29%. Figure 3B Disclosed is SEQ ID NO:6, with a full length of 485 amino acids. For details, see Example 1.

[0019] Figure 4A-4B Disclosed are the RagB-4 nucleic acid and amino acid sequences of Porphyromonas gingivalis. Figure 4A Disclosed is SEQ ID NO:7, with a full length of 1455 bases and an average C+G content of 51.00%. Figure 4B Disclosed is SEQ ID NO:8, with a full length of 480 amino acids. For details, see Example 1.

[0020] Figure 5A-5B Disclosed are the Cra4S1 nucleic acid and amino acid sequences of Porphyromonas gingivalis. Figure 5A Disclosed is SEQ ID NO:9, with a full length of 411 bases and an average C+G content of 52.55%. Figure 5B Disclosed is SEQ ID NO:10, with a full length of 132 amino acids. For details, see Example 1.

[0021] Figure 6 Disclosed is the polypeptide sequence alignment between four major outer membrane proteins including RagB-1, RagB-2, RagB-3 and RagB-4 by CLUSTALW(1.81) multiple sequence alignment. Figure 6 Disclosed are the sequences of SEQ ID NO:2 (new name RagB-1, old name W50RagB), SEQ ID NO:4 (new name RagB-2, old name ThaiRagB), SEQ ID NO:6 (new name RagB-3, old name QMULRagB) and SEQ ID NO:8 (new name RagB-4, old name 381RagB). For details, see Example 1.

[0022] Figure 7A-7B Disclosed is the expression, detection, purification and identification of the RagB-1 recombinant protein. Figure 7ARecombinant protein expression after induction was shown. Lane M was the protein molecular weight marker (15 - 120 kDa); Lanes 1 - 3 were 0.5 μg, 1 μg, 2 μg of BSA standards; Lanes 4 - 6 were total bacterial proteins (total bacterial lysates) of seed cells, after culturing for 7.4 hours and 24 hours; Lanes 7 - 9 were the centrifuged supernatants of total bacterial lysates of seed cells, after culturing for 7.4 hours and 24 hours; Lanes 10 - 12 were the centrifuged precipitates of total bacterial lysates of seed cells, after culturing for 7.4 hours and 24 hours. Figure 7B The purified recombinant protein detected by SDS - PAGE was shown. Lane M was the protein molecular weight marker (15 - 120 kDa); Lane 1 was the non - reduced recombinant protein RagB - 1; Lane 2 was the reduced recombinant protein RagB - 1. For details, see Example 1.

[0023] Figure 8A-8B The expression, detection and purification identification of the RagB - 2 recombinant protein were shown. Figure 8A Recombinant protein expression after induction was shown. Lane M was the protein molecular weight marker (15 - 120 kDa); Lanes 1 - 3 were 0.5 μg, 1 μg, 2 μg of BSA standards; Lanes 4 - 6 were total bacterial proteins (total bacterial lysates) of seed cells, without induction and after culturing for 22 hours; Lanes 7 - 9 were the centrifuged supernatants of total bacterial lysates of seed cells, without induction and after culturing for 22 hours. Figure 8B The purified recombinant protein detected by SDS - PAGE was shown. Lane M was the protein molecular weight marker (15 - 120 kDa); Lane 1 was the non - reduced recombinant protein RagB - 2; Lane 2 was the reduced recombinant protein RagB - 2. For details, see Example 1.

[0024] Figure 9A-9B The expression, detection and purification identification of the RagB - 3 recombinant protein were shown. Figure 9A Recombinant protein expression after induction was shown. Lane M was the protein molecular weight marker (15 - 120 kDa); Lanes 1 - 3 were 0.5 μg, 1 μg, 2 μg of BSA standards; Lanes 4 - 6 were total bacterial proteins (total bacterial lysates) of seed cells, without induction and after culturing for 22 hours; Lanes 7 - 9 were the centrifuged supernatants of total bacterial lysates of seed cells, without induction and after culturing for 22 hours. Figure 9B The purified recombinant protein detected by SDS - PAGE was shown. Lane M was the protein molecular weight marker (15 - 120 kDa); Lane 1 was the non - reduced recombinant protein RagB - 3; Lane 2 was the reduced recombinant protein RagB - 3. For details, see Example 1.

[0025] Figure 10A-10B The expression, detection and purification identification of the RagB - 4 recombinant protein were shown.Figure 10A Shows the induced recombinant protein expression. Lane M is the protein molecular weight marker (15 - 120 kDa); Lanes 1 - 3 are 0.5 μg, 1 μg, 2 μg of BSA standard; Lanes 4 - 6 are the whole bacterial proteins (whole bacterial lysates) of the seed cells, cells cultured for 6 hours and cells cultured for 15.5 hours; Lanes 7 - 9 are the centrifuged supernatants of the whole bacterial lysates of the seed cells, cells cultured for 6 hours and cells cultured for 15.5 hours; Lanes 10 - 12 are the centrifuged precipitates of the whole bacterial lysates of the seed cells, cells cultured for 6 hours and cells cultured for 15.5 hours. Figure 10B Shows the purified recombinant protein detected by SDS - PAGE. Lane M is the protein molecular weight marker (15 - 120 kDa); Lane 1 is the non - reduced recombinant protein RagB - 4; Lane 2 is the reduced recombinant protein RagB - 4. See Example 1 for details.

[0026] Figure 11A-11B Shows the expression, detection and purification identification of the recombinant protein Cra4S1. Figure 11A Shows the induced recombinant protein expression. Lane M is the protein molecular weight marker (15 - 120 kDa); Lanes 1 - 3 are 0.5 μg, 1 μg, 2 μg of BSA standard; Lanes 4 - 6 are the whole bacterial proteins (whole bacterial lysates) of the seed cells, cells cultured for 14 hours and cells cultured for 23 hours; Lanes 7 - 9 are the centrifuged supernatants of the whole bacterial lysates of the seed cells, cells cultured for 14 hours and cells cultured for 23 hours; Lanes 10 - 12 are the centrifuged precipitates of the whole bacterial lysates of the seed cells, cells cultured for 14 hours and cells cultured for 23 hours. Figure 11B Shows the purified recombinant protein detected by SDS - PAGE. Lane M is the protein molecular weight marker (15 - 120 kDa); Lane 1 is the non - reduced recombinant protein Cra4S1; Lane 2 is the reduced recombinant protein Cra4S1. See Example 1 for details.

[0027] Figure 12A-12B Shows the antibody distribution against different outer membrane proteins of Porphyromonas gingivalis in 51 serum samples from healthy individuals. Figure 12AIt was shown that the average OD value of the samples in the lowest quintile (10 / 51) was set as the negative value. The positive rate was set as the OD value greater than 2.5 times the negative value. The positive rate of the RagB-1 antibody was 9.8% (5 / 51), and the high-titer antibody (set as the OD value greater than 4 times the negative value) was 2% (1 / 51); the positive rate of the RagB-2 antibody was 0% (0 / 51); the positive rate of the RagB-3 antibody was 0% (0 / 51), but the high-titer antibody was 2% (1 / 51); the positive rate of the RagB-4 antibody was 17.6% (9 / 51), and the high-titer antibody was 13.7% (7 / 51); the positive rate of the Cra4S1 antibody was 9.8% (5 / 51), and the high-titer antibody was 0% (0 / 51); the positive rate of the non-related protein GST antibody was 0% (0 / 51). Figure 12B It shows the proportion of the antibody distribution in the analyzed samples. See Example 5 for details.

[0028] Figure 13 It shows the distribution of the Porphyromonas gingivalis ragB gene in the gingival crevicular fluid samples of 107 periodontal disease patients. The results showed that the Porphyromonas gingivalis 16s RNA of 28 patients was detected as negative by PCR (28 / 107, 26.2%). Among the 16s RNA positive samples, by PCR ragB amplification detection, 7 patients were positive for the RagB-1 subtype (7 / 107, 6.6%), 30 patients were positive for the RagB-2 subtype (30 / 107, 28.0%), 32 patients were positive for the RagB-3 subtype (32 / 107, 29.9%), 6 patients were positive for the RagB-4 subtype (6 / 107, 5.6%), and 4 patients had an unknown ragB subtype (4 / 107, 3.7%). See Example 5 for details.

[0029] Figure 14A-14B It shows the antibody distribution against different outer membrane proteins of Porphyromonas gingivalis in 62 serum samples from geriatric patients. Figure 14AIt was shown that the average OD value of the samples in the lowest quintile (12 / 62) was set as the negative value. The positive rate was set as 2.5 times greater than the negative value. The positive rate of the RagB-1 antibody was 25.8% (16 / 62), and the high-titer antibody (set as the OD value greater than 4 times the negative value) was 22.6% (14 / 62); the positive rate of the RagB-2 antibody was 24.2% (15 / 62), and the high-titer antibody was 24.2% (15 / 62); the positive rate of the RagB-3 antibody was 22.6% (14 / 62), and the high-titer antibody was 24.2% (15 / 62); the positive rate of the RagB-4 antibody was 16.1% (10 / 62), and the high-titer antibody was 22.6% (14 / 62); the positive rate of the Cra4S1 antibody was 22.6% (14 / 62), and the high-titer antibody was 21% (13 / 62); the positive rate of the non-related protein GST antibody was 19.4% (12 / 62), and the high-titer antibody was 21% (13 / 62). Figure 14B It shows the proportion of antibody distribution in the analyzed samples. For details, see Example 5.

[0030] Figure 15A-15B It shows the antibody distribution against different outer membrane proteins of Porphyromonas gingivalis in 49 serum samples from patients in the Department of Cardiology. Figure 15A It was shown that the average OD value of the samples in the lowest quintile (10 / 49) was set as the negative value. The positive rate was set as 2.5 times greater than the negative value. The positive rate of the RagB-1 antibody was 36.7% (18 / 49), and the high-titer antibody (OD value greater than 4 times the negative value) was 8.2% (4 / 49); the positive rate of the RagB-2 antibody was 32.7% (16 / 49), and the high-titer antibody was 8.2% (4 / 49); the positive rate of the RagB-3 antibody was 38.8% (19 / 49), and the high-titer antibody was 8.2% (4 / 49); the positive rate of the RagB-4 antibody was 46.9% (23 / 49), and the high-titer antibody was 10.2% (5 / 49); the positive rate of the Cra4S1 antibody was 36.7% (18 / 49), and the high-titer antibody was 4.1% (2 / 49); the positive rate of the non-related protein GST antibody was 49% (24 / 49), and the high-titer antibody was 4.1% (2 / 49). Figure 15B It shows the proportion of antibody distribution in the analyzed samples. For details, see Example 5.

[0031] Figure 16Shows the protein sequence alignment between Porphyromonas gingivalis RagB-4 and Porphyromonas gulae strain COT-052OH2179, outer membrane protein. The outer membrane protein sequence (PubMed ID: 25858832, GenBank: JRAJ01000005.1) was aligned with RagB-4 (SEQ ID NO: 8), and the two proteins have 93% similarity. See Example 5 for details.

[0032] Figure 17 A-17E shows the study on the passive immunization function using a combination of two target-specific antibodies. The photo was taken on the 12th day after Porphyromonas gingivalis challenge. Figure 17 A shows that mice in group G1 received monoclonal antibody (MAb) 1D2-2-1-3. Figure 17 B shows that mice in group G2 received MAb 1D2-2-1-3 and a murine polyclonal antibody against Cra4S1. Figure 17 C shows that mice in group G3 received a murine polyclonal antibody against Cra4S1. Figure 17 D shows that mice in group G4 received normal mouse serum. Figure 17 E shows that mice in group G5 received PBS as a control group for bacterial challenge. See Example 6 for details.

[0033] Figure 18A-18B Shows the passive immunoprotection function of the combined antibody. See Example 6 for details. The X-axis represents the groups of test animals, and the Y-axis represents the area of skin and soft tissue lesions (mm 2 ). Figure 18A Shows the results on the 5th day after challenge. Figure 18B Shows the results on the 12th day after challenge. See Example 6 for details. One-way ANOVA statistical analysis was used, *P<0.05.

[0034] Figure 19 A-19D shows the sequence of monoclonal antibody RagB-2-1A4-3-7-7. Figure 19 A shows the variable region (VH) DNA sequence of the heavy chain (mouse IgG1) confirmed by sequencing (SEQ ID NO: 128). Figure 19 B shows the VH amino acid sequence (SEQ ID NO: 129). Figure 19 C shows the variable region (VL) DNA sequence of the light chain (mouse Kappa) confirmed by sequencing (SEQID NO: 133). Figure 19 D shows the VL amino acid sequence (SEQ ID NO: 134). See Example 7 for details.

[0035] Figure 20A-20F shows the sequence of monoclonal antibody RagB-3-1D2-2-1-3 and the detection of the recombinant antibody expressed and purified in eukaryotic HEK293 cells. Figure 20 A shows the variable heavy chain (mouse IgG2a) (VH) DNA sequence (SEQ ID NO:138) confirmed by sequencing. Figure 20 B shows the VH amino acid sequence (SEQ ID NO:139). Figure 20 C shows the variable light chain (mouse kappa) (VL) DNA sequence (SEQ ID NO:143) confirmed by sequencing. Figure 20 D shows the VL amino acid sequence (SEQ ID NO:144). Figure 20 E shows the purification of the recombinant antibody in HPLC, with the peak collected at 280 nm. Figure 20 F shows the purified recombinant antibody confirmed by SDS-PAGE. Lane M: Protein molecular weight standard (15 - 120 kDa); Lane 1: Purified RagB-3-1D2-2-1-3. For details, see Example 7.

[0036] Figure 21 A-21B shows the sequence of monoclonal antibody RagB-4-1B11-4-4 and the detection of the recombinant antibody RagB-4-1B11-4-4 expressed and purified in eukaryotic HEK293 cells. Figure 21 A shows the variable heavy chain (mouse IgG1) (VH) DNA sequence (SEQ ID NO:148) confirmed by sequencing. Figure 21 B shows the VH amino acid sequence (SEQ ID NO:149). Figure 21 C shows the variable light chain (mouse kappa) (VL) DNA sequence (SEQ ID NO:153) confirmed by sequencing. Figure 21 D shows the VL amino acid sequence (SEQ ID NO:154). Figure 21 E shows the purification of the recombinant antibody in HPLC, with the peak collected at 280 nm. Figure 21 F shows the purified recombinant antibody confirmed by SDS-PAGE. Lane M: Protein molecular weight standard (15 - 120 kDa); Lane 1: Purified RagB-4-1B11-4-4. For details, see Example 7.

[0037] Figure 22 A-22B shows the sequence of monoclonal antibody RagB-4-1C3-7-8 and the detection of the recombinant antibody RagB-4-1C3-7-8 expressed and purified in eukaryotic HEK293 cells. Figure 22A shows the heavy chain (mouse IgG1) variable region (VH) DNA sequence (SEQ ID NO: 158) confirmed by sequencing. Figure 22 B shows the VH amino acid sequence (SEQ ID NO: 159). Figure 22 C shows the light chain (mouse Kappa) variable region (VL) DNA sequence (SEQ ID NO: 163) confirmed by sequencing. Figure 22 D shows the VL amino acid sequence (SEQ ID NO: 164). Figure 22 E shows the purification of the recombinant antibody in HPLC, the peak collected at 280 nm. Figure 22 F shows the purified recombinant antibody confirmed by SDS-PAGE, lane M: protein molecular weight standard (15 - 120 kDa); lane 1: RagB-4-1C3-7-8. See Example 7 for details.

[0038] Figure 23A-23H Shows the designed sequence of the bispecific antibody 1B11-1C3 and the detection of the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23A Shows the recombinant heavy chain variable region (VH) DNA sequence (SEQ ID NO: 168) confirmed by sequencing. Figure 23B Shows the VH amino acid sequence (SEQ ID NO: 169). Figure 23C Shows the sequence map of the bispecific antibody heavy chain, the bold and underlined parts represent the original specific MAb. Figure 23D Shows the light chain (mouse Kappa) variable region (VL) DNA sequence (SEQ ID NO: 173) confirmed by sequencing. Figure 23E Shows the VL amino acid sequence (SEQ ID NO: 174). Figure 23F Shows the sequence map of the bispecific antibody light chain, the bold and underlined parts represent the original specific MAb. Figure 23G Shows the purification of the recombinant bispecific antibody in HPLC, the peak collected at 280 nm. Figure 23H Shows the purified recombinant antibody confirmed by SDS-PAGE, lane M: protein molecular weight standard (15 - 120 kDa); lane R: reduced 1B11-1C3, 76 kDa (heavy chain) / 24 kDa (light chain); lane N-R: non-reduced 1B11-1C3, 200 kDa. See Example 8 for details.

[0039] Although the present invention shows and describes various different ways of implementing the invention, these embodiments are provided only as examples for those skilled in the art. Various changes, modifications, and substitutions may be made by those skilled in the art within the scope of the invention. Without departing from the principles of the present invention, several improvements and refinements may also be made, and these improvements and refinements should also be regarded as within the scope of protection of the present invention.

[0040] Term Definition

[0041] Guided by the foregoing description and the associated drawings, many modifications and other embodiments will come to the mind of those skilled in the art. Therefore, it should be understood that the disclosure of the present invention is not limited to the specific embodiments, and the modifications and other embodiments are intended to be included within the scope of the appended claims. Those skilled in the art will recognize many variations and adaptations. These variations and adaptations are intended to be included in the teachings disclosed by the present invention and are covered by the claims.

[0042] Although specific terms are used in the present invention, these terms are used only in a general and descriptive sense, not in a limiting sense.

[0043] After reading the information disclosed by the present invention, those skilled in the art will perceive that each specific embodiment described and exemplified by the present invention has independent combinations and features, and these combinations and features can be separated from or combined with the features of multiple other embodiments without departing from the scope or spirit disclosed by the present invention.

[0044] Any recited method may be carried out in the order of the recited events or in any other order that is logically possible. That is, unless otherwise expressly stated, no step of any method or aspect of the present invention is required to be carried out in a particular order. Accordingly, when a method claim does not specifically state that the steps are limited to a particular order, no order should be inferred. This rule applies to any possible basis of non-explicit interpretation, including logical issues regarding the arrangement of steps or the process flow, the ordinary meaning arising from the grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0045] All published publications and patents cited in this specification are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials associated with such publications. Each such publication or patent is specifically and individually designated for incorporation by reference. Such citation is limited to the methods and / or materials described in the cited publications and patents and does not extend to any glossary definitions in the cited publications and patents. Any glossary definitions provided in the cited publications and patents that are not expressly repeated in the present invention shall not be considered definitions and shall not be construed as defining any term in the appended claims. Any citation of a publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure should not be prior to that publication due to prior disclosure. Further, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

[0046] The associated published publications and patents cited for the methods and / or materials described in this specification are incorporated herein by reference. Each such individual publication or patent is specifically and individually indicated in the form of citation. Such citation is expressly limited to the methods and / or materials described in the cited publications and patents and does not extend to any glossary definitions in the cited publications or patents. Any glossary definitions in the cited publications and patents that are not expressly repeated in this application shall not be considered glossary definitions and shall not be construed as defining any term appearing in the appended claims. Citing any publication prior to the filing date should not be construed as an admission that this disclosure has no priority. Further, the publication dates provided by the present invention may differ from the actual publication dates and may need to be independently confirmed.

[0047] Although the subject matter disclosed herein may be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and those skilled in the art will understand that each aspect of the subject matter disclosed herein may be described and claimed in any statutory class.

[0048] It should also be understood that the terminology used in this specification is for the purpose of describing particular aspects and is not intended to be limiting. Unless otherwise agreed, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Further, terms as defined in a common dictionary shall be interpreted as having a consistent meaning in the context of the specification and the relevant technical field and shall not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0049] The various aspects of the present invention will employ techniques such as molecular biology, microbiology, organic chemistry, biochemistry, physiology, cell biology, vascular biology, etc., which are well established in the art unless otherwise indicated. These techniques are described in detail in the specification.

[0050] Before describing various aspects of the present invention, the following definitions are provided and used without further specification. Additional terms may be defined elsewhere in the present invention.

[0051] As used in the present invention, "comprising" shall be construed as specifying the presence of the recited features, integers, steps, or components, but not precluding the presence or addition of one or more features, integers, components, or groups thereof. Further, each of the terms "consisting of", "comprising", "including", "consisting essentially of", "including,", "includes,", "comprising,", "relating to", "relating to,", and "such as" is used in an open, non-limiting sense and may be used interchangeably. Additionally, the term "comprising" is intended to include examples and aspects covered by the terms "consisting essentially of" and "consisting of". Similarly, the term "consisting essentially of" is intended to include examples covered by the term "consisting of".

[0052] As used in the present invention, the term "and / or" includes any and all combinations of one or more of the related listed items. Before a list of elements, an expression such as "at least one" modifies the entire list of elements, rather than modifying a single element in the list.

[0053] The singular forms "a", "an", and "the" as used in the specification and claims include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a therapeutic agent" includes one or more therapeutic agents and / or combinations of one or more therapeutic agents, and the like.

[0054] Reference to "a / an" compound, therapeutic agent, and pharmaceutical composition each refers to one or more molecules of the compound, therapeutic agent, and pharmaceutical composition, rather than being limited to the compound, therapeutic agent, and pharmaceutical composition. One or more molecules may be the same or different, so long as they belong to the class of the compound, therapeutic agent, and pharmaceutical composition. For example, "a" therapeutic agent is construed to include one or more therapeutic agent molecules, which may be the same or different (e.g., including different isotope abundances and / or different degrees of hydration or equilibration with different conjugate bases or conjugate acid forms).

[0055] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It will be further understood that each endpoint of each range is significant with respect to the other endpoint and independent of the other endpoint. It should also be understood that many values are disclosed herein, and in addition to the value itself, each value is also disclosed herein as "about" that particular value. For example, if the value "10" is disclosed, then "about 10" is also disclosed. A range may be expressed herein as from "about" a particular value and / or to "about" another particular value. Similarly, when a value is expressed as an approximation, by use of the antecedent "about", it is understood that the particular value forms another aspect. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0056] In the case of expressing a range, it specifically refers to from a particular value and / or to another particular value. In the case where a series of values is provided, it can be understood that unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range and any other specified or intervening value within the range, down to one-tenth of the lower limit unit, is included within the scope of the present invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also included within the present invention, subject to any specifically excluded limitations within the stated range. If the range includes one or both of the limitations, ranges that do not include one or both of these limitations are also included within the present invention. For example, if the range includes one or both of the limitations, ranges that do not include one or both of these limitations are also included within the present invention. For example, the phrase "x to y" includes the range from "x" to "y" and ranges greater than "x" and less than "y". The range may also be expressed as an upper limit, such as "about x, y, z or less", and should be interpreted as including the specific ranges of "about x", "about y", and "about z", as well as ranges "less than x", "less than y", and "less than z". Similarly, the phrase "about x, y, z or greater" should be interpreted as including the specific ranges of "about x", "about y", and "about z", as well as ranges "greater than x", "greater than y", and "greater than z". In addition, the phrase "about 'x' to 'y'", where 'x' and 'y' are numerical values, includes "about 'x' to about 'y'".

[0057] It should be understood that this range format is used for convenience and brevity and should, therefore, be interpreted in a flexible manner to include not only the explicitly recited values as range limitations but also all individual values or sub-ranges included within that range as if each value and sub-range were explicitly recited. By way of illustration, a numerical range of "about 0.1% to 5%" should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also the individual values within the indicated range (e.g., about 1%, about 2%, about 3%, and about 4%) and sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges).

[0058] As used herein, the terms "about", "substantially", "essentially", etc., when associated with a numerical variable, generally refer to the value of that variable and all values within the experimental error range (e.g., within a 95% confidence interval) or within the range of + / - 10% of the indicated value. Herein, "about", "substantially", "or about", and "essentially" can mean that the relevant quantity or value can be an exact value or a value that provides an equivalent result or effect. That is, it is understood that quantities, dimensions, formulations, parameters, and other quantities and characteristics need not be exact but can be approximate and / or larger or smaller, as desired, reflecting factors such as tolerances, conversion factors, rounding, measurement errors, etc., so as to obtain an equivalent result or effect. In some cases, the value providing an equivalent result or effect cannot be reasonably determined. Generally speaking, whether or not explicitly stated, a quantity, dimension, formulation, parameter, or other quantity or characteristic preceded by "about", "substantially", or "or about" is "about", "substantially", or "or about", which also includes the particular magnitude itself, unless otherwise specifically stated.

[0059] As used in this specification, when used in conjunction with a numerical variable, "about", "approximately", "substantially", etc., can generally refer to the value of the variable and all values of the variable within the experimental error range (e.g., within the 95% confidence interval of the mean) or within + / -10% of the indicated value, whichever is greater. As used herein, the terms "about", "approximately", "equal to or about", and "substantially" can mean that the amount or value in question can be an exact value or a value of an equivalent result or effect as described in the claims or indicated in this specification. That is, it is understood that amounts, volumes, formulas, parameters, and other quantities and features do not need to be precise, but can be approximate and / or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art, so as to obtain equivalent results or effects. In some cases, it is not possible to reasonably determine the value that provides an equivalent result or effect, and generally speaking, it is not possible to clearly state that an amount, volume, formula, parameter, or other quantity or feature is "about", "approximately", or "equal to or about". It is understood that where the words "about," "approximately," or "equal to or approximately" are used before a quantitative value, the parameter also includes the specific quantitative value itself unless specifically stated otherwise.

[0060] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0061] As used in this specification, "administration" may refer to oral, topical, intravenous, subcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intradural, intravitreal, intracerebral and lateral ventricles, intratympanic, intracochlear, rectal, intravaginal, by inhalation, catheter, stent or by implanted reservoir or other device actively or passively (e.g., by diffusion) forming a route of administration through the perivascular space and the adventitia of the blood vessels. For example, medical devices such as stents may include a composition or preparation disposed on its surface, which may then be dissolved or otherwise distributed to surrounding tissues and cells. The term "enteral" may include subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Administration may be continuous or intermittent. In various aspects, the preparation may be administered therapeutically; i.e., for the treatment of an existing disease or condition. Further, the formulations may be administered prophylactically; that is, to prevent a disease or condition.

[0062] As used in this specification, "therapeutic agent" can refer to any substance, compound, molecule, etc. that may have biological activity or can produce pharmacological, immunogenic, biological, and / or physiological effects on a subject through local and / or systemic action. A therapeutic agent can be the primary active agent, or in other words, the component to which all or part of the effect of the composition is attributed. A therapeutic agent can be a secondary therapeutic agent, or in other words, the component to which additional parts and / or other actions of the composition are attributed. Thus, the term includes compounds or chemical substances that are traditionally regarded as drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, etc.

[0063] Examples of therapeutic agents are described in well-known reference works such as the Merck Index (14th Edition), the Physician's Desk Reference (64th Edition), and Goodman & Gilman's The Pharmacological Basis of Therapeutics (12th Edition), and include but are not limited to drugs; vitamins; mineral supplements; substances for the treatment, prevention, diagnosis, cure, or alleviation of disease or affliction; substances that affect the structure or function of the body, or prodrugs, which become biologically active or more active upon placement in a physiological environment. For example, the term "therapeutic agent" includes all major compounds or compositions used in the field of therapy, including but not limited to adjuvants; anti-infective agents such as antibiotics and antiviral agents; analgesics and analgesic combinations, anorectics, anti-inflammatory agents, antiepileptic agents, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuronal blockers, anticholinergic and cholinergic agents, antimuscarinic and muscarinic receptor agonists, adrenergic antagonists, antiarrhythmic agents, antihypertensive agents, hormones and nutrients, anti-arthritis agents, anti-asthmatic agents, anticonvulsants, antihistamines, analgesics, antineoplastic agents, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta blockers, beta agonists, and antiarrhythmic agents), antihypertensive agents, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic agents; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressive agents; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized, or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, ribonucleotides (RNA) or deoxyribonucleotides (DNA), including double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (such as doxorubicin), and other bioactive macromolecules such as proteins and enzymes. Therapeutic agents can be bioactive agents for use in medicine, including veterinary applications and agriculture (e.g., plants), as well as other fields. The term "therapeutic agent" also includes but is not limited to drugs; vitamins; mineral supplements; substances for the treatment, prevention, diagnosis, cure, or alleviation of disease or affliction; or substances that affect the structure or function of the body; or prodrugs, which become biologically active or more active upon entry into a particular physiological environment.

[0064] As used herein, a "kit" refers to a packaging box containing at least two components. These components together constitute a functional unit for a specific purpose. The individual components may be physically packaged together or separately packaged. For example, the instructions included in the kit may or may not be physically packaged together with the other individual components. The instructions can be separate from the other components, can be in paper form or electronic form, can be stored on a computer storage device or downloaded from an Internet website, or can be a presentation record.

[0065] As used herein, the term "instructions" refers to a document that describes the materials or methods associated with a kit. Such materials may include any combination of the following: background information, a list of components and information regarding their availability (such as purchasing information), a brief or detailed protocol for using the kit, troubleshooting, references, technical support, and other related documents. The instructions may be provided with the kit, or as a separate component, and may be in paper form, electronic form, provided on a computer-readable storage device, downloadable from an Internet website, or as a recorded presentation. The instructions may consist of one or more documents and include future updates.

[0066] The terms "subject", "individual", or "patient", which may be used interchangeably herein, can refer to a vertebrate, such as a mammal (e.g., a human). A "subject" can refer to a cell, cell population, tissue, organ, or organism, with a preference for humans and their components.

[0067] As used herein, the terms "treat" and "therapeutic" generally can refer to obtaining the desired pharmacological and / or physiological effect. The effect can be (but is not necessarily) prophylactic, to prevent or partially prevent a disease, symptom, or condition associated therewith. The effect can also be therapeutic, i.e., to partially or completely cure a disease, symptom, or condition associated therewith. The term "treat" as used herein can include any treatment of inflammation associated with any disease in a subject, particularly a human, and can include one or more of the following: (a) preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not been diagnosed as having the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., reducing or improving the disease and / or its symptoms or condition. The term "treat" as used herein can refer to treatment only, prophylactic treatment only, or both treatment and prophylactic treatment. A person in need of treatment (a subject in need of treatment) can include those who already have the disease and / or those in need of preventing the disease. The term "treat" as used herein can include inhibiting a disease, discomfort, or disorder. For example, hindering its progression; and alleviating a disease, discomfort, or disorder, such as causing the regression of the disease, discomfort, and / or disorder. Treating a disease, discomfort, or disorder can include improving at least one symptom of a particular disease, discomfort, or disorder, even if the underlying pathophysiology is not affected. For example, treating pain in a subject by administering an analgesic, even if the drug does not treat the cause of the pain.

[0068] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve the desired therapeutic result or to have an effect on an undesired symptom, but generally not sufficient to cause adverse side effects. The specific therapeutically effective dose level will vary for different patients, depending on a variety of factors, including the disease being treated and its severity; the specific composition being used; the age, weight, general health, gender and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound being used; the duration of the treatment; the medications used in combination with or concurrently with the specific compound being used, and similar factors within the knowledge and expertise of the healthcare practitioner, which may be well known in the medical field. In the case of treating a particular disease or disorder, in some instances, the desired response may be to inhibit the progression of the disease or disorder. This may only temporarily slow the progression of the disease. However, in other instances, it may be necessary to permanently halt the progression of the disease. This can be monitored by conventional diagnostic methods known to those of ordinary skill in the art for any particular disease. The expected response to the treatment of a disease or disorder may also be to delay the onset of the disease or disorder, or even to prevent its onset.

[0069] For example, those skilled in the art are fully capable of arranging the dosage of the drug required for the therapeutic effect, starting from a low level and gradually increasing the dosage until the desired effect is achieved. If necessary, the therapeutically effective daily dose can be divided into multiple doses. Thus, a single-dose composition can contain these amounts or multiples thereof to constitute the daily dose. When contraindications occur, the dosage can be adjusted by a specialist doctor. It is generally preferred to use the maximum dose of the pharmacological reagent of the present invention (alone or in combination with other therapeutic agents), i.e., the highest safe dose based on reasonable medical judgment. However, those of ordinary skill in the art will understand that patients may adhere to a lower or tolerable dose for medical reasons, psychological reasons, or almost any other reason.

[0070] For example, the response to a therapeutically effective dose of the disclosed compound and / or pharmaceutical composition can be measured by determining the physiological effects of the treatment or drug, such as the reduction or disappearance of disease symptoms after the administration of the treatment or pharmacological reagent. Those of ordinary skill in the art are aware of the levels at which to measure the response. The amount of the treatment can vary, for example, by increasing or decreasing the amount of the disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition being administered, by changing the route of administration, by changing the dosing time, etc. The dose can vary and can be administered once or multiple times a day for one or several days. Appropriate dosage guidelines for a given class of drugs can be found in the literature.

[0071] As used herein, the term "prophylactically effective amount" refers to an amount effective to prevent the onset or initiation of a disease or disorder.

[0072] As used herein, the terms "prevent" or "prevention" mean to preclude, avoid, eliminate, forestall, hinder, or inhibit the occurrence of something by prior action. It should be understood that unless specifically stated otherwise, the use of the terms reduce, inhibit, or prevent also expressly discloses the use of the other two terms herein.

[0073] As used herein, the term "pharmaceutically acceptable" describes a substance that does not produce biological or other adverse reactions, i.e., a substance that does not cause unacceptable levels of adverse biological effects or interact in a harmful manner.

[0074] As used herein, the term "pharmaceutically acceptable salt" refers to salts prepared from acids or bases that are tolerated by the body, or salts prepared from acids or bases of an active agent that are tolerated by a subject or the body when used in the effective therapeutic dose range. When the compounds described in the present invention contain relatively acidic functional groups, base addition salts can be obtained by bringing these compounds into contact in neutral form or with a sufficient amount of base, either in pure form or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include, but are not limited to: sodium salts, potassium salts, calcium salts, ammonium salts, organic amine salts, magnesium salts, lithium salts, strontium salts, or similar salts. When the compounds described in the present invention contain relatively basic functional groups, acid addition salts can be obtained by bringing the neutral form of these compounds into contact with a sufficient amount of the desired acid, either in pure form or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to: those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, hydrogen phosphoric acid, dihydrogen phosphoric acid, sulfuric acid, hydrogen sulfide, hydroiodic acid, phosphorous acid, etc., and those derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, sebacic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids, such as arginine salts, etc., and salts of organic acids, such as glucuronates or galacturonates, etc.

[0075] As used in the specification, the term "pharmaceutically acceptable ester" refers to esters of the compounds disclosed in the present invention that are hydrolyzed in vivo, including those that are readily decomposed in the human body to leave the parent compound or its salts. Examples of pharmaceutically acceptable non-toxic esters disclosed in the present invention include C1-C6 alkyl esters and C5-C7 cycloalkyl esters, although C1-C4 alkyl esters are preferred. The esters of the compounds disclosed in the present invention can be prepared according to conventional methods. Pharmaceutically acceptable esters can be attached to a hydroxyl group by reacting a compound containing a hydroxyl group with an acid and an alkyl carboxylic acid such as acetic acid or with an acid and an aryl carboxylic acid such as benzoic acid. In the case of a compound containing a carboxylic acid group, a pharmaceutically acceptable ester is prepared from the compound containing a carboxyl group by reacting the compound with a base such as triethylamine and an alkyl halide, such as methyl iodide, benzyl iodide, cyclopentyl iodide or an alkyl trifluoromethanesulfonate. They can also be prepared by reacting the compound with an acid such as hydrochloric acid and an alcohol such as ethanol or methanol.

[0076] The term "pharmaceutically acceptable amide" refers to non-toxic amides disclosed in the present invention that are derived from ammonia, primary C1-C6 alkylamines and secondary C1-C6 dialkylamines. In the case of secondary amines, the amine can also be in the form of a 5- or 6-membered heterocycle containing one nitrogen atom. Preferred amides are those derived from ammonia, primary C1-C3 alkyl amides and secondary C1-C2 dialkyl amides. The amides of the compounds disclosed in the present invention can be prepared according to conventional methods. Pharmaceutically acceptable amides can be prepared from a compound containing a primary or secondary amine group by reacting a compound containing an amino group with an alkyl anhydride, an aryl anhydride, an acyl halide or an aroyl halide. In the case of a compound containing a carboxylic acid group, a pharmaceutically acceptable amide is prepared from the compound containing a carboxylic acid group by reacting the compound with a base (such as triethylamine), a dehydrating agent (such as dicyclohexylcarbodiimide or carbonyldiimidazole) and an alkylamine, a dialkylamine (such as methylamine, diethylamine and piperidine). They can also be prepared by reacting the compound with an acid such as sulfuric acid and an alkyl carboxylic acid such as acetic acid, or with an acid and an aryl carboxylic acid such as benzoic acid under dehydrating conditions, such as by adding molecular sieves. The composition can contain a compound disclosed in the present invention in a pharmaceutically acceptable prodrug form.

[0077] The term "pharmaceutically acceptable prodrug" or "prodrug" refers to a prodrug of a compound disclosed in the present invention which, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals, is not unduly toxic, irritating, allergic, etc., is commensurate with a reasonable benefit / risk ratio, and is effective for its intended use. The prodrugs disclosed in the present invention can be rapidly converted in vivo into the parent compound having the structure of the disclosed compound, for example, by hydrolysis in the blood. This is discussed in depth in T. Higuchi and V. Stella, Prodrugs as Novel Drug Delivery Systems, A.C.S. Symposium Series No. 14, and Bioreversible Carriers in Drug Design, edited by Edward B. Roche, American Pharmaceutical Association and Pergamon Press (1987).

[0078] As used in this specification, the term "derivative" refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed in the present invention), the structure of which is sufficiently similar to the structure disclosed in this specification such that, based on this similarity, it can be expected that a compound having the same or similar activity and use as the compound, or a compound that can induce the same or similar activity and use as a precursor, can be made within the skills of a person skilled in the art. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound. These are also intended to be within the scope of the protection of this patent.

[0079] As used herein, the nomenclature of compounds, including organic compounds, can be given using the naming recommendations of IUPAC, IUBMB, or CAS common names. When there are one or more stereochemical features, the Cahn-Ingold-Prelog stereochemical rules can be used to specify stereochemical priorities, E / Z specifications, etc. To define the name of a compound, a person skilled in the art can systematically reduce the compound structure using naming conventions, or by using commercially available software such as CHEMDRAW TM (Cambridgesoft Corporation, U.S.A.), and can easily determine the structure of the compound.

[0080] It should be noted that unless otherwise specified, the temperatures referred to herein are based on atmospheric pressure (i.e., one atmosphere).

[0081] Therapeutic and / or prophylactic methods

[0082] The present invention discloses a method for treating and / or preventing various diseases and / or related pathological conditions caused by Porphyromonas gingivalis infection in patients in need thereof, comprising administering to the patient a therapeutically effective amount of one or more monoclonal and / or bispecific antibodies disclosed in the present invention. In certain embodiments, the present invention provides a method for reducing the level of infection caused by Porphyromonas gingivalis. Methods for measuring the degree of infection are well known in the art. In one embodiment, the level of infection is reduced by about 5% to about 100%. In another embodiment, the level of infection in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100%.

[0083] Monoclonal antibodies and bispecific antibodies

[0084] As disclosed in the present invention, in certain embodiments, nucleotide and amino acid sequences of certain specific antibodies are provided as follows:

[0085] 1. The RagB-2-1A4-3-7-7 monoclonal antibody comprises a heavy chain (mouse IgG1) variable region (VH) with a nucleotide sequence (357 bases) as shown in SEQ ID NO:128 and an encoded amino acid sequence (119 amino acids) as shown in SEQ ID NO:129; and a light chain (mouse κ type) variable region (VL) with a nucleotide sequence (282 bases) as shown in SEQ ID NO:133 and an encoded amino acid sequence (94 amino acids) as shown in SEQ ID NO:134.

[0086] 2. The RagB-3-1D2-2-1-3 monoclonal antibody comprises a heavy chain (mouse IgG2a) variable region (VH) with a nucleotide sequence (360 bases) as shown in SEQ ID NO:138 and an encoded amino acid sequence (120 amino acids) as shown in SEQ ID NO:139; and a light chain (mouse κ type) variable region (VL) with a nucleotide sequence (336 bases) as shown in SEQ ID NO:143 and an encoded amino acid sequence (112 amino acids) as shown in SEQ ID NO:144.

[0087] 3. The monoclonal antibody RagB-4-1B11-4-4 comprises a heavy chain (mouse IgG1) variable region (VH) with a nucleotide sequence (375 bases) as shown in SEQ ID NO:148 and an encoded amino acid sequence (125 amino acids) as shown in SEQ ID NO:149; and a light chain (mouse κ type) variable region (VL) with a nucleotide sequence (336 bases) as shown in SEQ ID NO:153 and an encoded amino acid sequence (112 amino acids) as shown in SEQ ID NO:154.

[0088] 4. The monoclonal antibody RagB-4-1C3-7-8 comprises a heavy chain (mouse IgG1) variable region (VH) with a nucleotide sequence (357 bases) as shown in SEQ ID NO:158 and an encoded amino acid sequence (119 amino acids) as shown in SEQ ID NO:159; and a light chain (mouse κ type) variable region (VL) with a nucleotide sequence (321 bases) as shown in SEQ ID NO:163 and an encoded amino acid sequence (107 amino acids) as shown in SEQ ID NO:164.

[0089] 5. The bispecific antibody 1B11-1C3 comprises a heavy chain (mouse IgG1) with a nucleotide sequence (2232 bases) as shown in SEQ ID NO:168 and an encoded amino acid sequence (728 amino acids) as shown in SEQ ID NO:172; and a light chain (mouse κ type) with a nucleotide sequence (744 bases) as shown in SEQ ID NO:173 and an encoded amino acid sequence (232 amino acids) as shown in SEQ ID NO:174.

[0090] Administration and Use

[0091] Although the active ingredient can be administered alone, it may be more desirable to present it as a pharmaceutical formulation or composition. As described below, the disclosed formulations, whether for veterinary or human use, comprise at least one active ingredient, and one or more acceptable carriers and optionally other therapeutic ingredients. The carrier must be "acceptable", i.e., compatible with the other ingredients in the formulation and physiologically harmless to the recipient.

[0092] Each active ingredient can be formulated with conventional carriers and excipients, which will be selected according to conventional practices. Tablets can contain excipients, glidants, fillers, binders, etc. Aqueous formulations are prepared in a sterile form and are usually isotonic when used for delivery by means other than oral administration. All formulations are used with reference to the Handbook of Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, etc. The pH range of the formulations is from about 3 to about 11, but is usually from about 7 to 10. A therapeutically effective amount of the active ingredient can be readily determined by a skilled clinician using conventional dose escalation studies. Generally, the dosage of the active ingredient is from 0.01 mg to 2 g. In one embodiment, the dosage is from about 10 mg to 450 mg. In another embodiment, the dosage is from about 25 to about 250 mg. In another embodiment, the dosage will be about 50 or 100 mg. By way of example, in one example, the dosage is about 100 mg, and it is contemplated that the active ingredient can be administered once, twice, or three times a day. In addition, the active ingredient can be administered once or twice a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks.

[0093] Compositions of the pharmaceutically active ingredient include suitable routes of administration. The formulations can be presented in unit dosage form or can be prepared by any method known in the pharmaceutical art. Techniques and formulations are referenced in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). These methods include the step of associating the active ingredient with a carrier, which constitutes one or more accessory ingredients. Generally, the formulations are finally shaped by uniformly and intimately combining the active ingredient with a liquid carrier or a finely divided solid carrier or both.

[0094] Formulations suitable for oral administration can be presented in discrete packages, such as capsules, coated tablets, or tablets containing a predetermined amount of the active pharmaceutical ingredient; can be in powder or granular form; can be in an aqueous or non-aqueous liquid or suspension; or can be a water-in-oil liquid emulsion or an oil-in-water liquid emulsion. The active ingredient can also be administered as a bolus, an ointment, or a paste. In certain embodiments, the active ingredient can be administered by subcutaneous injection.

[0095] Tablets can be made by compression or molding, optionally containing one or more accessory ingredients. Compressed tablets can be made by compressing the active ingredient in a free-flowing form, such as a powder or granules, in a suitable machine, and then optionally mixing with a binder, a lubricant, an inert diluent, a preservative, or a surfactant. Molded tablets can be made by mixing the active pharmaceutical ingredient powder with an inert liquid diluent in a suitable machine. Tablets can be optionally coated or scored and can be formulated in a sustained-release or controlled-release form to provide slow release of the active ingredient.

[0096] The active ingredient can be administered by a route suitable for the treatment condition. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), etc. It should be understood that the preferred route of administration may vary depending on the condition of the recipient. In certain embodiments, the active ingredient has high oral bioavailability and thus can be administered orally. In an embodiment, the patient is human.

[0097] Drug ingredient

[0098] The drug ingredient disclosed in the present invention includes one or more effective doses of antibodies.

[0099] For oral use, for example, tablets, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft gelatin capsules, syrups, or elixirs can be prepared. The compositions for oral use can be prepared by any method known in the pharmaceutical art, and such compositions can contain one or more excipients, including sweetening agents, flavoring agents, coloring agents, and preservatives, to provide a palatable preparation. Tablets containing the active ingredient admixed with non-toxic, pharmaceutically acceptable excipients are acceptable. These excipients can be, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, lactose monohydrate, crospovidone sodium, polyvinylpyrrolidone, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as cellulose, microcrystalline cellulose, starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets can be uncoated or can be coated by known techniques (including microencapsulation techniques) to delay disintegration and absorption in the gastrointestinal tract and thus provide a longer-lasting effect. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used, alone or in combination with waxes.

[0100] The preparations for oral use can also be presented in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium phosphate or kaolin, or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oily medium such as peanut oil, liquid paraffin, or olive oil.

[0101] The aqueous suspensions disclosed by the present invention contain an active substance mixed with excipients suitable for preparing aqueous suspensions. These excipients include suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, gum arabic and tragacanth; and dispersing agents or wetting agents such as naturally occurring phospholipids (such as lecithin), condensation products of fatty acids and alkylene oxides (such as polyoxyethylene stearate), condensation products of ethylene glycol and long-chain fatty alcohols (such as heptadecaoxyethanol), and condensation products of partial esters of fatty acids and hexitol (such as polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives such as ethyl or propyl p-hydroxybenzoate, and one or more colorants, flavoring agents and sweetening agents (such as sucrose or saccharin).

[0102] The oily suspensions can be formulated by suspending the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin). A thickening agent such as beeswax, hard paraffin or cetyl alcohol can be added to the oily suspensions. Sweetening agents and flavoring agents can also be added to provide a palatable oral preparation. These compositions can be preserved by adding an antioxidant (such as ascorbic acid).

[0103] The dispersion powders and granules disclosed by the present invention are suitable for preparing aqueous suspensions by adding water, providing a composition in which the active ingredient is mixed with a dispersing agent, a wetting agent, a suspending agent and one or more preservatives. Suitable dispersing agents and suspending agents are as described above. In addition, excipients such as sweetening agents, flavoring agents and colorants may also be present.

[0104] The pharmaceutical ingredients disclosed by the present invention may also be in the form of water-in-oil emulsions. The oil phase can be a vegetable oil (such as olive oil or peanut oil), a mineral oil (such as liquid paraffin) or a mixture thereof. Suitable emulsifying agents include natural gums (such as gum arabic and tragacanth), naturally occurring phospholipids (such as soybean lecithin), esters or partial esters of fatty acids and hexitol (such as sorbitan monooleate), and condensation products of these partial esters and ethylene oxide (such as polyoxyethylene sorbitan monooleate). Sweetening agents and flavoring agents can also be included in the emulsions. Syrups and elixirs can be formulated with sweetening agents such as glycerol, sorbitol or sucrose. In addition, these preparations may also contain mucosal protectants, preservatives, flavoring agents or colorants.

[0105] The pharmaceutical compositions disclosed by the present invention may be sterile injectable preparations, such as aqueous or oleaginous suspensions for sterile injection. Such suspensions may be formulated according to known techniques using the appropriate dispersing or wetting agents and suspending agents mentioned above. The sterile injectable preparations may also be sterile injectable solutions or suspensions, formulated using non-toxic, acceptable injectable diluents or solvents (such as 1,3-butanediol solution), or prepared as lyophilized powders. Acceptable carriers and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any mild fixed oil may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid may also be used in injectable preparations.

[0106] The amount of the active ingredient combined with the carrier material in a single dosage form will vary depending on the host being treated and the particular mode of administration, such as oral administration or subcutaneous injection. For example, a human sustained-release formulation for oral administration may contain from about 1 to 1000 milligrams of the active substance, mixed with a suitable and convenient carrier material in a proportion that may account for about 5% to about 95% (weight: weight) of the total composition. The pharmaceutical composition may be prepared in dosage amounts that are easy to measure. For example, an aqueous solution suitable for intravenous infusion may contain from about 3 to 500 micrograms of the active ingredient per milliliter of solution, so that an appropriate volume of the solution may be infused at a rate of about 30 milliliters per hour. For subcutaneous injection formulations, administration is typically once every two to four weeks for two to four months.

[0107] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents.

[0108] These formulations may be presented in single-dose or multi-dose containers, such as sealed ampoules or vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. The injectable solutions and suspensions prepared temporarily are prepared from the sterile powders, granules, and tablets described above. Preferred unit-dose formulations are those that contain the active ingredient in the daily dose or daily unit sub-dose (or an appropriate fraction thereof) as described above.

[0109] In some embodiments, the antibodies disclosed by the present invention can be formulated in any suitable dosage form for appropriate modes of administration. In some embodiments, the provided methods include administering a pharmaceutical composition comprising one or more antibodies disclosed by the present invention and a pharmaceutically acceptable carrier or excipient. The combination formulations and / or treatment regimens herein include the antibodies disclosed by the present invention, used in combination with one or more pharmaceutically acceptable carriers or excipients, and optionally other therapeutic agents, whether existing or to be developed in the future, for treating and / or preventing infections. The combination formulations containing the active ingredients can exist in any form suitable for the intended mode of administration.

[0110] In some embodiments, the vaccines disclosed by the present invention can be formulated in any suitable dosage form for appropriate modes of administration. In some embodiments, the provided methods include administering a pharmaceutical composition comprising one or more vaccines disclosed by the present invention and a pharmaceutically acceptable carrier or excipient. The combination formulations and / or treatment regimens disclosed by the present invention include the vaccines disclosed by the present invention, used in combination with one or more pharmaceutically acceptable carriers or excipients, and optionally other therapeutic agents (whether existing or to be developed in the future), for treating and / or preventing infections. The combination formulations containing the active ingredients can exist in any form suitable for the intended mode of administration.

[0111] According to the technology disclosed in PCT / GB2005 / 001976 and PCT / CN2019 / 124433, the rag gene locus encoding the outer membrane protein of Porphyromonas gingivalis consists of two co-transcribed and independent genes, ragA and ragB. Data shows that there are four major different subtypes of the outer membrane protein encoded by the ragB gene. These four subtypes account for more than 95% of the clinically collected samples from 197 periodontal disease patients in a global collaboration. According to their RagB outer membrane proteins, the four subtypes of Porphyromonas gingivalis are named W50ragB protein, ThairagB protein, QMLragB protein, and 381ragB protein, respectively.

[0112] These four allotypic proteins have significant differences in sequence, and the similarity rate of pairwise alignment is only 42 - 59% ( Figure 6 ). RagA was found to have allotypic proteins as well as RagB. However, Cra4S1 is a polypeptide fragment near the N-terminus of the RagA protein encoded by the ragA gene and is a conserved domain, which means that the Cra4S1 polypeptide exists in all allotypic strains of Porphyromonas gingivalis.

[0113] In some embodiments, the present invention discloses the genetic modification sequence of the Porphyromonas gingivalis outer membrane protein RagB, and successfully completes the pilot-scale production of untagged RagB and Cra4S1 recombinant proteins (Example 1, Figures 1-5 and Figures 7-11).

[0114] Different RagB outer membrane protein subtypes each have their own unique antigenic properties. However, studies have found that polyclonal antibody serum produced by immunizing animals with a single recombinant RagB protein has obvious cross-reactions with other RagB protein isoforms under specific dilution conditions (Example 2).

[0115] Mouse monoclonal antibodies against four different RagB and Cra4S1 proteins have been prepared, some of which react not only with homologous proteins but also cross-react with proteins of other subtypes (Example 3).

[0116] Although the sequence similarity of RagB isoforms is only 42-59%, the cross-reactivity between different RagB proteins suggests the existence of common antigens or compatible epitopes. Are these cross-reactivity caused by linear protein sequences, two-dimensional planar structures and / or multidimensional structures? What is the molecular basis of these cross-reactivity? Where is the target antigen? Do monoclonal antibodies have cross-immune protection?

[0117] In an embodiment of the present invention, a method for establishing an artificial synthetic peptide library is provided. These peptide libraries contain peptide segments of five different subtypes of Porphyromonas gingivalis outer membrane proteins RagB and Cra4S1, which are used to screen the target antigens of monoclonal antibodies. The antigenic target is found by utilizing the immune reaction between monoclonal antibodies and peptide segments at different positions in the corresponding protein. In the embodiment, several monoclonal antibodies with cross-reaction characteristics have found corresponding antigenic targets in the peptide library. This important discovery has far-reaching significance for the production process and production cost of future product development. In the past, the idea was to use specific monoclonal antibodies to target homologous bacteria according to the different strains of Porphyromonas gingivalis, while the new method is to use an antibody product that can simultaneously target four subtypes of Porphyromonas gingivalis (Example 4).

[0118] More specifically, the present invention is based on the research methods and results of the pathogenic mechanism of Porphyromonas gingivalis, and clarifies the pathogenic mechanism theory of Porphyromonas gingivalis. In the embodiments, the heterophilic antigen characteristics and individual heterophilic antibodies of Porphyromonas gingivalis are considered to be the cause of the chronic persistence and multi-tissue and organ damage of the disease.

[0119] In the embodiments, the serological research results of clinical specimens indicate that the outer membrane proteins of Porphyromonas gingivalis (including RagB and Cra4S1) have heterophilic antigen characteristics. The research results show that the local lesions and systemic damages caused by Porphyromonas gingivalis infection are closely related to heterophilic antigens and / or heterophilic antibodies (Embodiment 5).

[0120] In the embodiments, the present invention also discloses an animal model for establishing Porphyromonas gingivalis infection. This animal model is used to evaluate the systemic damages caused by pathogenic bacteria infection and the treatment methods for Porphyromonas gingivalis infection.

[0121] The experimental results of the combined antibodies on the mouse reproductive model show broad significance and promote the development of preventive and therapeutic products for Porphyromonas gingivalis and its associated chronic systemic diseases. Particularly importantly, two monoclonal antibodies with adjacent antigen targets are discovered. These antibodies are respectively directed against different subtypes of bacteria and identify the positions and sequences of the antigen targets in each outer membrane protein. As the research in this field continues to expand, further indications for treatment are established, and the antibodies and their usage methods disclosed in the present invention open up a broad market prospect for preventive and / or therapeutic products for various diseases associated with periodontal diseases. Therefore, the present invention provides stable and efficient biological products, solves the treatment problems of different subtypes of pathogenic bacteria, simplifies the production process, reduces the production cost, and ultimately brings overall benefits to public health (Embodiment 6).

[0122] In the embodiments, the present invention discloses the nucleotide and protein sequences of the mouse monoclonal antibody genes generated by immunizing with the outer membrane protein RagB of Porphyromonas gingivalis. The recombinant antibody plasmids constructed using these antibody sequences are successfully expressed in eukaryotic cells. They retain the ability of the original monoclonal antibodies to bind antigens, providing a molecular basis for the development of therapeutic drugs and the industrial production of products.

[0123] The present invention further provides a humanized mutation scheme for one or more monoclonal antibodies and a large-scale production process for these antibodies (Embodiment 7).

[0124] In the embodiments, the present invention provides two specific monoclonal antibodies, whose antigen targets are closely linked and belong to the same subtype of Porphyromonas gingivalis. Importantly, both of these antibodies exhibit cross-reactivity with different isoform outer membrane proteins, and the polypeptide sequences and positions of these cross-antigens on the outer membrane proteins are different. This discovery greatly simplifies the production process and effectively improves the production efficiency of the products.

[0125] In an embodiment, the present invention discloses the design and operation of bispecific antibodies against different subtypes of Porphyromonas gingivalis, successfully constructs recombinant antibody plasmids using the designed antibody sequences, and successfully expresses them in eukaryotic cells. The data presented in the present invention indicate that the recombinant bispecific antibodies not only retain the antigen-binding capabilities of the original different monoclonal antibodies but also compensate for the deficiencies of the original monoclonal antibodies. These data establish the feasibility and potential for the development of bispecific antibody products (Example 8).

[0126] In an embodiment, the present invention also discloses a diagnostic kit for diagnosing, treating, and predicting diseases associated with Porphyromonas gingivalis infection, including but not limited to periodontal diseases, cardiovascular diseases, rheumatoid arthritis, oral and digestive tract cancers, ulcerative colitis, neurological diseases, autoimmune encephalomyelitis, lung cancer, adverse pregnancy, pancreatic cancer, diabetes, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease.

[0127] The present invention discloses diagnostic kit reagents for diagnosing, treating, and predicting diseases associated with Porphyromonas gingivalis infection. In some embodiments, the amino acid sequences of the diagnostic reagents are shown in SEQ ID NO.2, 4, 6, 8, and 10. The diagnostic kit also provides instructions for reacting with the reagent to detect specific antibodies in a biological sample, which includes but is not limited to body fluids such as blood, gingival crevicular fluid, urine, saliva, cerebrospinal fluid, pleural effusion, ascites, and amniotic fluid. The methods for preparing, using, and analyzing the results of the diagnostic kit are described in detail in Example 9 of the present invention.

[0128] The present invention also discloses a polypeptide vaccine that can mobilize the individual's active immune function. In an embodiment, the sequence of the polypeptide vaccine is determined by the target of the neutralizing antibody, retains the antigenic characteristics of the polypeptide vaccine, and removes irrelevant and harmful components. The polypeptide vaccine disclosed in the present invention reduces the side effects of commonly used vaccines, can be administered repeatedly, and provides efficient and long-lasting protection against Porphyromonas gingivalis infection.

[0129] The present invention further provides polypeptide vaccines and combination antibodies for active and passive immunization of infected animals. The methods for preparing, using, and the results of these polypeptide vaccines and / or combination antibodies are described in detail in Example 10 of the present invention.

[0130] The above generally describes various aspects of the present invention. The following examples describe some additional and / or more detailed aspects of the present invention. Although the various aspects of the present invention are presented in combination with the following examples and corresponding written descriptions and illustrations, the present invention is not limited to these descriptions. On the contrary, the meaning of this aspect is to cover all alternatives, modifications, and equivalent solutions within the spirit and scope of the present invention. Detailed Description of the Invention

[0131] The following examples are intended to provide a complete illustration and description to those of ordinary skill in the art on how the compounds, compositions, materials, setups, and / or methods claimed herein are made and evaluated. The content disclosed in these examples is not limited to the scope of the inventors' disclosure. Efforts have been made to ensure the accuracy of numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be taken into account. Unless otherwise specified, the components are by weight, the temperature is in °C or at ambient temperature, and the pressure is at or near atmospheric pressure.

[0132] Example 1 Expression of RagB and Cra4S1 in Escherichia coli

[0133] The outer membrane protein RagB of Porphyromonas gingivalis mainly exists in four isoforms in clinical periodontal samples. These four isoforms of the outer membrane protein were previously named W50RagB, ThaiRagB, QMLRagB, and 381RagB.

[0134] In the present invention, the names of the four RagB subtypes are simplified. W50RagB is simplified to RagB-1, ThaiRagB is simplified to RagB-2, QMLRagB is simplified to RagB-3, and 381RagB is simplified to RagB-4. As Figure 6 shown in Table 1, the protein sequence similarity between RagB isoforms is only 42%-57%. Among them, the similarity between the RagB-1 and RagB-2 sequences is 47%, the similarity between RagB-1 and RagB-3 is 57%, the similarity between RagB-1 and RagB-4 is 49%, the similarity between RagB-2 and RagB-3 is 45%, the similarity between RagB-2 and RagB-4 is 42%, and the similarity between RagB-3 and RagB-4 is 49%.

[0135] The above four different outer membrane proteins (RagBs) of Porphyromonas gingivalis and the Cra4S1 protein-encoding genes, as well as the recombinant plasmids constructed by vectors, were all transfected into the host Escherichia coli (E. coli). After induction, the recombinant proteins were all successfully expressed.

[0136] Table 1, Multiple Sequence Alignment of RagB

[0137]

[0138]

[0139] The original nucleotide sequence of the target protein was modified according to the codon preference of the host Escherichia coli. After nucleotide sequence optimization, the encoded protein sequence was consistent with the original target protein, thus enabling accurate expression of the target protein.

[0140] Specifically, the nucleic acid sequences and amino acid sequences of artificially synthesized codon-optimized RagB-1, RagB-2, RagB-3, RagB-4, and Cra4S1 are shown in SEQ ID NO: 1-10 and Figure 1A-1B (specifically for RagB-1, SEQ ID NO: 1-2), Figure 2A-2B (specifically for RagB-2, SEQ ID NO: 3-4), Figure 3A-3B (specifically for RagB-3, SEQ ID NO: 5-6), Figure 4A-4B (specifically for RagB-4, SEQ ID NO: 7-8), and Figure 5A-5B (specifically for Cra4S1, SEQ ID NO: 9-10).

[0141] Table 2. Expression of Recombinant Outer Membrane Proteins in Escherichia coli

[0142] Recombinant Protein Name RagB-1 RagB-2 RagB-3 RagB-4 Cra4S1 Gene Length(bp) 1461 1455 1470 1455 411 Cloning Strategy NdeI-HindIII NdeI-HindIII NdeI-HindIII NdeI-HindIII NdeI-HindIII Resistant Antibiotic Ampicillin / Kanamycin Kanamycin Kanamycin Kanamycin Ampicillin Kanamycin Expected MW(kDa) ～54.40 ～55 ～55 ～55 ～13.67 CRO GenScrpt,USA DetaBio,China DetaBio,China DetaBio,China GenScrpt,USA Contract Number C7997FI160 DT1872 DT1868 DT1780 C0904FK060

[0143] Five outer membrane proteins of Porphyromonas gingivalis, including RagB-1 (W50 RagB), RagB-2 (ThaiRagB), RagB-3 (QML RagB), RagB-4 (381 RagB), and Cra4S1, were successfully expressed in the host Escherichia coli. Specifically, after the plasmid was successfully constructed, the cloned gene (including the optimized nucleotide sequence) was confirmed by sequencing to ensure the accuracy of the protein sequence. After the plasmid was transformed into Escherichia coli, the expression of the recombinant protein was promoted by induction. The bacteria expressing the recombinant protein were collected by centrifugation to obtain the precipitate, and the bacterial paste was broken by mechanical method. Then, the supernatant was purified by ion exchange chromatography to obtain the target protein. All recombinant proteins were tag-free and soluble proteins (see Table 2).

[0144] The pilot-scale production of the above five tag-free recombinant proteins was completed by a third-party contract research organization (CRO) company (GenScript USA, contract number C9458FG140). Specifically, the recombinant plasmid and / or seed bacteria were sent to the CRO, and after confirmation and identification, fermentation production was carried out by it. During the high-density fermentation process, the culture medium did not contain animal-derived proteins and antibiotics. After fermentation, the protein was purified by five-step chromatography. Each of the five recombinant protein products was accompanied by a certificate of analysis. Figure 7A-7B (specifically for RagB-1), Figure 8A-8B (specifically for RagB-2), Figure 9A-9B (specifically for RagB-3), Figure 10A-10B (specifically for RagB-4), and Figure 11A-11B (specifically for Cra4S1) show the induction expression and purification results of the five tag-free target proteins.

[0145] Table 3, Pilot-scale production of recombinant protein

[0146] Plasmid Name Contract Number Production batch Mw(kDa) Concentration Purity Endotoxin detection Certificate of Analysis Cra4S1 C9458FG140-1 Cra4S1F210321FE 13KDa 6.0mg / ml 98％ N / A Yes RagB-1 C9458FG140-2 RagB-1F201020F101 54.1KDa 3.1mg / ml 98％ <1EUmg Yes RagB-2 C9458FG140-3 RagB-2F200815F1-01-01 51.5KDa 3.4mg / ml 93％ <1EUmg Yes RagB-3 C9458FG140-4 RagB-3F200815F2-01 51.3KDa 10.7mg / ml 96％ <1EUmg Yes RagB-4 C9458FG140-5 RagB-4F201103F2 49.8KDa 1.0mg / ml 99％ <1EUmg Yes

[0147] Pilot-scale production of 5 different outer membrane proteins of Porphyromonas gingivalis has been successfully achieved, marking the feasibility of product industrialization.

[0148] Example 2: Cross-reactivity of polyclonal antibodies against outer membrane proteins of Porphyromonas gingivalis

[0149] Although the protein sequences of different subtypes of RagB of Porphyromonas gingivalis are different, in animal serological studies, it was found that the antibodies cross-react with RagB proteins of different subtypes. Therefore, a step for cross-reaction of antibodies against different RagB subtypes was added to the detection experimental design. In this study, the enzyme-linked immunosorbent assay (ELISA) method was mainly used to detect the different affinities of antibodies for antigens.

[0150] Specifically, the preparation methods of mouse and rabbit antibody sera are briefly described as follows: Immunize mice and rabbits with antigen proteins (RagB / Cra4S1) three to four times, and determine the titers of specific antibodies in the sera by the indirect ELISA method. When the antibody level reaches the predetermined standard, collect the whole blood samples of the animals, incubate and centrifuge to obtain sera, and store them frozen.

[0151] ELISA is an enzyme-linked immunosorbent assay. Its basic principle is to immobilize a certain concentration of antigen on the surface of a polystyrene microplate through physical adsorption, add diluted serum (host antibody) to the reaction plate, leave enough time for the antigen and antibody to bind, and then wash away the unbound free antibody molecules. Then, add an enzyme-linked secondary antibody (anti-host antibody), and wash away the free molecules again. Finally, add an enzyme substrate solution, leave a certain time for the enzymatic reaction, and indirectly reflect the qualitative and semi-quantitative characteristics of the substrate by measuring the color reaction of the substrate. The specific steps are as follows:

[0152] 1. Coating antigen: Prepare a protein (antigen) solution with PBS at a concentration of 4 μg / ml, add 100 μl of the antigen solution to each well, seal the plate, incubate overnight at room temperature (about 20 °C), wash the plate 4 times with PBS buffer, 300 μl / well, pat dry the residual buffer in the wells with absorbent paper, add 100 μl of blocking buffer (1% BSA in PBS) to each well and incubate at room temperature (20 °C) for 2 hours, wash the plate 4 times with PBS buffer, 300 μl / well, pat dry the residual buffer in the wells with absorbent paper. The coated ELISA plate can be stored in a -20 °C freezer for 4 - 6 weeks.

[0153] 2. Add primary antibody: Dilute the cell culture supernatant or serum antibody with sample dilution buffer (1% BSA in PBS) to the required working concentration. Add 100 μl of the primary antibody solution to each well, seal the plate, incubate at 37 °C for 60 - 90 minutes, wash the plate 4 times with PBS buffer, 300 μl / well, and pat dry the residual buffer in the wells on absorbent paper.

[0154] 3. Add enzyme-labeled secondary antibody: Dilute the secondary antibody with sample dilution buffer according to the manufacturer's instructions. Add 100 μl of the enzyme-labeled secondary antibody to each well, incubate at 37 °C for 40 - 60 minutes, wash the plate 3 times with PBS buffer, 300 μl / well, pat dry the residual buffer in the wells on absorbent paper, and finally wash the plate with deionized water, 300 μl / well, and pat dry the residual liquid in the wells on absorbent paper.

[0155] 4. Reaction and color development: Prepare the substrate solution; dissolve disodium 4-nitrophenyl phosphate hexahydrate in 1 M Tris-MgCl2, 1 mg / ml. Add 100 μl of the substrate solution to each well, incubate at room temperature in the dark for 50 - 180 minutes, set the dual-wavelength (405 nm and 630 nm) program on the microplate reader, place the microplate reader on the preheated one to read the values, save the data, and perform analysis.

[0156] In the traditional ELISA method, many different detection systems are available for selection. In this example, an alkaline phosphatase-conjugated secondary antibody is selected for enzyme labeling because of its long stability and low false positive error. Specifically, for the ELISA plate numbered 17003 shown in Table 4, the antigen layout described here is as follows: Columns 1 and 7 are coated with RagB-1 recombinant protein, columns 2 and 8 are coated with RagB-2, columns 3 and 9 are coated with RagB-3, columns 4 and 10 are coated with RagB-4, columns 5 and 11 are coated with Cra4S1 recombinant protein, and columns 6 and 12 are coated with GST recombinant protein as a negative irrelevant protein control. The sera of the experimental group and the control group are both diluted 1:2000, and each serum sample has the opportunity to detect 6 wells of antigens, including RagB-1, RagB-2, RagB-3, RagB-4, Cra4S1, and GST. The optical density (OD) values of the ELISA plate numbered 17003 are shown in Table 4, with PBS and normal serum as negative controls.

[0157] Table 4. Detection of polyclonal antibodies in animals infected with Porphyromonas gingivalis

[0158]

[0159] ELISA results showed that antibodies against the corresponding outer membrane proteins could be detected in the sera of animals infected with homologous strains of Porphyromonas gingivalis. For example, among the mice infected with the RagB-2 subtype of Porphyromonas gingivalis in Table 5, there were a total of 8 mice (G6-1, G6-2, G7-1, G7-2, G8-1, G8-2, G8-3, and G8-4). Their sera reacted with the RagB-2 recombinant protein, confirming that the RagB-2 protein is a major antigen on the bacterial surface. The results also showed that the more times of infection, the higher the corresponding antibody level. For example, 4 mice in group G8 (G8-1, G8-2, G8-3, G8-4) were only infected with Porphyromonas gingivalis once, while the other 4 mice (G6-1, G6-2, G7-1, G7-2) were infected with Porphyromonas gingivalis 3 times. The OD values of antibody detection levels in mice with multiple infections were higher than those in mice with only one infection. In addition, animals with a history of Porphyromonas gingivalis infection produced high-titer specific antibodies after inoculation with the RagB-2 protein (Table 4 and Table 5, G6-3, G6-4, G7-3, and G7-4). Compared with animals receiving only the vaccination protocol, the OD values of antibodies in animals with prior bacterial infection were significantly increased after vaccination. This phenomenon indicates that bacterial infection renders animals in an immune-sensitive state, suggesting that the vaccine product has an immune-promoting function for patients with a history of previous infection.

[0160] Table 5, Information of experimental animals

[0161] Received RagB-2Vaccine Number of attacks of P.gingivalis RagB-2 ♂G6-1 No 3 ♂G6-2 No 3 ♂G6-3 Yes 3 ♂G6-4 Yes 3 ♂G7-1 No 3 ♂G7-2 No 3 ♂G7-3 Yes 3 ♂G7-4 Yes 3 ♂G8-1 No 1 ♂G8-2 No 1 ♂G8-3 No 1 ♂G8-4 No 1

[0162] The results showed that animals pre-infected with Porphyromonas gingivalis RagB-2 produced not only a strong immune response of serum antibodies against RagB-2 but also cross-reactions against RagB proteins of different subtypes (including RagB-1, RagB-3, and RagB-4) and a weak but visible cross-reaction against the Cra4S1 protein after inoculation with the RagB-2 protein (G6-3, G6-4, G7-3, and G7-4), and no reaction against the negative control GST protein.

[0163] Table 6, Detection of polyclonal antibodies in animals infected with Porphyromonas gingivalis

[0164]

[0165] Table 7, Information of experimental animals

[0166] Received RagB-3 Vaccine Number of attacks of P.gingivalis RagB-3 ♂G9-1 No 3 ♂G9-2 No 3 ♂G9-3 Yes 3 ♂G9-4 Yes 3 ♂G11-1 No 3 ♂G11-2 No 3 ♂G11-3 Yes 3 ♂G11-4 Yes 3 ♂G12-1 No 1 ♂G12-2 No 1 ♂G12-3 No 1 ♂G12-4 No 1

[0167] Similarly, animals with prior infection also produced high-intensity specific antibodies after inoculation with the RagB-3 protein, and the serum antibodies had cross-reactions with RagB-1, RagB-2, RagB-4, and Cra4S1 (Table 6 and Table 7).

[0168] This example demonstrated that recombinant outer membrane proteins of Porphyromonas gingivalis, including RagB and Cra4S1, have good antigenicity and can stimulate animals to produce high-titer antibodies. In addition, cross-reactivity of polyclonal serum antibodies with other subtype RagB proteins was also observed. Although there are significant differences in the gene sequences and protein sequences of the outer membrane protein RagB of Porphyromonas gingivalis among different subtypes, they are homologous alleles. These results suggest that there may be common antigens or compatible antigenic determinants among Porphyromonas gingivalis subtypes. In particular, the antibodies produced by immunization with RagB protein can cross-react with Cra4S1 protein, and the results are surprising and unexpected.

[0169] Example 3: Cross-reactivity of monoclonal antibodies against RagB

[0170] Monoclonal antibodies against the outer membrane proteins of Porphyromonas gingivalis, including RagB and Cra4S1 proteins, were prepared by a third-party CRO (Contract Research Organization) company respectively. Specifically, the general process of preparing mouse hybridoma cells to obtain monoclonal antibodies is briefly described as follows: First, animals were immunized five times with the antigen protein at intervals of 1-2 weeks each. Mouse spleen cells were fused with well-growing SP2 / 0 myeloma cells, and the fused cells were allowed to grow in the medium for seven days. Then, positive clones were screened with the immunizing antigen and further subcloned three times to confirm that the positive cell line continuously and stably produced antibodies. Finally, the hybridoma seed cell line was stored in liquid nitrogen.

[0171] Table 8, RagB hybridoma cell lines

[0172]

[0173] Table 9, Cra4S1 hybridoma cell lines

[0174]

[0175] Five to ten monoclonal antibody-positive hybridoma cell lines were provided for each antigen item (Tables 8 and 9). Monoclonal antibodies were mainly purified from cell culture supernatants and ascites produced after implantation of mouse hybridoma cells into the abdominal cavity. The immunoglobulin subclasses of the purified monoclonal antibodies were detected, and positive results were marked as "P" in the table.

[0176] Table 10, Cross-antigen detection of monoclonal antibodies

[0177]

[0178] Table 11, Cross-antigen detection of monoclonal antibodies

[0179]

[0180] Tables 10 and 11 show the original results of the cross-reaction of monoclonal antibodies with the recombinant RagB protein. The ELISA operation was as described in Example 2. The antigen adsorption plate was designed as follows: Columns 1, 5, and 9 adsorbed the RagB-1 recombinant protein; Columns 2, 6, and 10 adsorbed RagB-2; Columns 3, 7, and 11 adsorbed RagB-3; Columns 4, 8, and 12 adsorbed RagB-4. The antibodies were from hybridoma culture supernatants or purified antibodies, and the dilution factors were 1:1000 to 1:4000. Each antibody sample was able to react with the four-well antigen proteins RagB-1, RagB-2, RagB-3, and RagB-4.

[0181] Compared with the normal serum negative control group, if the OD reading value exceeded twice that of the control group, it was defined as positive. The data presented in this example indicate that all the tested monoclonal antibodies showed positive immunoreactivity against the antigen, except for one hybridoma (well B5-8 listed in Table 11, cell name RagB-4-1C11-1-5 showed a negative reaction). In addition, several hybridoma antibodies were identified that not only reacted with the corresponding RagB protein but also had cross-reactivity with other subtypes of the RagB protein.

[0182] For example, in Table 10, RagB-2-1A4-3-7-7 at plate positions G1-G4 was a specific monoclonal antibody against the RagB-2 protein but also showed cross-reactivity with the RagB-1 protein; RagB-3-1D2-2-1-3 at plate positions E5-E8 was a specific monoclonal antibody against the RagB-3 protein but also showed cross-reactivity with the RagB-1 and RagB-2 proteins. In Table 11, the 1B11-4-4 antibody and the 1C3-7-8 antibody at positions H1-H4 and A5-A8 were specific monoclonal antibodies against the RagB-4 protein, but they showed cross-reactivity with the RagB-1, RagB-2, and RagB-3 proteins, respectively.

[0183] Since the nucleic acid and protein sequence differences between the RagB subtypes of Porphyromonas gingivalis are significant and there is no obvious continuous common sequence in pairwise alignments, previous bacterial immunotherapy focused on precision, first identifying the subtype and then selecting the corresponding specific monoclonal antibody. However, this example discloses some monoclonal antibodies that can cross-react with different subtypes of the RagB protein, indicating that different subtypes of Porphyromonas gingivalis share common targets or compatible epitopes. Then, what exactly are these common targets and / or compatible epitopes? Where are they located?

[0184] Example 4: Searching for the targets of monoclonal antibodies in a peptide library

[0185] To determine the antigenicity of the outer membrane protein RagB of Porphyromonas gingivalis and the antigen targets that bind to monoclonal antibodies, five peptide libraries against RagB-1, RagB-2, RagB-3, RagB-4, and Cra4S1 were established. The synthetic peptides were produced by a third-party CRO company (GenScript, USA). Specifically, based on the amino acid sequence of each protein, starting from the N-terminus, a peptide chain of 18-25 amino acids was artificially synthesized. The next peptide chain would contain five amino acids from the C-terminus of the previous peptide chain, forming sequence overlap, and so on. Except for the peptides at both ends of each protein (each peptide chain has only a single overlapping sequence), all other peptide chains have five-amino-acid overlaps with the upstream and downstream peptide chains on both sides to avoid missing any antigenic determinants. The peptide sequences are shown in Tables 12-16. Each RagB subtype protein has 24 peptide chains. The peptides of RagB-1 are named RA plus a serial number, such as RA-1, RA-2, etc. The peptides of RagB-2 are named RB plus a serial number, the peptides of RagB-3 are named RC plus a serial number, and the peptides of RagB-4 are named RD plus a serial number. The peptides of Cra4S1 are named Cra and New Cra plus a serial number. Most of the artificially synthesized peptide chain fragments are soluble in ultrapure water, PBS, and / or DMSO. The peptide chain sequences and the physicochemical properties of the peptide chains are listed in Tables 12-16.

[0186] After the peptide libraries of the outer membrane proteins of Porphyromonas gingivalis were constructed, all monoclonal antibodies reacted with the fragments in the peptide libraries to search for the antigen-antibody binding targets and key sequence fragments. Specifically, according to the solubility of the peptides, a soluble solution of 1 mg / ml was prepared, and then the polypeptide solution was further diluted to 20 μg / ml and adsorbed onto ELISA plates. The subsequent antibody detection steps were as described in Example 2. Each monoclonal antibody reacted with the corresponding RagB subtype outer membrane protein polypeptide fragment. To avoid missing antigens and determine the binding positions, each synthetic peptide chain has overlapping parts with the upstream and downstream polypeptides, as described above.

[0187] Four RagB subtype peptide libraries were established to capture antigenic epitopes and antigen targets that cross-react with monoclonal antibodies. As described in Example 3, some monoclonal antibodies against specific RagB subtypes showed cross-reactivity with another RagB subtype. These antibodies were screened in the peptide libraries to localize the antibody-antigen targets.

[0188] Table 12, Peptide Library of RagB-1 (SEQ ID NO:2) (GenScript, USA, C4965GB230)

[0189]

[0190] Table 13, RagB-2 (SEQ ID NO:4) Peptide Library (GenScript, USA, C4965GB230)

[0191]

[0192] Table 14, RagB-3 (SEQ ID NO:6) Peptide Library (GenScript, USA, C4965GB230)

[0193]

[0194] Table 15, RagB-4 (SEQ ID NO:8) Peptide Library (GenScript, USA, C4965GB230)

[0195]

[0196] Table 16, RagB-4 (SEQ ID NO:10) Peptide Library

[0197]

[0198] The antigen-antibody binding reaction was determined by the OD value of the indirect ELISA experiment. An OD value higher than twice that of the negative serum control was considered a positive result.

[0199] Table 17 shows the peptide library of RagB-1 (SEQ ID NO:2) and the situation of monoclonal antibodies capturing antigen targets, including monoclonal antibodies against RagB-1 and monoclonal antibodies recognized as reacting with other RagB subtypes. ELISA results showed that 3 hybridoma antibodies against RagB-1 detected peptide antigen targets. For example, monoclonal antibodies 1A1-7-7 and 1A3-3-8 reacted with the RA-1 peptide chain, and 1E3-4-7-1 reacted with the RA-9 peptide chain (Table 17). The mouse serum of polyclonal antibodies produced by inoculating RagB-1 protein was named 2019-G85-1 serum (P), which reacted with the RA-1 peptide chain. Interestingly, monoclonal antibodies of other subtypes cross-reacted with RagB-1 protein, and target antigens were also found in the RagB-1 peptide library. For example, 1A4-3-7-7 (specific for RagB-2 protein) bound to the RA-19 peptide chain; 1D2-2-1-3 (specific for RagB-3 protein) bound to the RA-17 peptide chain, 2C1-4-6-2 (specific for RagB-3 protein) bound to RA-9 and RA-17, 2B7-4-6-7 (specific for RagB-3 protein) bound to RA-9; while 1B11-4-4 (specific for RagB-4 protein) bound to the RA-21 peptide chain.

[0200] Table 17, Screening of Peptide Library of RagB-1 (SEQ ID NO:2) for Target Antigens

[0201]

[0202] Table 18 shows the peptide library of RagB-2 (SEQ ID NO:4) and the capture of antigen targets by monoclonal antibodies, including monoclonal antibodies against RagB-2 and monoclonal antibodies recognized as reacting with other RagB subtypes. ELISA results showed that 3 hybridoma antibodies against RagB-2 detected peptide antigen targets. For example, monoclonal antibodies 1A4-3-7-7, 1A6-3-3, and 1B3-1-5 reacted with peptide chains RB-18, RB-21, and RB-7 respectively (Table 18). The mouse serum of polyclonal antibodies produced by inoculation with RagB-2 protein was named 2019-G65-1, which reacted with peptide chain RB-17.

[0203] Monoclonal antibodies that cross-react with RagB-2 protein also found peptide targets in the RagB-2 peptide library. For example, 1D2-2-1-3 (specific for RagB-3 protein) bound to peptide chain RB-17, while 1C3-7-8 (specific for RagB-4 protein) bound to peptide chain RB-21.

[0204] Table 18, Screening of Peptide Library of RagB-2 (SEQ ID NO:4) for Target Antigens

[0205]

[0206] Table 19, Screening of Peptide Library of RagB-3 (SEQ ID NO:6) for Target Antigens

[0207]

[0208] Table 20, Screening of Peptide Library of RagB-3 (SEQ ID NO:6) for Target Antigens

[0209]

[0210] Tables 19 and 20 show the peptide library of RagB-3 (SEQ ID NO:6) and the capture of antigen targets by monoclonal antibodies, including monoclonal antibodies against RagB-3 and monoclonal antibodies recognized as reacting with other RagB subtypes. ELISA results showed that almost all tested RagB-3 hybridoma antibodies detected peptide antigen targets, and 15 out of 16 hybridoma antibodies reacted with the peptide chains in the RagB-3 peptide library.

[0211] Surprisingly, the RagB-3 peptide library data also showed that several monoclonal antibodies reacted with multiple peptide chains at different positions. In addition, four different mouse polyclonal antibodies generated by a single protein vaccine (Table 20, 2019-G75-2) and multivalent RagB vaccines (Table 20, 2019-G160-2, 2019-G160-4, and 2019-G250-3) failed to successfully detect a clear polypeptide target in the peptide library. The 1D2-2-1-3 recombinant plasmid antibody showed a highly specific reaction with the RC-17 peptide chain, but no reaction with other polypeptide fragments was observed.

[0212] In the RagB-3 peptide library, certain polypeptide targets also interacted with the tested monoclonal antibodies. For example, the 1B11-4-4 antibody specific for the RagB-4 protein was found to react with the RC-21 peptide, and the 1C3-7-8 antibody specific for the RagB-4 protein reacted with the RC-22 peptide chain.

[0213] Table 21, RagB-4 (SEQ ID NO:8) Peptide Library Screening of Target Antigens

[0214]

[0215] Table 21 shows the peptide library of RagB-4 (SEQ ID NO:8) and the situation of monoclonal antibodies capturing antigen targets, including monoclonal antibodies against RagB-4 and monoclonal antibodies recognized as reacting with other RagB subtypes. ELISA results showed that the hybridoma antibodies against RagB-4 detected polypeptide antigen targets, and 6 out of 7 hybridoma antibodies reacted with the polypeptides in the RagB-4 peptide library. Two RagB-3 monoclonal antibodies, 1E4-6-1-7 and 1H11-2-2-4, cross-reacted with the RagB-3 protein, but no cross-reaction of these antibodies with RagB-4 polypeptide fragments was found. The mouse serum from which polyclonal antibodies were generated by inoculating RagB-4 protein was named G90 serum (P) and reacted with the RD-1 peptide chain.

[0216] In the antigen target capture study of the Cra4S1 monoclonal antibody, the culture supernatants of more than 40 positive hybridoma cell lines were tested to screen for antigen targets. Although these antibodies had a high-titer reaction with the recombinant protein, they did not react with the synthetic peptide antigens created in the Cra4S1 peptide library. Only a few clones showed a weak antigen-antibody reaction. Table 16 provides two peptide libraries of Cra4S1. After adjusting the length and position of the peptide chain, no antigen-antibody binding reaction was detected.

[0217] As mentioned above, several monoclonal antibodies showed cross-reactivity with another RagB protein and its fragments in the peptide library. The important cross-antigen targets are as follows:

[0218] 1. Binding sites of monoclonal antibody RagB-2-1A4-3-7-7 to polypeptides:

[0219] RA-19: and / or

[0220] RB-18:

[0221] Monoclonal antibody 1A4-3-7-7 specifically reacts with RagB-2 protein and reacts with RA-19 and RB-18 peptide chains. The underlined amino acids are the parts where the two sequences are exactly the same, indicating that the specific antigen-binding site of the RagB-2-1A4-3-7-7 monoclonal antibody is located in these prominent epitopes in RA-19 (SEQ ID NO:29) and / or RB-18 (SEQ ID NO:52).

[0222] 2. Binding sites of monoclonal antibody RagB-3-1D2-2-1-3 to polypeptides (1):

[0223] RA-17&18: and / or

[0224] RC-17&18:

[0225] Binding sites of monoclonal antibody RagB-3-1D2-2-1-3 to polypeptides (2):

[0226] RB-17: and / or

[0227] RC-17&18:

[0228] The specific antigen-binding site of the RagB-3-1D2-2-1-3 monoclonal antibody is located in RA-17&18 (SEQ ID NO:123) and / or RB-17 (SEQ ID NO:51), and / or RC-17&18 (SEQ ID NO:124). The underlined amino acids are the parts where the two sequences are exactly the same, indicating that the RagB-3-1D2-2-1-3 monoclonal antibody has cross-reactivity with two other RagB subtypes, but the epitopes and lengths of the antibody-antigen binding sites are not the same.

[0229] 3. Binding sites of monoclonal antibody RagB-4-1B11-4-4 to polypeptides (1):

[0230] RA-21: and / or

[0231] RD-21:

[0232] Binding site (2) of monoclonal antibody RagB-4-1B11-4-4 to polypeptide:

[0233] RB-21: and / or

[0234] RD-21:

[0235] Binding site (3) of monoclonal antibody RagB-4-1B11-4-4 to polypeptide:

[0236] RC-21: and / or

[0237] RD-21:

[0238] The specific antigen-binding site of the RagB-4-1B11-4-4 monoclonal antibody is located in RA-21 (SEQ ID NO:31), RB-21 (SEQ ID NO:55), RC-21 (SEQ ID NO:79), and RD-21 (SEQ ID NO:103). The underlined amino acids are the parts where the two sequences are exactly the same, indicating that the RagB-4-1B11-4-4 monoclonal antibody shows cross-reactivity to the other three RagB subtypes, but the epitopes and lengths of the antibody-antigen binding sites are not the same.

[0239] 4. Binding site (1) of monoclonal antibody RagB-4-1C3-7-8 to polypeptide:

[0240] RA-21&22: and / or

[0241] RD-21&22:

[0242] Binding site (2) of monoclonal antibody RagB-4-1C3-7-8 to polypeptide

[0243] RB-21: and / or

[0244] RD-21:

[0245] Binding site (3) of monoclonal antibody RagB-4-1C3-7-8 to polypeptide

[0246] RC-22: and / or

[0247] RD-21&22:

[0248] The specific antigen-binding sites of the RagB-4-1C3-7-8 monoclonal antibody are located in RA-21&22 (SEQ ID NO: 125), RB-21 (SEQ ID NO: 55), RC-22 (SEQ ID NO: 80), and RD-21&22 (SEQ ID NO: 126). The underlined amino acids are the parts where the two sequences are exactly the same, indicating that the monoclonal antibody RagB-4-1C3-7-8 exhibits cross-reactivity to the other three RagB subtypes, but the epitopes and lengths of the antibody-antigen binding sites are not the same.

[0249] It is surprising and unexpected to find polyclonal antibodies that can cross-react with RagB proteins of different subtypes, as well as monoclonal antibodies with multiple antigen targets. Two monoclonal antibodies with multiple cross-reactivities, 1B11-4-4 (SEQ ID NO: 148-149 and SEQ ID NO: 153-154) and 1C3-7-8 (SEQ ID NO: 158-159 and SEQ ID NO: 163-164), obtained from immunization with RagB-4, have data confirming that their antigen targets are located in the RD-21 (SEQ ID NO: 103) and RD-21&22 (SEQ ID NO: 126) regions. Although the positions of their antigen targets are close to each other, each antigen target has its unique position and sequence. Importantly, they share common antigens with antigenic determinants at different positions in RagB-1, RagB-2, and RagB-3 proteins.

[0250] In summary, the antigen-antibody reaction of the monoclonal antibody with the peptide chains in the RagB peptide library confirmed the antigenicity of the RagB polypeptide fragments, providing a molecular basis for the development of antibodies and vaccines that can react with these antigenic fragments, and contributing to the development of therapeutic drugs for controlling diseases caused by Porphyromonas gingivalis infection. The monoclonal antibodies 1B11-4-4 (SEQ ID NO: 148-149 and SEQ ID NO: 153-154) and 1C3-7-8 (SEQ ID NO: 158-159 and SEQ ID NO: 163-164) have their respective antigen targets within the adjacent RD-21 (SEQ ID NO: 103) and RD-21&22 (SEQ ID NO: 126) peptide fragments, and they can both recognize antigens from four different RagB subtypes. The adjacent antigen targets provide ideal conditions for mixing monoclonal antibodies and preparing bispecific antibodies.

[0251] Example 5: Heterophilic Antigen and Pathogenic Mechanism of Porphyromonas gingivalis

[0252] In recent years, clinical studies and statistical surveys have shown that periodontal diseases are associated with a variety of systemic diseases, especially chronic diseases and tumors. Although extensive research has been carried out, it is not clear whether there is a direct association or pathogenic mechanism between Porphyromonas gingivalis and chronic diseases.

[0253] In Example 4, when screening for monoclonal antibodies against the Cra4S1 protein, although the antibodies showed a strong reaction with the full-length recombinant protein of Cra4S1, they failed to identify specific antigen target fragments of the protein. The polyclonal antibody serum of Cra4S1 also failed to reveal the major polypeptide targets in the Cra4S1 peptide library. These data suggest that there may be no significant dominant antigen fragments in Cra4S1, or the antigenicity of Cra4S1 is diffuse and presented only through its three-dimensional spatial structure. In addition, in Example 4, monoclonal antibodies against the RagB-3 protein identified multiple targeted antigen polypeptide sequence fragments in the RagB-3 peptide library. However, studies using polyclonal antibody serum failed to reveal the major peptide fragments and / or epitopes of RagB in the peptide library. Nevertheless, animals immunized against the RagB-1 and RagB-4 proteins and the resulting polyclonal antibodies were able to recognize antigen polypeptide fragments / targets RA-1 and RD-1 at the N-terminus of each peptide, respectively, while the RagB-2 polyclonal antibody was able to react with the peptide RB-17. Overall, these results indicate that the antigenic characteristics of different outer membrane protein subtypes of Porphyromonas gingivalis are not the same.

[0254] Reviewing the study of the Cra4S1 recombinant protein, Example 2 showed that the sera of animals infected with Porphyromonas gingivalis did not react with Cra4S1 but reacted with RagB, indicating that the antigenicity of the Cra4S1 protein may not be dominant. The data in Example 2 also showed that when the RagB protein was used to immunize animals with a history of bacterial infection, the serum antibodies reacted with Cra4S1 but not with the irrelevant protein GST negative control, indicating that the RagB antibody had a cross-reaction with the Cra4S1 protein. In some examples, it was found that the RagB-4-1B11-4-4 monoclonal antibody did have a cross-reaction with Cra4S1.

[0255] Surprisingly, when using human serum samples to detect antibodies against Porphyromonas gingivalis, a significant proportion of antibodies against RagB and Cra4S1 were detected in the specimens. Specifically, the human serum samples collected in this example included healthy individuals, cardiovascular disease patients, and geriatric patients. Tables 22, 26, and 29 list the ELISA results of these human serum samples, detecting antibodies against the outer membrane proteins of Porphyromonas gingivalis. The design of the antigen adsorption on the ELISA plate was the same as described in Example 2, and the antigen layout design is briefly described as follows: Column 1 and Column 7 adsorbed RagB-1 recombinant protein, Column 2 and Column 8 adsorbed RagB-2, Column 3 and Column 9 adsorbed RagB-3, Column 4 and Column 10 adsorbed RagB-4, Column 5 and Column 11 adsorbed Cra4S1, and Column 6 and Column 12 adsorbed GST recombinant protein as an irrelevant protein negative control.

[0256] The first group (healthy subjects) of these serum samples had a dilution of 1:300. Each serum sample was tested with 6-well antigen proteins, including RagB-1, RagB-2, RagB-3, RagB-4, Cra4S1, and GST. The secondary antibody was alkaline phosphatase-labeled mouse anti-human IgG for human samples and goat anti-mouse IgG for the control. Each ELISA plate was equipped with a PBS negative control, and the positive control (labeled M-PC) was from the immune mouse serum immunized with the pentavalent protein, and the vaccine components included RagB-1, RagB-2, RagB-3, RagB-4, and Cra4S1.

[0257] Since this was the first time to use human serum to detect antibodies against the outer membrane proteins of Porphyromonas gingivalis, it was pre-specified that the average OD value of at least one-fifth (at least 20%) of all samples was negative, and the initial cut-off value was set at 2.5 times this value. If the OD value exceeded 2.5 times this negative value, it was considered positive. Table 22 shows the ELISA results of the serum samples of 51 healthy subjects. Table 23 shows the lowest OD values of each group of proteins detected from 10 subjects (20% of the 51 samples). Table 23 also lists the number of samples with OD values 2.5 times higher than the negative OD value (considered positive results), as well as the data of the suspected positive subjects with OD values between 1.8 and 2.5 times the negative value. OD values exceeding four times (4 times) the negative value were considered strong positive results and quantitatively reflected higher protein titers.

[0258] The first group of human serum samples consisted of 51 healthy individuals (blood tests of young adults, numbered from H1 to H51). The positive rate of the RagB-1 antibody test was 11.8% (6 / 51), among which the strong positive rate was 2% (1 / 51), and its OD value exceeded 4 times that of the negative value; the positive rate of the RagB-2 antibody test was 0% (0 / 51); the positive rate of the RagB-3 antibody test was 2% (1 / 51); the positive rate of the RagB-4 antibody was 31.3% (16 / 51), among which the strong positive rate was 13.7% (7 / 51). Weak positive results (1.8 - 2.5 times the OD value of negative serum) accounted for 29.4% (15 / 51), indicating that RagB-4 antibody-positive samples accounted for a relatively large proportion of the total samples (15 / 51 weak positive, 9 / 51 positive, 7 / 51 strong positive). The positive rate of the Cra4S1 antibody was 9.8% (5 / 51), while the positive rate of the irrelevant protein GST antibody was 0% (0 / 51).

[0259] The detection rates of RagB-1 and Cra4S1 antibodies were lower than that of RagB-4, and some RagB-1 antibodies were positively correlated with RagB-4 antibodies, that is, when RagB-4 antibodies were detected, the higher the OD value, the higher the OD value of RagB-1 antibodies. The detection rates of RagB-2, RagB-3 and GST antibodies were very low ( Figure 12A-12B , Table 22, Table 23 and Table 24). In this group of human serum specimens, is it because the antigenicity of RagB-2 and RagB-3 subtypes is low, so they do not cause a significant antibody response?

[0260] From 2002 to 2004, we conducted an etiological study on periodontal disease patients visiting the dental outpatient department of the Royal London Hospital in the UK. A total of 107 samples were used to detect Porphyromonas gingivalis and its subtypes from the gingival crevicular fluid samples of periodontal disease patients using the PCR method. The PCR nucleic acid detection results showed that the ragB-2 and ragB-3 alleles of Porphyromonas gingivalis were the most common groups in the periodontal disease patient population. The detailed method is described in patent application PCT / GB2005 / 001976 and is hereby incorporated by reference. Briefly, total DNA was extracted from the gingival crevicular fluid samples, and the 16S RNA of Porphyromonas gingivalis was identified by PCR. Four pairs of probes were used to detect the ragB alleles using ragB primers specifically designed for each RagB outer membrane protein subtype of Porphyromonas gingivalis. The results showed that the RagB-2 and RagB-3 subtypes accounted for approximately 58% of the total samples ( Figure 13 , Table 25). The results of these clinical specimens showed that the RagB-2 and RagB-3 subtypes of Porphyromonas gingivalis were relatively high in the local East London periodontal patient population.

[0261] Virulence studies using animal models of soft tissue injury with different bacterial strains have shown that no outer membrane protein subtype is significantly more or less virulent than others. The mechanisms by which bacterial virulence and immune responses cause local and systemic damage to the body remain a mystery and an urgently desired milestone.

[0262] The same ELISA method was also used to detect human serum antibodies in patients with cardiovascular diseases and geriatric diseases. Table 26 shows the serum test results of geriatric patients. Briefly, the antigen adsorption and detection procedures were the same as those in the foregoing embodiments. For plates 20-05-P5, 20-05-P6, and 20-05-P7, the primary antibody (patient serum) dilution was 1:1000, and for plate 20-05-P8, the primary antibody (patient serum) dilution was 1:500. To avoid false positive results, the sample dilution was increased from 1:300 to 1:500 and 1:1000.

[0263] Table 22, Survey of antibodies against Porphyromonas gingivalis in healthy human serum

[0264]

[0265] Table 23, Data analysis of the survey of healthy human serum

[0266]

[0267] Table 24, Analysis of the distribution of outer membrane protein antibodies

[0268] Individual Negative <1.8X 1.8-2.5X 2.5-4X >4X Total Anti-RagB-1 10 23 12 5 1 51 Anti-RagB-2 10 37 4 0 0 51 Anti-RagB-3 10 39 1 0 1 51 Anti-RagB-4 10 10 15 9 7 51 Anti-Cra4S1 10 27 9 5 0 51 Anti-GST 10 39 2 0 0 51 Percentage Negative <1.8X 1.8-2.5 2.5-4X >4X Total Anti-RagB-1 19.6％ 45.1％ 23.5％ 9.8％ 2.0％ 100.0％ Anti-RagB-2 19.6％ 72.5％ 7.8％ 0.0％ 0.0％ 100.0％ Anti-RagB-3 19.6％ 76.5％ 2.0％ 0.0％ 2.0％ 100.0％ Anti-RagB-4 19.6％ 19.6％ 29.4％ 17.6％ 13.7％ 100.0％ Anti-Cra4S1 19.6％ 52.9％ 17.6％ 9.8％ 0.0％ 100.0％ Anti-GST 19.6％ 76.5％ 3.9％ 0.0％ 0.0％ 100.0％

[0269] Table 25, Analysis of the distribution of RagB subtypes in periodontal disease patients

[0270] Negative ragB-1 ragB-2 ragB-3 ragB-4 unknown Total Individuals 28 7 30 32 6 4 107 Percentage 26.2％ 6.5％ 28.0％ 29.9％ 5.6％ 3.7％ 100.00％

[0271] Table 26, Survey of antibodies against Porphyromonas gingivalis in the serum of geriatric patients

[0272]

[0273] Table 27 shows the data analysis of the serum sample survey of elderly patients (samples 201 - 255 in Table 26; samples 256 - 262 in Table 29), a total of 62 samples. According to the preset criteria, Table 28 shows the data analysis of the serum sample survey of elderly patients. The positive rate of RagB-1 antibody detection is 48.4% (30 / 62), of which the strong positive rate is 22.6% (14 / 62); the positive rate of RagB-2 is 48.4% (30 / 62), of which the strong positive rate is 24.2% (15 / 62); the positive rate of RagB-3 antibody is 46.8% (29 / 62), of which the strong positive rate is 24.2% (15 / 62); the positive rate of RagB-4 antibody is 38.7% (24 / 62), of which the strong positive rate is 22.6% (14 / 62); the positive rate of Cra4S1 antibody is 43.5% (27 / 62), of which the strong positive rate is 21% (13 / 62); while the positive rate of the irrelevant protein GST antibody is 40.3% (25 / 62), of which the strong positive rate is 21% (13 / 62), see Attachment Figure 14A-14B 。

[0274] Table 27, Data Analysis of the Survey Results of Elderly Patients

[0275]

[0276] Table 28, Analysis of the Distribution of Outer Membrane Protein Antibodies

[0277] Individual Negative <2.5X 2.5-4X >4X Total specimen Anti-RagB-1 12 20 16 14 62 Anti-RagB-2 12 20 15 15 62 Anti-RagB-3 12 21 14 15 62 Anti-RagB-4 12 26 10 14 62 Anti-Cra4S1 12 23 14 13 62 Anti-GST 12 25 12 13 62 Percentage Negative <2.5X 2.5-4X >4X Total specimen Anti-RagB-1 19.4％ 32.3％ 25.8％ 22.6％ 100.0％ Anti-RagB-2 19.4％ 32.3％ 24.2％ 24.2％ 100.0％ Anti-RagB-3 19.4％ 33.9％ 22.6％ 24.2％ 100.0％ Anti-RagB-4 19.4％ 41.9％ 16.1％ 22.6％ 100.0％ Anti-Cra4S1 19.4％ 37.1％ 22.6％ 21.0％ 100.0％ Anti-GST 19.4％ 40.3％ 19.4％ 21.0％ 100.0％

[0278] Table 29, Survey of Antibodies against Porphyromonas gingivalis in Serum of Cardiovascular Patients

[0279]

[0280] Table 30. Data Analysis of the Survey Results of Cardiovascular Patients

[0281]

[0282] Table 29 shows the original ELISA results of the serum tests of cardiovascular patients (and some elderly patients, see the above for details). Briefly, the antigen adsorption and detection procedures are the same as described above, and the dilution ratio of the patient serum in the reaction plate is 1:500.

[0283] Table 30 shows the data analysis of the serum samples of cardiovascular patients (numbers 101 - 149), a total of 49 samples. According to the preset criteria, the positive rate of RagB-1 antibody detection was 44.9% (22 / 49), of which the strong positive rate was 8.2% (4 / 49); the positive rate of RagB-2 was 40.8% (20 / 49), of which the strong positive rate was 8.2% (4 / 49); the positive rate of RagB-3 antibody was 46.9% (23 / 49), of which the strong positive rate was 8.2% (4 / 49); the positive rate of RagB-4 antibody was 57.1% (28 / 49), of which the strong positive rate was 10.2% (5 / 49); the positive rate of Cra4S1 antibody was 40.8% (20 / 49), of which the strong positive rate was 4.1% (2 / 49); while the positive rate of the irrelevant protein GST antibody was 53.1% (26 / 49), of which the strong positive rate was 4.1% (2 / 49); see Attachment Figure 15A-15B and Table 31.

[0284] Compared with healthy individuals, the serological surveys of cardiovascular and geriatric patients had three significant characteristics. First, the detection rates of RagB-2 and RagB-3 antibodies increased significantly; second, the detection rate of GST protein antibodies (i.e., antibodies against irrelevant antigens) increased substantially; third, the proportion of patients with high antibody titers in the samples of cardiovascular and geriatric patients was much higher than that in the healthy population.

[0285] Table 31. Analysis of the distribution of outer membrane protein antibodies

[0286] Individual Negative <2.5X 2.5-4X >4X Total specimen Anti-RagB-1 10 17 18 4 49 Anti-RagB-2 10 19 16 4 49 Anti-RagB-3 10 16 19 4 49 Anti-RagB-4 10 11 23 5 49 Anti-Cra4S1 10 19 18 2 49 Anti-GST 10 13 24 2 49 Percentage Negative <2.5X 2.5-4X >4X Total specimen Anti-RagB-1 20.4％ 34.7％ 36.7％ 8.2％ 100.0％ Anti-RagB-2 20.4％ 38.8％ 32.7％ 8.2％ 100.0％ Anti-RagB-3 20.4％ 32.7％ 38.8％ 8.2％ 100.0％ Anti-RagB-4 20.4％ 22.4％ 46.9％ 10.2％ 100.0％ Anti-Cra4S1 20.4％ 38.8％ 36.7％ 4.1％ 100.0％ Anti-GST 20.4％ 26.5％ 49.0％ 4.1％ 100.0％

[0287] The "antibody" against the irrelevant antigen GST was not detected in healthy individuals, while the detection rate of GST antibody in cardiovascular disease patients and geriatric patients increased significantly. In addition, the detection of GST antibody was directly related to the levels of RagB and Cra4S1 antibodies and only occurred in patients positive for these antibodies. Individuals with higher OD values of anti-RagB and Cra4S1 antibodies also had higher OD values of "GST antibody". No independent "GST antibody" positive cases were found.

[0288] The serological antibody study of the outer membrane proteins of Porphyromonas gingivalis found that a large proportion of patients with cardiovascular and geriatric diseases had antibodies against the outer membrane proteins of Porphyromonas gingivalis in their sera. Especially in sera with high titers of RagB-2, RagB-3, and Cra4S1 antibodies, the incidence (and high antibody titers) of antibodies against the irrelevant protein GST also increased. These data provide a scientific basis indicating that Porphyromonas gingivalis can trigger diseases through heterophilic antigen characteristics.

[0289] Multiple lines of evidence indicate that the outer membrane proteins of Porphyromonas gingivalis, including RagB and Cra4S1, have heterophilic antigen characteristics. Heterophilic antigens refer to a class of common antigens that are completely unrelated to the antigens present in host tissues but are structurally similar. Many scientific literature reports have described diseases caused by heterophilic antigens. For example, the cell membrane of Streptococcus hemolyticus shares antigens with the glomerular basement membrane and myocardial tissue, so tubulointerstitial nephritis or myocarditis may occur after streptococcal infection. Similarly, the lipopolysaccharide of Escherichia coli (E. coli) shares antigens with human colonic mucosa, and the infection of the former is associated with the development of ulcerative colitis.

[0290] Heterophilic antibodies refer to multispecific immunoglobulins with a certain titer induced by known or unknown antigenic substances (heterophilic antigens), which can bind to various proteins, but the affinity may not be strong. These antibodies can be endogenous and naturally occurring and can react with molecules unrelated to the original antigen. These immunoglobulins with different chemical structures but with multiple binding abilities to the test substance are called heterophilic antibodies.

[0291] Heterophilic antibodies can be understood as multispecific or nonspecific immune enhancement and may have immune protection functions. However, the multiple immune responses of heterophilic antibodies to heterophilic antigens and antigen components shared in different tissues may lead to immunopathology.

[0292] Serological surveys found that a large proportion of the overall samples of cases positive for RagB, Cra4S1, and GST antibodies were among patients with cardiovascular diseases and geriatric diseases. Combining with the epidemiological survey data, the outer membrane proteins of Porphyromonas gingivalis, including RagB and Cra4S1, exhibit the characteristics of heterophilic antigens.

[0293] Since the first disclosure of the RagB-4 (381RagB) DNA / protein sequence (SEQ ID NO:8) in 2004, scientists have found a high similarity of protein sequences similar to RagB-4 in other species. For example, Porphyromonas gulae (the main periodontal pathogen of dogs and cats), whose outer membrane protein (WP_039431148) has 93% similarity to RagB-4 (see Figure 16 ), further supports the characteristics of the heterophilic antigen of Porphyromonas gingivalis and its wide application value. In addition, RagB-4 has no molecular sequence relationship with Cra4S1, but as described in Example 4 and subsequent Example 7, the RagB-4-1B11-4-4 monoclonal antibody has a cross-reaction with the Cra4S1 protein. This reaction to the full-length Cra4S1 protein rather than specific polypeptides indicates the possible existence of diffuse or weak binding, a characteristic commonly observed in heterophilic antigens.

[0294] Periodontal disease is a very common disease in humans. Porphyromonas gingivalis is the main pathogen. Detecting antibodies against the outer membrane proteins of Porphyromonas gingivalis in human serum means that the subject has natural antibodies or a history of Porphyromonas gingivalis infection. Periodontal disease is usually chronic and progressive. The body's immune response to Porphyromonas gingivalis varies, but usually cannot completely eliminate the infection.

[0295] This example shows that the detection rate of antibodies against the outer membrane proteins of Porphyromonas gingivalis in the sera of patients with cardiovascular diseases and geriatric diseases is relatively high. In addition, a large proportion of these patients also showed reactions between immunoglobulins and irrelevant proteins in the serum. This indicates that a considerable proportion of these patients are in an immune-sensitive state. These antibodies are likely to be heterophilic antibodies and have a strong correlation with chronic diseases and pathological damage, and may even play a pathogenic role in the etiology of certain diseases.

[0296] As shown by the experimental results, Porphyromonas gingivalis seems to be less toxic to the young. It can be speculated that heterophilic antibodies have not accumulated to harmful levels in the young population. Human serum samples also showed that in all three groups (including healthy people, cardiovascular patients, and geriatric patients), some individuals did not detect any antibodies against the outer membrane proteins of Porphyromonas gingivalis (Tables 22, 26, and 29). These individuals either have natural / non-specific resistance to Porphyromonas gingivalis or have never been infected by Porphyromonas gingivalis. This finding emphasizes that Porphyromonas gingivalis may be one of the multi-factorial contributors to complex systemic diseases, and individual immune response differences play an important role in determining whether the infection will be cleared or persist.

[0297] Detecting antibodies against RagB and Cra4S1 antigens, as well as GST or other tissue antigens, in the serum is a simple and easy method for predicting Porphyromonas gingivalis infection and the development of associated diseases. This can prompt timely attention to the disease development and provide great benefits for the early treatment of patients, reducing potential morbidity and mortality.

[0298] Further research can deepen the understanding of the association between Porphyromonas gingivalis infection and chronic diseases. The data provided in this example show that the heterophilic antigen characteristics of Porphyromonas gingivalis, combined with the characteristics of individuals, can explain heterophilic antibodies and / or hypersensitivity reactions, which are important factors in chronic Porphyromonas gingivalis infection and systemic diseases. Therefore, by eliminating pathogenic bacteria infection, reducing the release of heterophilic antigens, and / or reducing the level of heterophilic antibodies in the circulation, tissue damage caused by harmful heterophilic antibodies can be reduced, related diseases can be actively treated, and further deterioration of multiple tissues can be controlled and prevented.

[0299] Example 6: Application of combined antibodies in an animal reproduction model

[0300] Animal experiment data indicate that the combined vaccine of RagB protein and Cra4S1 protein is superior to the single-protein vaccine in terms of local tissue protection effect. However, the combined vaccine does not show cross-protection against other subtypes of Porphyromonas gingivalis. For example, when animals are inoculated with a mixed vaccine containing RagB-1, RagB-3, and Cra4S1, they are protected against infection with RagB-1 and RagB-3 subtypes of Porphyromonas gingivalis, but no protection is provided against infection with the RagB-2 subtype. Passive immunization confirms that a combination of antibodies, such as a mixture of anti-RagB and anti-Cra4S1, exhibits the best protection effect compared to any individual antibody and the control group. Details of the protection are described in PCT / CN2019 / 124433, which is hereby incorporated by reference.

[0301] Conventional antibody drug therapy focuses on using specific antibodies to provide precise treatment based on the diagnosis of RagB subtypes of the outer membrane protein of Porphyromonas gingivalis. As described in Example 3, monoclonal antibodies against four different RagB proteins and Cra4S1 protein have been identified and described.

[0302] To design a combined antibody experiment, in this example, the monoclonal antibody 1D2-2-1-3 specific for RagB-3 was combined with the polyclonal antibody of Cra4S1 derived from mouse serum. Specifically, male Balb / c mice aged 5-6 weeks and weighing 15-18 g were used, with six mice in each group. Four days and one day before bacterial infection, 100 μl of the antibody solution was injected into the abdominal cavity of the mice (see Table 32). The injection solution contained 50 μg of the monoclonal antibody, 200 μg of mouse serum (Pab in Table 32 is the mouse polyclonal antibody), or a mixture of 50 μg of the monoclonal antibody and 200 μg of mouse serum, and then the animals were challenged with bacteria.

[0303] The preparation of the bacterial suspension is briefly described as follows: Porphyromonas gingivalis, including various subtypes (RagB-1, RagB-2, RagB-3, and RagB-4), was grown on a selective anaerobic agar (FAA) medium containing 5% defibrinated horse blood and placed in an anaerobic incubator at 37°C, with a culture gas of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide. Bacterial colonies were transferred to a newly prepared brain heart infusion (BHI) medium (containing 5 μg / ml of heme) and cultured for 18-24 hours until the OD600nm reached 1-1.2. Then the culture was centrifuged and washed twice, and after washing, bacterial suspensions with different concentrations (2-8x10 10 CFU / ml) were prepared for animal experiments.

[0304] Table 32. Animal information participating in the passive immunization experiment

[0305]

[0306] After bacterial infection, the general condition of the animals and the development of local injuries were observed, and photographs were taken. The records are shown in Tables 33 and 34, and Figure 17 A-17E and Figure 18A-18B .

[0307] The data showed that the animals receiving the combination of RagB-3 monoclonal antibody 1D2-2-1-3 and Cra4S1 polyclonal antibody obtained better local immune protection at the site of bacterial infection. Compared with other groups of animals not receiving the combined antibody, they had less soft tissue damage and a faster healing rate. The data also showed that the antibody combination achieved a local effect in the humoral circulation. In this example, intraperitoneal injection (systemic administration technique) achieved a local subcutaneous protective effect in subsequent Porphyromonas gingivalis bacterial infection.

[0308] Table 33. Record of experimental data on monoclonal antibody immune protection

[0309]

[0310] Table 34. Data summary

[0311]

[0312] Periodontal diseases caused by Porphyromonas gingivalis can present as visible local injuries. Clinicians can judge the severity of periodontal diseases by detecting the depth of the periodontal pocket and the susceptibility to gingival bleeding. However, the association between Porphyromonas gingivalis infection and a series of systemic diseases suggests that periodontal diseases may only be the tip of the iceberg of the harm caused by Porphyromonas gingivalis infection.

[0313] Currently, conventional animal models of Porphyromonas gingivalis infection mainly focus on soft tissue destruction and alveolar bone resorption. The virulence of different strains of Porphyromonas gingivalis is not consistent. Local tissue damage caused by Porphyromonas gingivalis infection can be observed in almost all animals, and the degree of damage depends on the infection dose. Generally, the higher the bacterial concentration the animals are infected with, the more severe the local tissue damage. In most cases, the infected animals will heal on their own, but some abscesses may reappear after the initial wound healing. If only infected with bacteria, animals usually do not have a high mortality rate. The pathogenic mechanism of Porphyromonas gingivalis has never been proven conclusively, and the scientific community has not yet successfully established an animal model related to Porphyromonas gingivalis infection and systemic diseases.

[0314] For many years, clinical and epidemiological data have shown that pregnant women infected with Porphyromonas gingivalis are associated with the birth of premature infants and low birth weight infants. Inspired by these clinical findings, we established an animal model to study the effect of Porphyromonas gingivalis infection on mouse reproduction.

[0315] For the newly established animal model, three groups were set up: in the first group, both male and female mice were simultaneously administered the antibody combination and infected with Porphyromonas gingivalis; in the second group, male mice were the control group, while female mice were inoculated with the antibody combination and infected with Porphyromonas gingivalis; in the third group, male mice were inoculated with the antibody combination and infected with Porphyromonas gingivalis, and female mice were the control group. The antibodies were from rabbit serum, and the details of the antibody combination are shown in Table 35. Specifically, the mice were injected with the antibody intraperitoneally twice, with a 4-day interval, and then the mice were infected with Porphyromonas gingivalis. During the recovery period, male and female mice in each group were randomly paired; in each cage, 1 male was paired with 2 female mice for the mice receiving the PBS control group, and 1 male was paired with 1 female mouse in the cage.

[0316] In the initial practice, the number of female mice in the control group was limited (i.e., not receiving the antibody and not infected with Porphyromonas gingivalis), so younger female mice (11 - 12 weeks old instead of 18 - 20 weeks old) were used, as shown in Table 35. In contrast, control female mice of a similar age were paired with control male mice, showing that the number of offspring produced by female mice at 11 - 12 weeks old was lower.

[0317] The results showed that when both male and female mice were injected with the antibody and then infected with Porphyromonas gingivalis, the ratio of live-born mice was lower and the stillbirth rate was higher (Table 36). In contrast, when mice not injected with the antibody (PBS group) were infected with Porphyromonas gingivalis, compared with the mice inoculated with the antibody combination, the number of live-born pups increased and no stillbirth was observed in terms of their reproductive ability.

[0318] In Table 37, female mice inoculated with the combined antibody and then infected with Porphyromonas gingivalis were paired with normal control male mice. The results showed that compared with the previous group where both male and female were inoculated with the antibody combination and infected, the number of live pups in this group increased. The control group was female mice inoculated with PBS and infected with Porphyromonas gingivalis paired with normal control male mice. In fact, the average number of live-born mice of female mice inoculated with the antibody combination and infected (51 / 13, 3.92 live-born mice per female mouse on average) was slightly higher than that of the PBS control mice. Although the data may not have a large enough volume to show statistical significance, these results indicate that antibody administration plus Porphyromonas gingivalis infection plays an important role in the offspring reproduction of male and female mice.

[0319] In Table 38, male mice that received the antibody combination and were subsequently infected with Porphyromonas gingivalis were paired with normal control female mice. The results showed that the number of live-born mice improved compared to the previous situation where both male and female received the antibody combination and Porphyromonas gingivalis infection. The control group was male mice inoculated with PBS and infected with Porphyromonas gingivalis paired with normal control female mice. The average number of live-born mice in male mice that received the antibody combination and were infected was similar to the number in mice that received the PBS control. Interestingly, the number of live pups and survival rate in Table 38 were lower than those in Table 37, and the stillbirth rate was higher. These data indicate that the male infection status after antibody administration and infection has a greater impact on the live birth rate than the infection status of female mice.

[0320] Table 35. Information on Experimental Animals Involved

[0321]

[0322] Table 36. Reproductive Experiment (1) - Both Male and Female Animals Received Antibodies and Infection (plus Antibody-Free Control)

[0323]

[0324] Table 37. Reproductive Experiment (2) - Female Mice Received Antibodies and Infection (plus Antibody-Free Control) Paired with Antibody-Free and Infected Control Male Mice

[0325]

[0326] Table 38. Reproductive Experiment (3) - Male Mice Received Antibodies and Infection (plus Antibody-Free Control) Paired with Antibody-Free and Infected Control Female Mice

[0327]

[0328] Table 39. Investigation of the Immunoprotective Function of the Combined Monoclonal Antibodies

[0329]

[0330] Based on the above results, the next step was to evaluate the effect of receiving the RagB monoclonal antibody on these mouse models. As described in Example 4, three monoclonal antibodies showed potential as a vaccine against Porphyromonas gingivalis due to their cross-reactivity with multiple RagB subtypes. They are RagB-3-1D2-2-1-3 (abbreviated as 1D2), RagB-4-1B11-4-4 (abbreviated as 1B11), and RagB-4-1C3-7-8 (abbreviated as 1C3). Since the effect of passive immunity provided by a single antibody is limited, as described in PCT / CN2019 / 124433, this example aimed to evaluate the effect of testing passive immunity using a combination of two (or more) monoclonal antibodies disclosed in the present invention.

[0331] Animal experiments involving the administration of the antibody combination and infection with Porphyromonas gingivalis were conducted as described above. Specifically, mice were injected intraperitoneally with the antibody twice, with a 4-day interval between injections. Then, the animals were infected with Porphyromonas gingivalis, and the survival rate and local damage were observed and recorded.

[0332] Table 39 shows the results of passive immunization in animals infected with Porphyromonas gingivalis after receiving the antibody combination. Male and female mice were inoculated with the two monoclonal antibody combinations listed in Table 39. The control group received normal mouse IgG or PBS, and then they were infected with the RagB-3 subtype of Porphyromonas gingivalis. One mouse (1 / 8) that received normal mouse IgG died after bacterial infection, and two mice (2 / 8) in each of the G942 and G944 groups died. None of the female mice died.

[0333] During the recovery period, male and female mice were paired; there was 1 male and 3 female mice in each cage. In each breeding group, two female mice were challenged immunologically, and the other female mouse did not receive the antibody or infection. The body weight of the female mice and the number of live and stillborn pups were recorded. When the body weight of the female mouse suddenly decreased and no pups were found, it might indicate a miscarriage. Due to the shortage of female mice compared to male mice, resulting in a shortage of female mice in each group, some female mice from other studies with records of antibody injection and Porphyromonas gingivalis infection were used. The specific details are shown in Table 40.

[0334] Table 40. Immunization profiles of female mice added to the reproductive model

[0335]

[0336] Table 41. Effects of combined monoclonal antibodies on reproduction (1) - male and female mice received mouse IgG and infection, with female mice without antibody and without infection as controls in the experimental groups

[0337]

[0338] G941 / G951 (Table 41) and G945 / G955 (Table 45) were the control groups, which were given normal mouse IgG and PBS respectively. None of these animals received any antibodies with protective properties before being infected with Porphyromonas gingivalis, so they represent the baseline expected reproductive success rate of animals recovering from infection. The number of live-born pups observed in the two groups was similar, but in the G941 / G951 group, a high proportion of female mice in four cages completely failed to become pregnant (4 / 7), while a high proportion of stillbirths was observed in the G945 / G955 group.

[0339] Table 42. Effects of combined monoclonal antibodies on reproduction (2) - male and female mice received the combination of monoclonal antibodies 1B11 and 1D2 plus infection, and the experimental group included female mouse controls without antibodies and without infection

[0340]

[0341] In the G942 / G952 group (Table 42), animals received the combination of monoclonal antibodies 1D2 and 1B11 before infection with Porphyromonas gingivalis. As previously mentioned, the mortality rate of male mice after bacterial infection was 25% (2 / 8). The subsequent reproductive success rate of this group was also greatly affected. Five out of six male mice failed to impregnate female mice (including control female mice), and only one male mouse was able to reproduce. One normal female mouse gave birth to 4 live pups and 1 stillborn in this group, and the other two female mice were suspected of having miscarriages.

[0342] In the G943 / G953 group (Table 43), animals received the combination of monoclonal antibodies 1B11 and 1C3 before infection with Porphyromonas gingivalis. All animals, including males and females, survived after infection with Porphyromonas gingivalis. Males and females recovered well, although two males failed to impregnate females (including control females). In the remaining six cages, 18 females were observed to give birth to a total of 48 live pups, and a small number of stillborns (4) were recorded.

[0343] In the G944 / G954 group (Table 44), animals received the combination of monoclonal antibodies 1D2 and 1C3 before infection with Porphyromonas gingivalis. As previously mentioned, the mortality rate of male mice after bacterial infection was 25% (2 / 8). The subsequent reproductive success rate was similar to that of the G945 / G955 control group that received PBS before infection. Out of the remaining six male mice, one failed to impregnate any female (including control females). The number of live births observed: 15 females gave birth to a total of 31 pups. A higher number of stillborns (10) were observed, and 3 female mice were suspected of having miscarriages.

[0344] Table 43. Effects of combined monoclonal antibodies on reproduction (3) - male and female mice received the combination of monoclonal antibodies 1B11 and 1C3 plus infection, and the experimental group included female mouse controls without antibodies and without infection

[0345]

[0346] Table 44. Effects of combined monoclonal antibodies on reproduction (4) - male and female mice received the combination of monoclonal antibodies 1C3 and 1D2 plus infection, and the experimental group included female mouse controls without antibodies and without infection

[0347]

[0348] Table 45. Effects of combined monoclonal antibodies on reproduction (5) - male and female mice received monoclonal antibody PBS plus infection, and the experimental group included female mouse controls without antibody and without infection

[0349]

[0350] Notably, the Rag-3 1D2 monoclonal antibody showed a local protective effect in animal experiments of Porphyromonas gingivalis infection (as shown in Tables 33 and 34). In multiple ELISA assays, 1D2 showed high affinity for the antigen. However, the effect of the 1D2 antibody (in different combinations) on animal reproduction may pose a safety hazard, as a 25% male mouse mortality rate, a decrease in the live birth rate within the group, an increase in the abortion and stillbirth rates, a decrease in the fertilization success rate, or a combination of these indicators were observed in groups G942 and G944.

[0351] The results of the animal experimental model disclosed in this example indicate that Porphyromonas gingivalis infection causes reproductive damage. Literature reports that pregnant women with periodontal disease have a 3 - 7 times higher chance of giving birth to premature and low birth weight babies than pregnant women without periodontal disease. For many years, researchers have been concerned about the infection of the maternal reproductive system and fetal characteristics, studying genetics, endocrine characteristics, and tissue and organ infections.

[0352] This example clarifies that Porphyromonas gingivalis infection causes reproductive damage. The worst results occur when both males and females are infected, with a high fertilization failure rate, high stillbirth rate, and high abortion rate. The data disclosed in this example also show that when infected females are paired with non-infected males, their reproduction remains in a stable state, but conversely, when infected males are paired with non-infected females, the outcome is worse. These results clearly show that male Porphyromonas gingivalis infection and immune status have an adverse effect on reproductive outcomes, as the reproductive outcomes of infected males are consistently poor in these animal models. The possible mechanism of action is through the heterophilic antigen on the surface of Porphyromonas gingivalis, which is similar to host tissues such as sperm or tissues of the male reproductive system.

[0353] In addition, Porphyromonas gingivalis - specific antibodies can provide an immune protection function, but immune - mediated pathological damage may also occur. For example, the 1D2 monoclonal antibody has a strong antigen - antibody reaction against multiple RagB subtypes, with antigen targets including RA - 17, RB - 17, and RC - 17 peptides (Example 4), and shows local immune protection, but it was also noted that this monoclonal antibody led to a high mortality rate and a decrease in reproductive success rate in this example.

[0354] The data disclosed in this example also show that two other monoclonal antibodies, namely 1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3), which specifically target RagB-4, can cross-react with multiple RagB subtypes and are safe without causing harm to the reproductive system of animals. These combined antibodies effectively improve the physical conditions of male and female animals and directly or indirectly restore / cure reproductive ability. The data suggest that these combined antibodies can be used for other diseases and / or systemic diseases associated with Porphyromonas gingivalis infection, including but not limited to periodontal disease, cardiovascular disease, rheumatoid arthritis, oral gastrointestinal cancer, ulcerative colitis, nervous system diseases, autoimmune encephalomyelitis, lung cancer, adverse pregnancy, pancreatic cancer, diabetes, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease. One of the treatment strategies is to remove harmful antigens and / or antibodies associated with the occurrence and development of the disease, and / or increase the titer or concentration of beneficial antibodies in the body.

[0355] Example 7: Mouse Monoclonal Antibodies and Humanized Mutations

[0356] Four monoclonal antibody hybridoma cell lines: RagB-2-1A4-3-7-7, RagB-3-1D2-2-1-3, RagB-4-1B11-4-4, and RagB-4-1C3-7-8 all showed cross-reactivity with outer membrane proteins of different subtypes of Porphyromonas gingivalis. The nucleic acid sequences of these antibodies have been obtained. Briefly, total RNA was extracted from each cell line and reverse transcribed using antisense primers according to conventional RNA extraction and transcription methods. Next, the variable region sequences of the light and heavy chains were amplified, and the PCR fragments were inserted into the pUC19T vector. Then, PCR technology was used to screen the clones, and positive clones were selected for sequencing.

[0357] The nucleotide and amino acid sequences of the specific antibodies are shown below:

[0358] RagB-2-1A4-3-7-7 (Beyotime, China, B467701, also known as "1A4") contains a variable region (VH) of the heavy chain (mouse IgG1) with a nucleotide sequence shown in SEQ ID NO: 128 and an encoded amino acid sequence shown in SEQ ID NO: 129; and a variable region (VL) of the light chain (mouse Kappa) with a nucleotide sequence shown in SEQ ID NO: 133 and an encoded amino acid sequence shown in SEQ ID NO: 134 (see also Figure 19 A-19D). The expression of the recombinant antibody was not provided.

[0359] RagB-3-1D2-2-1-3 (Bio Ying, China, B467702, also known as "1D2") contains a variable region of the heavy chain (mouse IgG2a) (VH) with the nucleotide sequence shown in SEQ ID NO: 138 and the encoded amino acid sequence shown in SEQ ID NO: 139, and a variable region of the light chain (mouse Kappa) (VL) with the nucleotide sequence shown in SEQ ID NO: 143 and the encoded amino acid sequence shown in SEQ ID NO: 144 (see also Figure 20 A-20D). The expression of the recombinant antibody is as shown in Figure 20 E and 20F.

[0360] RagB-4-1B11-4-4 (Bio Ying, China, B583901, also known as "1B11") contains a variable region of the heavy chain (mouse IgG1) (VH) with the nucleotide sequence shown in SEQ ID NO: 148 and the encoded amino acid sequence shown in SEQ ID NO: 149, and a variable region of the light chain (mouse Kappa) (VL) with the nucleotide sequence shown in SEQ ID NO: 153 and the encoded amino acid sequence shown in SEQ ID NO: 154 (see also Figure 21 A-21D). The present invention also provides bispecific antibodies of the monoclonal antibody. The expression of the recombinant antibody is as shown in Figure 21 E and 21F.

[0361] RagB-4-1C3-7-8 (Bio Ying, China, B583902, also known as "1C3") contains a variable region of the heavy chain (mouse IgG1) (VH) with the nucleotide sequence shown in SEQ ID NO: 158 and the encoded amino acid sequence shown in SEQ ID NO: 159, and a variable region of the light chain (mouse Kappa) (VL) with the nucleotide sequence shown in SEQ ID NO: 163 and the encoded amino acid sequence shown in SEQ ID NO: 164 (see also Figure 22 A-22D). The present invention provides a detailed description of the monoclonal antibody and its bispecific antibody. The expression of the recombinant antibody is as shown in Figure 22 E and 22F.

[0362] Table 46. Physicochemical properties of recombinant antibodies

[0363]

[0364] Three RagB monoclonal antibodies of the recombinant plasmid, including 1D2, 1B11 and 1C3, were expressed in the eukaryotic cell HEK293 by a CRO company (Bio Ying, China). The purification results of the recombinant antibody are as shown in Figure 20As shown in E-20F, 21E-21F, and 22E-22F, the physical and chemical properties of each recombinant antibody are shown in Table 46. The binding function of the recombinant antibody against the antigen was detected by ELISA (see Table 47).

[0365] Table 47 shows the detection results of the recombinant antibody plasmids. The ELISA operation steps are as described in Example 2, and the antigen adsorption design is as follows: The first column: RA-21 peptide, the second column: RB-21 peptide, the third column: RC-21 peptide, the fourth column: RC-22 peptide, the fifth column: RD-21 peptide, the sixth column: RD-22 peptide, the seventh column: RagB-1 recombinant protein, the eighth column: RagB-2 recombinant protein, the ninth column: RagB-3 recombinant protein, the tenth column: RagB-4 recombinant protein, the eleventh column: Cra4S1 recombinant protein, the twelfth column: RC-17 peptide. The antibody was diluted to 100 μg / ml, and each antibody was added to a row of the ELISA plate for antigen detection in 12 wells, including RA-21 (SEQ ID NO.31), RB-21 (SEQ ID NO.55), RC-21 (SEQ ID NO.79), RC-22 (SEQ ID NO.80), RD-21 (SEQ ID NO.103), RD-22 (SEQ ID NO.104), RagB-1 (SEQ ID NO.2), RagB-2 (SEQ ID NO.4), RagB-3 (SEQ ID NO.6), RagB-4 (SEQ ID NO.8), Cra4S1 (SEQ ID NO.10), and RC-17 (SEQ ID NO.75).

[0366] Table 47. Detection of Monoclonal Antibody Target Polypeptides

[0367]

[0368] The data indicate that the binding reaction pattern of the recombinant antibody to its corresponding antigen is the same as that of the monoclonal antibody produced by hybridoma cells. Under the same antibody dilution conditions, the absorbance values and patterns of the ELISA reaction plates are very similar, indicating that the recombinant antibody retains the antigen-binding function and has the same stability as the original monoclonal antibody. These recombinant antibodies are candidates for further development into therapeutic drugs.

[0369] Example 6 discloses that the combination of two monoclonal antibodies 1B11-4-4 (1B11) and 1C3-7-8 (1C3) exhibits cross-immunoprotection against four different subtypes of Porphyromonas gingivalis. The 1B11-4-4 (1B11) antibody also cross-reacts with the Cra4S1 protein (Table 47), which is an epitope conserved among all Porphyromonas gingivalis subtypes. Since Cra4S1 is considered a heterophilic antigen, monoclonal antibody 1B11 can achieve synergy not only through cross-immunoprotection against other Porphyromonas gingivalis outer membrane protein subtypes but also by reducing the heterophilic antigen of Cra4S1.

[0370] Therefore, humanized sequence mutations were performed on the RagB-4-1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3) monoclonal antibodies, and were designed by a third-party CRO company (Genscript, USA) according to conventional methods. The present invention provides the analysis of murine chimeric antibodies, the synthesis of humanized genes, antibody expression and affinity screening, antibody production, and the establishment of seed cell banks to achieve the industrial production of antibodies.

[0371] The monoclonal antibodies disclosed in the present invention are used for the treatment of chronic diseases caused by Porphyromonas gingivalis infection, including but not limited to periodontal disease, cardiovascular disease, rheumatoid arthritis, oral and gastrointestinal cancer, ulcerative colitis, neurological diseases, autoimmune encephalomyelitis, lung cancer, adverse pregnancy, pancreatic cancer, diabetes, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease.

[0372] Example 8: RagB Bispecific Monoclonal Antibody

[0373] Bispecific monoclonal antibody (BsMAb) is an artificially synthesized protein that can bind two different antigens or two or more different epitopes simultaneously. Natural monoclonal antibodies usually target only one antigen. Through development, BsMAb can be designed into various different structural patterns. Through different mechanisms of action, BsMAb can be designed to recruit and activate immune cells, interfere with receptor signal transduction, inactivate signal transduction ligands, and bind to protein complexes.

[0374] The above data show that the antigen targets of two monoclonal antibodies, RagB-4-1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3), are two adjacent epitopes within the peptide segment RD-21&22 region of the outer membrane protein RagB-4 of Porphyromonas gingivalis. The combination of the two monoclonal antibodies exhibits stable systemic and local immune protection. Therefore, a bispecific monoclonal antibody 1B11-1C3 (Baiying, China, B745901) that combines RagB-4-1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3) was prepared. The antibody includes a heavy chain with a nucleotide sequence as shown in SEQ ID NO:168 and an encoded amino acid sequence as shown in SEQ ID NO:172, and a light chain with a nucleotide sequence as shown in SEQ ID NO:173 and an encoded amino acid sequence as shown in SEQ ID NO:174.

[0375] Specifically, the bispecific antibody 1B11-1C3 uses the scFv mode. The single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, linked by a commercially available short peptide. The fusion protein includes a heavy chain that contains the variable region of a specific monoclonal antibody and the constant region (CH) of the mouse IgG1 heavy chain linked by a short peptide; the light chain contains the variable region of a specific monoclonal antibody and the constant region of the mouse immunoglobulin kappa.

[0376] The heavy chain of the bispecific 1B11-1C3 antibody includes a nucleotide sequence and an amino acid sequence. The nucleotide sequence of the heavy chain variable region VH includes SEQ ID NO:169 (1C3 VH), SEQ ID NO:170 (1B11 VH), and SEQ ID NO:171 (1B11 VL), and the encoded amino acid sequences include SEQ ID NO:159 (1C3 VH), SEQ ID NO:149 (1B11 VH), and SEQ ID NO:154 (1B11 VL); the nucleotide sequence of the light chain variable region VL is SEQ ID NO:173, and the encoded amino acid sequence is the full-length light chain variable region of 1C3 as shown in SEQ ID NO:164 (see Figures 23A - 23F ).

[0377] Table 48. Physicochemical properties of recombinant bispecific antibodies

[0378]

[0379] The recombinant plasmid was transfected into the eukaryotic cell HEK293, and the recombinant antibody was induced to express and purified by protein A affinity chromatography (see Figure 23G and 23H)。The physical and chemical properties of the recombinant bispecific antibody protein are shown in Table 48. The antigen-antibody reaction was detected by ELISA method; Table 47 in Example 7 also provides data on bispecific antibodies. The experimental operation of ELISA was as described in Example 2, and the antigen adsorption design was as described in Example 7. The antibody was diluted to 100 μg / ml, and 12 wells of antigen were detected for each antibody.

[0380] The data provided by the present invention indicate that the bispecific 1B11-1C3 antibody exhibits antigen-antibody binding reactions to each corresponding target peptide fragment and recombinant proteins of different subtypes, and each monoclonal antibody also exhibits the same reaction to its corresponding peptide target. The bispecific antibody shows the same effect as the combination of monoclonal antibodies 1B11-4-4 and 1C3-7-8. Compared with the use of combination antibodies and single antibodies, the fusion protein retains the specificity of the original immunoglobulin and enhances the immune response to antigen targets. The bispecific monoclonal antibodies disclosed in the present invention are intended to treat chronic diseases caused by Porphyromonas gingivalis infection, including but not limited to periodontal disease, cardiovascular disease, rheumatoid arthritis, oral and gastrointestinal cancer, ulcerative colitis, neurological diseases, autoimmune encephalomyelitis, lung cancer, adverse pregnancy, pancreatic cancer, diabetes, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease.

[0381] If efficacy can be ensured and stable production can be achieved, bispecific antibodies represent a more efficient and simpler solution for developing new therapies.

[0382] Example 9: Serum antibody diagnostic kit

[0383] The data disclosed in Example 5 indicate that approximately 40-50% of patients with cardiovascular disease and geriatric patients have high-titer antibodies against the outer membrane proteins RagB and Cra4S1 of Porphyromonas gingivalis and unrelated proteins (such as GST recombinant protein). The relevant data illustrate that the qualitative and / or quantitative detection and / or measurement of these antibodies can provide early diagnosis of Porphyromonas gingivalis infection; evaluate the risk of developing chronic diseases associated with Porphyromonas gingivalis infection; and evaluate disease prognosis and treatment efficacy.

[0384] The present invention provides a method for diagnosing or evaluating the prognosis and / or treatment effect of Porphyromonas gingivalis infection or chronic diseases caused by or associated with Porphyromonas gingivalis. Specifically, antibodies against RagB proteins (including Rag-1, RagB-2, RagB-3, and RagB-4), Cra4S1 protein, and irrelevant proteins (including GST and / or other tissue proteins such as muscle proteins or mucosal proteins) can be screened in patients. ELISA technology is used to detect the presence or absence of antibodies in the individual serum of patients. If the amount of antibodies is high, especially at high titers, it indicates that the patient is in an immune-sensitive state, which will put them at a higher risk of developing certain chronic diseases. This screening allows for the stratification of patients and the identification of high-risk individuals, prompting the rapid initiation of treatment.

[0385] Example 10: PgingiVacRD1B11-1C3, polypeptide vaccine

[0386] Animal experimental model data show that most local injuries caused by the initial infection of Porphyromonas gingivalis in animals can self-repair. However, if the infection occurs repeatedly, the condition may gradually deteriorate and the local injury becomes difficult to heal. These phenomena indicate that host immune protection and bacterial invasion have entered a continuous imbalance process of repair and deterioration, and chronic Porphyromonas gingivalis infection usually cannot be completely cleared.

[0387] Some diseases require patients to use antibody drugs for a long time without interruption. However, a one-time vaccination may represent a more effective, simple, and cost-effective treatment method.

[0388] For the treatment of Porphyromonas gingivalis infection, after a period of specific antibody passive immunotherapy, patients can be vaccinated with an efficient polypeptide vaccine to stimulate their own production of effective neutralizing antibodies, which can persist in the patient's serum for a long time, thereby preventing the growth and spread of pathogenic bacteria. The polypeptide vaccine of the present invention is cost-effective and can be safely administered through conventional routes. In addition, the vaccine product does not contain full-length proteins, especially avoiding harmful, allergic, and irrelevant antigen fragments, thereby reducing the risk of heterophilic antibodies stimulated by heterophilic antigen stimulation.

[0389] The present invention discloses a polypeptide vaccine, namely PgingiVacRD1B11-1C3, which is a polypeptide fragment RD21 to RD22 based on the RagB-4-1B11-4-4 (1B11) and / or RagB-4-1C3-7-8 (1C3) antibody reactions. The two ends of the polypeptide can be appropriately optimized. The polypeptide of the PgingiVacRD1B11-1C3 vaccine can be synthesized, or an expression plasmid can be constructed using the recombinant nucleotide of the polypeptide and expressed under the induction conditions of a host cell. The protein expressed in the host cell can be purified by conventional methods. The PgingiVacRD1B11-1C3 polypeptide consists of 75 amino acids, and its amino acid sequence is shown in SEQ ID NO: 127.

[0390] The above display and description are only the preferred embodiments of the present invention, but those skilled in the art will understand that these examples are provided only by way of example. The present invention is not limited to the specific examples provided in the specification. Although the present invention has been described with reference to the above specification, the description and illustration of the examples herein are not intended to be construed in a limiting sense. Those skilled in the art can also make several changes, alterations, and substitutions without departing from the principles of the present invention. Therefore, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions described herein, which depend on various conditions and variables. It should be understood that various alternatives of the embodiments of the present invention described herein can be adopted when implementing the present invention. Therefore, the present invention should also cover any such alternatives, modifications, variations, or equivalent schemes. The following claims are intended to define the scope of the present invention and cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A monoclonal antibody targeting one or more outer membrane proteins of Porphyromonas gingivalis, comprising: (i) A light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO. 154, which comprises (a) CDR-L1, comprising the amino acid sequence shown in SEQ ID NO. 155; (b) CDR-L2, comprising the amino acid sequence set forth in SEQ ID NO. 156; and (c) CDR-L3, comprising the amino acid sequence set forth in SEQ ID NO. 157; and a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO. 149, which comprises: (a) CDR-H1, comprising the amino acid sequence shown in SEQ ID NO. 150; (b) CDR-H2, comprising the amino acid sequence set forth in SEQ ID NO. 151; and (c) CDR-H3, comprising the amino acid sequence set forth in SEQ ID NO. 152; (ii) A light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO. 164, which comprises (a) CDR-L1, comprising the amino acid sequence shown in SEQ ID NO. 165; (b) CDR-L2, comprising the amino acid sequence set forth in SEQ ID NO. 166; and (c) CDR-L3, comprising the amino acid sequence set forth in SEQ ID NO. 167; and a heavy chain variable domain (VH) comprising the amino acid sequence shown in SEQ ID NO. 159, which comprises: (a) CDR-H1, comprising the amino acid sequence shown in SEQ ID NO. 160; (b) CDR-H2, comprising the amino acid sequence set forth in SEQ ID NO. 161; and (c) CDR-H3, comprising the amino acid sequence set forth in SEQ ID NO. 162; (iii) A light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO. 144, which comprises (a) CDR-L1, comprising the amino acid sequence shown in SEQ ID NO. 145; (b) CDR-L2, comprising the amino acid sequence set forth in SEQ ID NO. 146; and (c) CDR-L3, comprising the amino acid sequence set forth in SEQ ID NO. 147; and a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO. 139, which comprises: (a) CDR-H1, comprising the amino acid sequence shown in SEQ ID NO. 140; (b) CDR-H2, comprising the amino acid sequence set forth in SEQ ID NO. 141; and (c) CDR-H3, comprising the amino acid sequence set forth in SEQ ID NO. 142; or (iv) A variable light chain domain (VL) comprising the amino acid sequence set forth in SEQ ID NO. 134, which comprises (a) CDR-L1, which comprises the amino acid sequence shown in SEQ ID NO. 135; (b) CDR-L2, which comprises the amino acid sequence set forth in SEQ ID NO. 136; and (c) CDR-L3, which comprises the amino acid sequence set forth in SEQ ID NO. 137; A variable heavy chain domain (VH) comprising the amino acid sequence shown in SEQ ID NO. 129, which comprises: (a) CDR-H1, which comprises the amino acid sequence set forth in SEQ ID NO. 130; (b) CDR-H2, which comprises the amino acid sequence set forth in SEQ ID NO. 131; and (c) CDR-H3, which comprises the amino acid sequence set forth in SEQ ID NO.

132.

2. The monoclonal antibody according to claim 1, which is in the form of Fab, Fab', F(ab)'2, single-chain Fv (scFv), Fv fragment or IgG.

3. The monoclonal antibody according to claim 1, which is a bifunctional antibody, a linear antibody or a bispecific or multispecific antibody.

4. The monoclonal antibody according to claim 3, wherein the bispecific antibody comprises a chimeric monoclonal antibody, the chimeric monoclonal antibody comprising: A variable light chain domain (VL) comprising the amino acid sequence set forth in SEQ ID NO. 174, which comprises (a) CDR-L1, which comprises the amino acid sequence shown in SEQ ID NO. 165; (b) CDR-L2, which comprises the amino acid sequence set forth in SEQ ID NO. 166; and (c) CDR-L3, which comprises the amino acid sequence set forth in SEQ ID NO. 167; A hybrid variable heavy chain domain (VH) comprising the amino acid sequence set forth in SEQ ID NO. 172, which comprises: (a) CDR-H, comprising the amino acid sequence set forth in SEQ ID NO. 160; (b) CDR-H2, which comprises the amino acid sequence set forth in SEQ ID NO. 161; and (c) CDR-H3, which comprises the amino acid sequence set forth in SEQ ID NO. 162; (d) CDR-H1, which comprises the amino acid sequence set forth in SEQ ID NO. 150; (e) CDR-H2, which comprises the amino acid sequence set forth in SEQ ID NO. 151; (f) CDR-H3, which comprises the amino acid sequence set forth in SEQ ID NO. 152; (g) CDR-L1, which comprises the amino acid sequence set forth in SEQ ID NO. 155; (h) CDR-L2, which comprises the amino acid sequence set forth in SEQ ID NO. 156; and (i) CDR-L3, which comprises the amino acid sequence set forth in SEQ ID NO.

157.

5. One or more isolated nucleic acid molecules encoding the monoclonal antibody of any one of claims 1-4.

6. One or more expression vectors comprising the isolated nucleic acid molecule of claim 5.

7. A cell or cell line consisting of the nucleic acid molecule of claim 5.

8. A method of treatment for a patient suffering from a Porphyromonas gingivalis infection or a chronic disease caused by a Porphyromonas gingivalis infection, the method comprising administering to the subject an effective dose of one or more of the monoclonal antibodies described in any one of claims 1-4.

9. The method according to claim 8, wherein the monoclonal antibodies are mixed prior to administration.

10. A method of treatment for a patient suffering from a Porphyromonas gingivalis infection or a chronic disease caused by a Porphyromonas gingivalis infection, the method comprising administering to the subject an effective dose of one or more of the isolated nucleic acid molecules of claim 5.

11. A method of treatment for a patient suffering from a Porphyromonas gingivalis infection or a chronic disease caused by a Porphyromonas gingivalis infection, the method comprising administering to the subject an effective dose of the cell or cell line of claim 7.

12. A composition comprising one or more of the monoclonal antibodies of any one of claims 1-4 and a pharmaceutically acceptable carrier.

13. A composition comprising one or more of the isolated nucleic acid molecules of claim 5 and a pharmaceutically acceptable carrier.

14. A composition comprising the cell or cell line of claim 7 and a pharmaceutically acceptable carrier.

15. A method of treatment for a patient suffering from a Porphyromonas gingivalis infection or a chronic disease caused by a Porphyromonas gingivalis infection, the method comprising administering to the subject an effective dose of the composition of any one of claims 12-14.

16. The method according to claim 15, wherein the chronic disease is periodontal disease, cardiovascular disease, rheumatoid arthritis, oral gastrointestinal cancer, ulcerative colitis, neurological disease, autoimmune encephalomyelitis, cancer, adverse pregnancy, pancreatic cancer, diabetes, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease.

17. A method for producing the monoclonal antibody of any one of claims 1-4, comprising culturing the cell or cell line of claim 7 and recovering the monoclonal antibody from the cultured cells.

18. An antigen of a Porphyromonas gingivalis outer membrane protein that reacts or cross-reacts with one or more of the monoclonal antibodies described in any one of claims 1-4.

19. The antigen of claim 18, wherein the antigen comprises an amino acid sequence selected from SEQ ID NO:2, 4, 6, 8, 10, and 11-127.

20. A vaccine comprising one or more of the antigens of claim 18 or 19.

21. An isolated nucleic acid encoding the vaccine of claim 20.

22. An expression vector comprising the isolated nucleic acid of claim 21.

23. A composition comprising the vaccine of claim 20 and a pharmaceutically acceptable carrier.

24. A composition comprising the isolated nucleic acid of claim 21 and a pharmaceutically acceptable carrier.

25. A method for preventing or treating Porphyromonas gingivalis infection or a chronic disease caused by Porphyromonas gingivalis infection, comprising administering to a subject an effective dose of the vaccine of claim 20.

26. A method for preventing or treating Porphyromonas gingivalis infection or a chronic disease caused by Porphyromonas gingivalis infection, comprising administering to a subject an effective dose of the composition of claim 23 or 24.

27. An animal model for evaluating the infection, treatment effect and prognosis of Porphyromonas gingivalis, wherein, An animal is infected with Porphyromonas gingivalis, and the monoclonal antibody, antibody-antigen reaction or cross-reaction according to any one of claims 1-3 is used for evaluating the reproductive effect on the animal.

28. A method for diagnosing and prognosticating Porphyromonas gingivalis infection, comprising: (a) identifying serum polyclonal antibodies against one or more outer membrane proteins of Porphyromonas gingivalis; (b) performing an antigen-antibody reaction or cross-reaction with one or more antigens of claim 18 or 19; and (c) evaluating the diagnosis and / or prognosis of an infection caused by Porphyromonas gingivalis or a chronic disease caused by Porphyromonas gingivalis infection.

29. A kit for diagnosing and prognosticating an infection caused by Porphyromonas gingivalis, comprising (a) a reagent of the antigen of claim 18 or 19, and (b) instructions for using the reagent to detect specific antibodies in a biological sample.

30. The kit of claim 29, wherein the biological fluid sample comprises blood, gingival crevicular fluid, urine, saliva, cerebrospinal fluid, pleural and peritoneal effusions, and amniotic fluid.

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