Antibodies against Porphyromonas gingivalis and methods of use

Abstract

The present invention provides monoclonal antibodies and methods for using them for diagnosing and / or treating P. gingivalis infection and / or chronic diseases caused by or associated with this infection. The present invention also identifies specific antigen-antibody reactive regions on P. gingivalis outer membrane proteins, which are used in the development of preventive and therapeutic vaccines. The monoclonal antibodies of the present invention cross-react with different antigenic regions of each P. gingivalis outer membrane protein. The present invention also provides animal models and diagnostic kits for serological evaluation, prognosis, and evaluation of therapeutic effects after P. gingivalis infection.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and biological preparations, in particular to a multi-target monoclonal antibody against Porphyromonas gingivalis infection.

Background Art

[0002] Periodontal disease is one of the most common oral infectious diseases worldwide. The global annual cost of treating oral diseases is about 442 billion US dollars, among which periodontal disease accounts for 10.5 to 12%. In the United States, 17 million people receive medical consultation for periodontal disease every year, with the cost exceeding 6 billion US dollars. According to the UK's National Health Service (NHS), it is estimated that more than 500 million pounds are spent annually on periodontal care in England and Wales. These costs only apply to routine acute periodontal disease treatment provided by NHS dentists, and do not include specialized treatment, follow-up and hospital services, as well as consultations with private dentists. In China, 80 to 97% of adults have varying degrees of periodontal problems.

[0003] Porphyromonas gingivalis is considered the causative bacterium of "adult" periodontal disease in the research field. Published literature has shown that periodontal disease caused by Porphyromonas gingivalis infection in children is not as severe as that in adults. The survival rate of dental implants is closely related to the health of periodontal tissues in the oral cavity. Porphyromonas gingivalis is a major pathogen causing peri-implantitis. In fact, most patients with periodontal disease follow a course of chronic, progressive and recurrent attacks, which means that bacterial infection cannot be eradicated.

[0004] Numerous clinical and statistical studies have shown that Porphyromonas gingivalis not only causes oral and periodontal lesions, but is also associated with cardiovascular disease, Alzheimer's disease, diabetes, and tumors of the gastrointestinal and respiratory systems. Porphyromonas gingivalis infection is closely linked to an increased incidence of low birth weight and premature infants, and is closely associated with many chronic systemic diseases. To date, the pathogenesis linking local infections with systemic diseases remains unclear, and experimental results may sometimes contradict the intended direction of research.

[0005] Porphyromonas gingivalis has many different subtypes, each with varying levels of bacterial toxicity. Bacterial toxicity and abnormal immune responses have been a focal point of debate in the scientific community when studying bacterial pathogenic mechanisms.

[0006] Currently, the primary clinical treatment for periodontal disease involves removing local lesions using broad-spectrum antibiotics and physical / mechanical methods, with surgical treatment performed as needed. In clinical practice, problems that need to be addressed comprehensively include how to prevent, treat, and control Porphyromonas gingivalis infection and mitigate local and systemic damage caused by bacterial infection, how to select biological products with immunoprotective effects, and how to evaluate the function of new drugs using animal models. [Overview of the project] [Problems that the invention aims to solve]

[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 disease).

[0008] The technical problem that this invention aims to solve is to provide evidence that antibodies against Porphyromonas gingivalis outer membrane protein are involved in local tissue and systemic damage in infected individuals.

[0009] A further technical problem that the present invention aims to solve is to provide the heterophilic antigenic properties of Porphyromonas gingivalis outer membrane proteins and the pathogenesis of local and systemic disorders caused by Porphyromonas gingivalis infection.

[0010] A further technical problem that this invention aims to solve is to provide a novel animal model for evaluating the protective function of a product. [Means for solving the problem]

[0011] In some embodiments, the present invention discloses the pathogenesis of Porphyromonas gingivalis, specific monoclonal antibodies, and methods for producing the same. The present invention also discloses specific antigen targets corresponding to monoclonal antibodies, and methods for identifying these antigen targets. In some embodiments, novel untagged RagB and Cra4S1 recombinant plasmids and their nucleotide and amino acid sequences are also disclosed (see SEQ ID NO: 1-10). The present invention also provides methods for generating recombinant proteins from plasmids, including the design of artificially synthesized genes, expression cassettes, and recombinant vectors or cells. In some embodiments, specific antigen target sequences of the above recombinant proteins are shown in SEQ ID NO: 11-126.

[0012] The monoclonal antibodies described in the present invention target a plurality of antigen targets disclosed herein. In some embodiments, the nucleotide and amino acid sequences of the monoclonal antibodies corresponding to the antigen targets are shown in SEQ ID NO: 128-167. In some embodiments of the present invention, bispecific antibodies and methods for producing the same are also provided. In some embodiments, the nucleotide and amino acid sequences of the bispecific antibodies are shown in SEQ ID NO: 168-174, and the present invention also provides methods for producing these bispecific antibodies.

[0013] Furthermore, the present invention provides a method for preventing and treating periodontal disease / peri-implantitis and other diseases caused by chronic infection with Porphyromonas gingivalis, using one or more monoclonal antibodies disclosed herein. In some embodiments, the combined antibody preparation has a mass ratio of RagB-4-1B11-4-7-7 monoclonal antibody to RagB-4-1C3-7-8 monoclonal antibody of 1:0.5–5. The present invention also provides expression cassettes, recombinant vectors, and eukaryotic cell applications for producing monoclonal and / or bispecific antibody pharmaceuticals for the prevention or treatment of periodontal disease / peri-implantitis and diseases caused by chronic Porphyromonas gingivalis infection. The monoclonal and / or bispecific antibodies in the present invention are used in patients to prevent the underlying disease, recurrent disease, and chronic infections and non-oral / dental diseases caused by Porphyromonas gingivalis infection.

[0014] The present invention also provides prophylactic and / or therapeutic vaccines for preventing and / or treating diseases associated with periodontal disease and / or peri-implantitis or Porphyromonas gingivalis infection. In some embodiments, the amino acid sequence of such vaccines is shown in SEQ ID NO. 127. The vaccines described in the present invention are used in patients to prevent the underlying disease, recurrence of the disease in patients with periodontal disease / peri-implantitis, and chronic infections and non-oral / dental diseases caused by Porphyromonas gingivalis infection.

[0015] The present invention also provides novel animal models. In some embodiments, animals are infected with Porphyromonas gingivalis and used to evaluate the effects of monoclonal antibodies and / or antibody-antigen reactions or cross-reactions according to the present invention on animal reproduction.

[0016] The present invention also provides a serological diagnostic kit for diagnosing and evaluating the therapeutic effect and prognosis of Porphyromonas gingivalis infection and related diseases using specific diagnostic reagents. In some embodiments, the amino acid sequences of these diagnostic reagents are as shown in SEQ ID NO. 2, 4, 6, 8, and 10. The diagnostic kit also provides instructions for use with reagents for testing specific antibodies in biological samples, including but not limited to bodily fluids such as blood, gingival crevicular exudate, urine, saliva, cerebrospinal fluid, pleural fluid / ascites, and amniotic fluid. [Brief explanation of the drawing]

[0017] The multifaceted content disclosed in this invention can be better understood by referring to the drawings. The drawings are not necessarily drawn to scale, and it is important that they clearly illustrate the principles of the invention. Furthermore, the parts corresponding to the reference numerals in the drawings have multiple meanings. The drawings (hereinafter abbreviated as "Figures") can be applied to the principles of the invention and, in combination with the detailed descriptions in the embodiments, can be better understood. [Figure 1A] The RagB-1 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 1A shows SEQ ID NO: 1, which has a total length of 1461 nucleotides and an average C+G content of 55.10%. [Figure 1B] The RagB-1 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 1B shows SEQ ID NO: 2, which has a total length of 482 amino acids. See Example 1 for details. [Figure 2A] Figure 2A shows the RagB-2 nucleic acid and amino acid sequence of Porphyromonas gingivalis. SEQ ID NO: 3 has a total length of 1449 nucleotides and an average C+G content of 51.62%. See Example 1 for details. [Figure 2B] The RagB-2 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 2B shows SEQ ID NO: 4, which has a total length of 480 amino acids. See Example 1 for details. [Figure 3A]The RagB-3 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 3A shows SEQ ID NO: 5, which has a total length of 1470 nucleotides and an average C+G content of 51.29%. See Example 1 for details. [Figure 3B] The RagB-3 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 3B shows SEQ ID NO: 6, which has a total length of 485 amino acids. See Example 1 for details. [Figure 4A] The RagB-4 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 4A shows SEQ ID NO: 7, which has a total length of 1455 bases and an average C+G content of 51.00%. See Example 1 for details. [Figure 4B] The RagB-4 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 4B shows SEQ ID NO: 8, which has a total length of 480 amino acids. See Example 1 for details. [Figure 5A] The Cra4S1 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 5A shows SEQ ID NO: 9, which has a total length of 411 nucleotides and an average C+G content of 52.55%. See Example 1 for details. [Figure 5B] The Cra4S1 nucleic acid and amino acid sequence of Porphyromonas gingivalis are shown. Figure 5B shows SEQ ID NO: 10, which has a full length of 132 amino acids. See Example 1 for details. [Figure 6] The polypeptide sequence alignment between four major outer membrane proteins, including RagB-1, RagB-2, RagB-3, and RagB-4, is shown using CLUSTALW(1.81) multiplex sequence alignment. Figure 6 discloses the sequences of SEQ ID NO: 2 (new name RagB-1, old name W50 RagB), 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). See Example 1 for details. [Figure 7A] Shows the expression, detection, purification and identification of RagB-1 recombinant protein. Figure 7A shows the expression of the recombinant protein after induction. Lane M is a protein molecular weight marker (15~120 kDa), lanes 1 to 3 are 0.5 μg, 1 μg, and 2 μg of BSA standard, respectively, lanes 4 to 6 are total bacterial proteins (total bacterial lysate) after seed cells were cultured for 7, 4 and 24 hours, respectively, lanes 7 to 9 are the centrifugation supernatant of total bacterial lysate after seed cells were cultured for 7, 4 and 24 hours, respectively, and lanes 10 to 12 are the centrifugation precipitate of total bacterial lysate after seed cells were cultured for 7, 4 and 24 hours, respectively. For details, please refer to Example 1. [Figure 7B] Shows the expression, detection, purification and identification of RagB-1 recombinant protein. Figure 7B shows the purified recombinant protein detected by SDS-PAGE. Lane M is a protein molecular weight marker (15~120 kDa), lane 1 is non-reduced recombinant protein RagB-1, and lane 2 is reduced recombinant protein RagB-1. For details, please refer to Example 1. [Figure 8A] Shows the expression, detection, purification and identification of RagB-2 recombinant protein. Figure 8A shows the expression of the recombinant protein after induction. Lane M is a protein molecular weight marker (15~120 kDa), lanes 1 to 3 are 0.5 μg, 1 μg, and 2 μg of BSA standard, respectively, lanes 4 to 6 are total bacterial proteins (total bacterial lysate) of seed cells obtained after uninduced culture and 22 hours of culture, respectively, and lanes 7 to 9 are the centrifugation supernatant of total bacterial lysate of seed cells obtained after uninduced culture and 22 hours of culture, respectively. For details, please refer to Example 1. [Figure 8B] Shows the expression, detection, purification and identification of RagB-2 recombinant protein. Figure 8B shows the purified recombinant protein detected by SDS-PAGE. Lane M is a protein molecular weight marker (15~120 kDa), lane 1 is non-reduced recombinant protein RagB-2, and lane 2 is reduced recombinant protein RagB-2. For details, please refer to Example 1. [Figure 9A]Shows the expression, detection, purification and identification of RagB-3 recombinant protein. Figure 9A shows the expression of the recombinant protein after induction. Lane M is protein molecular weight markers (15-120 kDa), Lanes 1-3 are 0.5 μg, 1 μg and 2 μg of BSA standard, Lanes 4-6 are whole bacterial proteins (whole bacterial lysate) obtained from uninduced culture and 22-hour culture of seed cells, and Lanes 7-9 are centrifugal supernatants of whole bacterial lysate obtained from uninduced culture and 22-hour culture of seed cells. Refer to Example 1 for details. [Figure 9B] Shows the expression, detection, purification and identification of RagB-3 recombinant protein. Figure 9B shows the purified recombinant protein detected by SDS-PAGE. Lane M is protein molecular weight markers (15-120 kDa), Lane 1 is non-reduced recombinant protein RagB-3, and Lane 2 is reduced recombinant protein RagB-3. Refer to Example 1 for details. [Figure 10A] Shows the expression, detection, purification and identification of RagB-4 recombinant protein. Figure 10A shows the expression of the recombinant protein after induction. Lane M is protein molecular weight markers (15-120 kDa), Lanes 1-3 are 0.5 μg, 1 μg and 2 μg of BSA standard, Lanes 4-6 are whole bacterial proteins (whole bacterial lysate) obtained after 6-hour and 15.5-hour culture of seed cells, Lanes 7-9 are centrifugal supernatants of whole bacterial lysate obtained after 6-hour and 15.5-hour culture of seed cells, and Lanes 10-12 are centrifugal precipitates of whole bacterial lysate obtained after 6-hour and 15.5-hour culture of seed cells. Refer to Example 1 for details. [Figure 10B] Shows the expression, detection, purification and identification of RagB-4 recombinant protein. Figure 10B shows the purified recombinant protein detected by SDS-PAGE. Lane M is protein molecular weight markers (15-120 kDa), Lane 1 is non-reduced recombinant protein RagB-4, and Lane 2 is reduced recombinant protein RagB-4. Refer to Example 1 for details. [Figure 11A]The expression, testing, purification, and identification of the Cra4S1 recombinant protein are shown. Figure 11A shows the expression of the recombinant protein after induction, with lane M representing protein molecular weight markers (15-120 kDa), lanes 1-3 representing 0.5 μg, 1 μg, and 2 μg of BSA standards, lanes 4-6 representing total bacterial proteins (total bacterial lysate) after culturing seed cells for 14 and 23 hours, lanes 7-9 representing the supernatant after centrifugation of total bacterial lysate after culturing seed cells for 14 and 23 hours, and lanes 10-12 representing the precipitate after centrifugation of total bacterial lysate after culturing seed cells for 14 and 23 hours. See Example 1 for details. [Figure 11B] The expression, testing, purification, and identification of the recombinant Cra4S1 protein are shown. Figure 11B shows the purified recombinant protein tested by SDS-PAGE, where lane M is the protein molecular weight marker (15-120 kDa), lane 1 is the non-reduced recombinant protein Cra4S1, and lane 2 is the reduced recombinant protein Cra4S1. See Example 1 for details. [Figure 12A] Figure 12A shows the distribution of antibodies against different outer membrane proteins of Porphyromonas gingivalis in 51 serum samples from healthy individuals. Figure 12A indicates that the mean OD value of the least quintile (10 / 51) samples is set as the negative value. The positive rate is set as an OD value greater than 2.5 times the negative value. The positive rate for RagB-1 antibody was 9.8% (5 / 51), with high-titer antibodies (OD value greater than 4 times the negative value) at 2% (1 / 51). The positive rate for RagB-2 antibody was 0% (0 / 51). The positive rate for RagB-3 antibody was 0% (0 / 51), with high-titer antibodies at 2% (1 / 51). The positive rate for RagB-4 antibody was 17.6% (9 / 51), with high-titer antibodies at 13.7% (7 / 51). The positive rate for Cra4S1 antibody was 9.8% (5 / 51), with high-titer antibodies at 0% (0 / 51). The positive rate for unrelated protein GST antibody was 0% (0 / 51). For details, please refer to Example 5. [Figure 12B]Figure 12B shows the distribution of antibodies against different outer membrane proteins of Porphyromonas gingivalis in 51 serum samples from healthy individuals. Figure 12B shows the proportion of antibody distribution in the analyzed samples. See Example 5 for details. [Figure 13] This shows the distribution of the ragB gene in Porphyromonas gingivalis in gingival crevicular exudate samples from 107 periodontal disease patients. As a result, 28 patients were negative for Porphyromonas gingivalis 16s RNA by PCR testing (28 / 107, 26.2%). Among the 16s RNA-positive samples, PCR ragB amplification testing revealed the following: 7 patients were positive for the RagB-1 subtype (7 / 107, 6.6%), 30 for the RagB-2 subtype (30 / 107, 28.0%), 32 for the RagB-3 subtype (32 / 107, 29.9%), 6 for the RagB-4 subtype (6 / 107, 5.6%), and 4 for an unknown ragB subtype (4 / 107, 3.7%). See Example 5 for details. [Figure 14A] Figure 14A shows the distribution of antibodies against different outer membrane proteins of Porphyromonas gingivalis in 62 serum samples from elderly patients. Figure 14A indicates that the mean OD value of the least quintile (12 / 62) samples is set as the negative value. The positive rate is set as an OD value greater than 2.5 times the negative value. The positive rate for RagB-1 antibody was 25.8% (16 / 62), with high-titer antibodies (OD value greater than 4 times the negative value) at 22.6% (14 / 62). The positive rate for RagB-2 antibody was 24.2% (15 / 62), with high-titer antibodies at 24.2% (15 / 62). The positive rate for RagB-3 antibody was 22.6% (14 / 62), with high-titer antibodies at 24.2% ( The positive rates were 15 / 62 for RagB-4 antibody (16.1%, 10 / 62), with high titer antibodies at 22.6%, for Cra4S1 antibody (22.6%, 14 / 62), with high titer antibodies at 21%, and for unrelated protein GST antibody (19.4%, 12 / 62), with high titer antibodies at 21%, for 21%, for 13%. See Example 5 for details. [Figure 14B]Figure 14B shows the distribution of antibodies against different outer membrane proteins of Porphyromonas gingivalis in 62 serum samples from elderly patients. The percentage of antibody distribution in the analyzed samples is shown. See Example 5 for details. [Figure 15A] Figure 15A shows the distribution of antibodies against different outer membrane proteins of Porphyromonas gingivalis in 49 serum samples from cardiovascular patients. Figure 15A indicates that the mean OD value of the least quintile (10 / 49) samples is set as the negative value. The positive rate is set as an OD value greater than 2.5 times the negative value. The positive rate for RagB-1 antibodies was 36.7% (18 / 49), with high titer antibodies (OD value greater than 4 times the negative value) at 8.2% (4 / 49); for RagB-2 antibodies, it was 32.7% (16 / 49), with high titer antibodies at 8.2% (4 / 49); and for RagB-3 antibodies, it was 38.8% (19 / 49), with high titer antibodies at 8.2% (4 / 4). 9) The positive rate for RagB-4 antibody was 46.9% (23 / 49), with high titer antibodies at 10.2% (5 / 49); the positive rate for Cra4S1 antibody was 36.7% (18 / 49), with high titer antibodies at 4.1% (2 / 49); and the positive rate for unrelated protein GST antibody was 49% (24 / 49), with high titer antibodies at 4.1% (2 / 49). For details, see Example 5. [Figure 15B] Figure 15B shows the distribution of antibodies against different outer membrane proteins of Porphyromonas gingivalis in 49 serum samples from cardiovascular patients. Figure 15B shows the percentage of antibody distribution in the analyzed samples. See Example 5 for details. [Figure 16] This shows the protein sequence alignment between the outer membrane proteins of Porphyromonas gingivalis RagB-4 and Porphyromonas gyae strain COT-052 OH2179. Aligning the outer membrane protein sequence (PubMed ID: 25858832, GenBank: JRAJ01000005.1) with RagB-4 (SEQ ID NO: 8) revealed a 93% similarity between the two proteins. See Example 5 for details. [Figure 17]This study explores the study of passive immunity function using a combination of two target-specific antibodies. The photographs were taken 12 days after Porphyromonas gingivalis attack. Figure 17A shows that mice in group G1 were administered the monoclonal antibody (MAb) 1D2-2-1-3. Figure 17B shows that mice in group G2 were administered MAb 1D2-2-1-3 and a mouse polyclonal antibody against Cra4S1. Figure 17C shows that mice in group G3 were administered a mouse polyclonal antibody against Cra4S1. Figure 17D shows that mice in group G4 were administered normal mouse serum. Figure 17E shows that mice in group G5 were administered PBS and used as a control group for bacterial attack. See Example 6 for details. [Figure 18A] The passive immune defense function of the composite antibody is shown. For details, please refer to Example 6. The X-axis represents the test animal group, and the Y-axis represents the lesion area (mm2) of the skin and soft tissue. Figure 18A shows the results 5 days after attack. For details, please refer to Example 6. Statistical analysis was performed using one-way ANOVA, and a P<0.05 indicates a statistically significant difference. [Figure 18B] The passive immune defense function of the composite antibody is shown. For details, please refer to Example 6. The X-axis represents the test animal group, and the Y-axis represents the lesion area (mm2) of the skin and soft tissue. Figure 18B shows the results 12 days after attack. For details, please refer to Example 6. Statistical analysis was performed using one-way ANOVA, and a P<0.05 indicates a statistically significant difference. [Figure 19] Figure 19A shows the sequence of the monoclonal antibody RagB-2-1A4-3-7-7. Figure 19A shows the DNA sequence (SEQ ID NO: 128) of the heavy chain (mouse IgG1) variable region (VH) confirmed by sequencing. Figure 19B shows the amino acid sequence (SEQ ID NO: 129) of VH. Figure 19C shows the DNA sequence (SEQ ID NO: 133) of the light chain (mouse Kappa) variable region (VL) confirmed by sequencing. Figure 19D shows the amino acid sequence (SEQ ID NO: 134) of VL. See Example 7 for details. [Figure 20]Figure 20A shows the sequence of the monoclonal antibody RagB-3-1D2-2-1-3 and the analysis of the recombinant antibody expressed and purified in eukaryotic HEK293 cells. Figure 20A shows the DNA sequence (SEQ ID NO: 138) of the heavy chain (mouse IgG2a) variable region (VH) confirmed by sequencing. Figure 20B shows the amino acid sequence (SEQ ID NO: 139) of VH. Figure 20C shows the DNA sequence (SEQ ID NO: 143) of the light chain (mouse Kappa) variable region (VL) confirmed by sequencing. Figure 20D shows the amino acid sequence (SEQ ID NO: 144) of VL. Figure 20E shows the peak at 280 nm obtained by collecting the eluate after the recombinant antibody was purified by HPLC. Figure 20F shows purified recombinant antibodies confirmed by SDS-PAGE, where lane M represents protein molecular weight markers (15-120 kDa) and lane 1 represents purified RagB-3-1D2-2-1-3. See Example 7 for details. [Figure 21] Figure 21A shows the sequence of the monoclonal antibody RagB-4-1B11-4-4 and the analysis of the recombinant RagB-4-1B11-4-4 antibody expressed and purified in eukaryotic HEK293 cells. Figure 21A shows the DNA sequence (SEQ ID NO: 148) of the heavy chain (mouse IgG1) variable region (VH) confirmed by sequencing. Figure 21B shows the amino acid sequence (SEQ ID NO: 149) of VH. Figure 21C shows the DNA sequence (SEQ ID NO: 153) of the light chain (mouse Kappa) variable region (VL) confirmed by sequencing. Figure 21D shows the amino acid sequence (SEQ ID NO: 154) of VL. Figure 21E shows the peak at 280 nm obtained by collecting the eluate after the recombinant antibody was purified by HPLC. Figure 21F shows purified recombinant antibodies confirmed by SDS-PAGE, where lane M represents protein molecular weight markers (15-120 kDa) and lane 1 represents purified RagB-4-1B11-4-4. See Example 7 for details. [Figure 22]Figure 22A shows the sequence of the monoclonal antibody RagB-4-1C3-7-8 and the analysis of the recombinant RagB-4-1C3-7-8 antibody expressed and purified in eukaryotic HEK293 cells. Figure 22A shows the DNA sequence (SEQ ID NO: 158) of the heavy chain (mouse IgG1) variable region (VH) confirmed by sequencing. Figure 22B shows the amino acid sequence (SEQ ID NO: 159) of VH. Figure 22C shows the DNA sequence (SEQ ID NO: 163) of the light chain (mouse Kappa) variable region (VL) confirmed by sequencing. Figure 22D shows the amino acid sequence (SEQ ID NO: 164) of VL. Figure 22E shows the peak at 280 nm obtained by collecting the eluate after purification of the recombinant antibody by HPLC. Figure 22F shows purified recombinant antibodies confirmed by SDS-PAGE, where lane M is the protein molecular weight marker (15-120 kDa) and lane 1 is the purified RagB-4-1C3-7-8. See Example 7 for details. [Figure 23A] The design sequence of the bispecific antibody 1B11-1C3 and the analysis of the recombinant plasmid expressing it in eukaryotic HEK293 cells are shown. Figure 23A shows the DNA sequence (SEQ ID NO: 168) of the recombinant heavy chain variable region (VH) confirmed by sequencing. See Example 8 for details. [Figure 23B] The design sequence of the bispecific antibody 1B11-1C3 and the analysis of the recombinant plasmid expressing it in eukaryotic HEK293 cells are shown. Figure 23B shows the amino acid sequence of VH (SEQ ID NO: 169). See Example 8 for details. [Figure 23C] The design sequence of the bispecific antibody 1B11-1C3 and the analysis of the recombinant plasmid expressing it in eukaryotic HEK293 cells are shown. Figure 23C shows the heavy chain sequence of the bispecific antibody, with bold and underlined parts indicating the original specific MAb. See Example 8 for details. [Figure 23D]Figure 23D shows the design sequence of the bispecific antibody 1B11-1C3 and the analysis of the recombinant plasmid expressing it in eukaryotic HEK293 cells. The DNA sequence of the light chain (mouse Kappa) variable region (VL) confirmed by sequencing (SEQ ID NO: 173) is shown. See Example 8 for details. [Figure 23E] The design sequence of the bispecific antibody 1B11-1C3 and the analysis of the recombinant plasmid expressing it in eukaryotic HEK293 cells are shown. Figure 23E shows the amino acid sequence of VL (SEQ ID NO: 174). See Example 8 for details. [Figure 23F] The design sequence of the bispecific antibody 1B11-1C3 and the analysis of the recombinant plasmid expressing it in eukaryotic HEK293 cells are shown. Figure 23F shows the sequence diagram of the light chain of the bispecific antibody, with bold and underlined parts indicating the original specific MAb. See Example 8 for details. [Figure 23G] The design sequence of the bispecific antibody 1B11-1C3 and the analysis of the recombinant plasmid expressing it in eukaryotic HEK293 cells are shown. Figure 23G shows the peak at 280 nm obtained by collecting the eluate after purification of the recombinant bispecific antibody by HPLC. See Example 8 for details. [Figure 23H] Figure 23H shows the design sequence of the bispecific antibody 1B11-1C3 and the analysis of the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23H shows the purified recombinant antibody confirmed by SDS-PAGE, where lane M represents the protein molecular weight marker (15-120 kDa), lane R represents the reduced form 1B11-1C3, 76 kDa (heavy chain) / 24 kDa (light chain), and lane NR represents the unreduced form 1B11-1C3, 200 kDa. See Example 8 for further details. [Modes for carrying out the invention]

[0018] This invention presents and describes various methods for carrying out the invention, but to those skilled in the art, these embodiments are provided only as examples. Those skilled in the art can make various changes, modifications and substitutions within the scope of the invention. Several improvements and modifications may be made without departing from the principles of the invention, and these improvements and modifications should also be considered to be within the scope of protection of the invention. Definition of Terms

[0019] With reference to the foregoing description and the associated drawings, many modifications and other embodiments will come to mind for those skilled in the art. Therefore, it should be understood that the disclosure of the present invention is not limited to any particular embodiment, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Many variations and adaptability will be recognized by those skilled in the art. These variations and adaptability are intended to be included within the teachings of the disclosure of the present invention and are protected by the claims.

[0020] Although this invention uses specific terminology, these terms are used only in a general and descriptive sense, not in a restrictive sense.

[0021] Those skilled in the art will, after reading the information disclosed in this invention, recognize that each specific embodiment described and illustrated in this invention has independent combinations and features without departing from the scope or spirit of the invention, and that these combinations and features can be separated or combined with features of several other embodiments.

[0022] Any of the listed methods may be performed in the order of the listed events, or in any other logically possible order. That is, unless otherwise specified, no method or embodiment of the present invention requires its steps to be performed in a particular order. Accordingly, no order should be presumed unless a claim for a method specifically states that the steps should be limited to a particular order. This rule applies to the basis of possible implicit interpretations, including logical issues relating to the arrangement or flow of operations of the steps, the usual meaning arising from grammatical structure or punctuation, or the number or type of embodiments described in the specification.

[0023] All published publications and patents cited herein are incorporated herein by reference to disclose and describe methods and / or materials relating to those publications. Each publication or patent is specifically and individually designated as to be incorporated by reference. Such references are limited to the methods and / or materials described in the cited publications and patents and do not extend to any definitions of terms in the cited publications and patents. No definition of a term provided in any cited publication or patent shall be considered a definition unless expressly repeated herein, and shall not be construed as defining any term in any appended claim. Citing any publication for disclosure prior to the filing date shall not be construed as acknowledging that the current disclosure does not precede such publication by prior disclosure. Furthermore, the provided publication dates may differ from the actual publication dates, and the actual publication dates may need to be verified individually.

[0024] Related publications and patents cited herein in relation to the methods and / or materials are incorporated herein by reference as references. Each individual publication or patent is specifically and individually indicated in its form of reference. Such citations are expressly limited to the methods and / or materials described in the cited publications and patents and do not extend to any dictionary definitions in the cited publications or patents. Dictionary definitions in cited publications and patents shall not be considered dictionary definitions unless expressly repeated herein and should not be construed as defining any term appearing in the appended claims. Citation of any publication prior to the filing date should not be construed as acknowledging that this disclosure does not have priority. Furthermore, publication dates provided herein may differ from actual publication dates and may require separate verification.

[0025] The disclosures of this invention may be described and claimed in certain legal categories, such as the System Legal Category, but this is for convenience only, and those skilled in the art will understand that each aspect of the disclosures of this invention may be described and claimed in any legal category.

[0026] It should be understood that the terms used herein are for the purpose of describing specific aspects and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Furthermore, like terms defined in general dictionaries, they should be interpreted as having a consistent meaning within the context of this specification and the relevant technical field, and should not be interpreted as idealized or overly formal unless explicitly defined herein.

[0027] Each aspect of the disclosure of this invention employs techniques from molecular biology, microbiology, organic chemistry, biochemistry, physiology, cell biology, vascular biology, and other fields, and unless otherwise specified, these techniques are mature in the art. These techniques are described in detail in the specification.

[0028] Before describing each aspect of the present invention, unless otherwise specified, the following definitions are provided and used. Additional terms may be defined elsewhere in the present invention.

[0029] As used in this invention, “including” should be interpreted as specifying the existence of the mentioned feature, integer, step, or component, and not as negating the existence or addition of one or more features, integers, components, or groups thereof. Furthermore, the terms “from,” “inclusion,” “including,” “consisting,” “including,” “concerning,” “concerning,” and “for example” are used in an open and non-restrictive sense and can be used interchangeably. Furthermore, the term “including” is intended to include examples and embodiments covered by the terms “essentially consisting of” and “consisting of.” Similarly, the term “essentially consisting of” is intended to include examples covered by the term “consisting of.”

[0030] As used in this invention, the term "and / or" includes any and all combinations of one or more of the listed items in question. Expressions such as "at least one" preceding a list of elements modify the entire list of elements, rather than the individual elements within the list.

[0031] As used in the specification and claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless otherwise indicated in the context. For example, a reference to “therapeutic drug” includes one or more therapeutic drugs and / or combinations of one or more therapeutic drugs.

[0032] Each reference to a compound, therapeutic agent, and pharmaceutical composition “one” refers to one or more molecules of that compound, therapeutic agent, and pharmaceutical composition, and is not limited to a single compound, therapeutic agent, or pharmaceutical composition. These one or more molecules may be identical or distinct, as long as they belong to the class of compound, therapeutic agent, and pharmaceutical composition. For example, “one” therapeutic agent is interpreted as including one or more therapeutic agents that may be identical or distinct (e.g., different isotopic abundances and / or different degrees of hydration, or equilibrium states with different conjugate base or conjugate acid forms).

[0033] It should be noted that ratios, concentrations, quantities, and other numerical data may be expressed in range form in this specification. Furthermore, it should be understood that each range endpoint is important in comparison to and independent of other endpoints. Also, it should be understood that many values ​​are disclosed in this specification, and in addition to the values ​​themselves, each value is disclosed in this specification "about" that particular value. For example, if the value "10" is disclosed, "about 10" is also disclosed. Ranges can be expressed here as "about" one particular value and / or "about" another particular value. Similarly, when values ​​are expressed as approximations, it should be understood that the antecedent "about" forms another aspect of the particular value. For example, if the value "about 10" is disclosed, "10" is also disclosed.

[0034] When a range is expressed, it specifically refers to a range from one particular value to and / or another particular value. When a range of values ​​is provided, unless the context otherwise explicitly indicates, it is understood that the upper and lower limits of that range, each intermediate value between them and any other specified value or intermediate value within that range, and up to one-tenth of the unit of the lower limit are included in the invention. These upper and lower limits of smaller ranges may be independently included in smaller ranges and are also included in the invention, subject to any restrictions specifically excluded within the described range. If a described range includes one or both limits, ranges excluding one or both of those limits are also included in the invention. For example, if a described range includes one or both limits, such as the phrase "from x to y" including not only the range from "x" to "y" but also the range greater than "x" and less than "y", ranges excluding one or both of those limits are also included in the invention. This range can also be expressed as an upper limit; for example, "approximately x, y, z or less" should be interpreted to include not only the specific ranges of "approximately x," "approximately y," and "approximately z," but also the ranges of "less than x," "less than y," and "less than z." Similarly, the phrase "approximately x, y, z or greater" should be interpreted to include not only the specific ranges of "approximately x," "approximately y," and "approximately z," but also the ranges of "greater than x," "greater than y," and "greater than z." Furthermore, when 'x' and 'y' are numerical values, the phrase "from approximately 'x' to 'y'" includes "from approximately 'x' to approximately 'y'."

[0035] Because such range formats are used for convenience and conciseness, it is important to understand that they should be interpreted flexibly to include not only the numbers explicitly listed as limits to the range, but also all individual numbers or subranges that fall within that range, as if each number and subrange were explicitly listed. For the sake of explanation, the numerical range "approximately 0.1% to 5%" should be interpreted to include not only the explicitly cited values ​​from approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (e.g., approximately 1%, approximately 2%, approximately 3%, and approximately 4%) and subranges (e.g., approximately 0.5% to approximately 1.1%, approximately 0.5% to approximately 2.4%, approximately 0.5% to approximately 3.2%, and approximately 0.5% to approximately 4.4%, as well as other possible subranges).

[0036] As used herein, terms such as “approximately,” “about,” and “substantially” typically refer to the value of a numerical variable, as well as all values ​​within the range of experimental error (e.g., within a 95% confidence interval) or within + / - 10% of the indicated value. In this specification, “approximately,” “about,” “or approximately,” and “substantially” mean that the quantity or value in question is either an exact value or a value that may produce an equivalent result or effect. That is, to obtain an equivalent result or effect, quantities, dimensions, formulations, parameters, and other quantities and characteristics do not need to be perfectly accurate and may reflect approximations and / or values ​​that are larger or smaller than necessary, taking into account factors such as tolerances, conversion factors, rounding, and measurement errors. In some cases, the value that produces an equivalent result or effect may not be reasonably determined. Generally, whether explicitly stated or not, quantities, sizes, formulations, parameters, or other quantities or characteristics preceding “approximately,” “about,” or “or approximately” are “approximately,” “about,” or “or approximately,” and unless otherwise specified, include the value of the specific quantity itself.

[0037] When used in conjunction with numerical variables as used herein, “about,” “approximate,” “substantially,” etc., may typically refer to the larger of the value of the variable and all values ​​of the variable within the range of experimental error (e.g., within the 95% confidence interval of the mean) or within + / - 10% of the indicated value. The terms “about,” “approximate,” “equal to or about,” and “substantially,” as used herein, may mean that the quantity or value under consideration is an exact value or a value that would produce an equivalent result or effect as stated in the claims or shown herein. That is, while quantities, volumes, formulations, parameters, and other quantities and characteristics do not necessarily need to be exact to obtain an equivalent result or effect, it can be understood that tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art may be approximated and / or greater or less as needed. In some cases, it may not be reasonably possible to determine a value that would produce an equivalent result or effect, and it may not be possible to clearly explain in general that a quantity, volume, formulation, parameter, or other quantity or characteristic is “about,” “approximate,” or “equal to or about.” When using "approximately," "approximately," or "equal to or approximately" before a quantitative value, it is understood that the parameter includes the specific quantitative value itself, unless otherwise specified.

[0038] As used herein, “optional” or “optional” means whether or not the event or situation described thereafter may or may not occur, and such description includes both cases in which the event or situation occurs and cases in which it does not occur.

[0039] As used herein, “administration” may refer to active or passive (e.g., by diffusion) routes of administration into perivascular spaces and adventitia by oral, topical, intravenous, subcutaneous, percutaneous, intramuscular, intra-articular, parenteral, arteriole, dermal, ventricular, intraosseous, intraocular, intracranial, intraperitoneal, intrafocal, intranasal, intracardiac, intraarticular, intracavernosal, intradural, intradural, intravitreous, intracerebral and lateral ventricles, intratympanic cavity, cochlea, rectum, vagina, by inhalation, via catheter, stent, or implantable reservoir or other device. For example, medical devices such as stents contain compositions or formulations placed on their surface that can be dissolved or otherwise distributed to surrounding tissues and cells. The term “parenteral” may include subcutaneous, intravenous, intramuscular, intra-articular, synovial, intrasternal, intrathecal, intrahepatic, intrafocal, and intracranial injection or infusion techniques. Administration may be continuous or intermittent. In various forms, the formulation may be administered therapeutically, i.e., used to treat an existing disease or condition. Furthermore, the formulation may be administered prophylactically, i.e., used to prevent a disease or condition.

[0040] As used herein, “therapeutic agent” may refer to any substance, compound, molecule, etc. that may have biological activity or may produce pharmacological, immunogenic, biological, and / or physiological effects in a subject through local and / or systemic action. A therapeutic agent may be a primary activator, in other words, a component that produces all or part of the effect of the composition. A therapeutic agent may also be a secondary therapeutic agent, in other words, a component that produces an additional part and / or other effect of the composition. Accordingly, this term includes compounds or chemical substances traditionally considered drugs, vaccines, and biological preparations, including molecules such as proteins, peptides, hormones, nucleic acids, and gene constructs.

[0041] Examples of therapeutic agents are listed in well-known references such as the Merck Index (14th edition), the Physician's Desk Reference (64th edition), and the Fundamentals of Therapeutic Science (12th edition), and include, but are not limited to, drugs, vitamins, mineral supplements, substances used to treat, prevent, diagnose, cure or alleviate diseases or conditions, substances that affect the structure or function of the body, or prodrugs that become biologically active or more active when placed in a physiological environment. For example, the term "therapeutic drugs" includes anti-infective drugs such as adjuvants, antibiotics and antivirals, analgesics and combinations of analgesics, appetite suppressants, anti-inflammatory drugs, antiepileptic drugs, topical and general anesthetics, hypnotics, sedatives, antipsychotics, nerve blockers, antidepressants, anxiolytics, antagonists, neuroleptics, anticholinergics and cholinergic mimetic drugs, antimuscarinic drugs and muscarinic receptor agonists, antiadrenaline drugs, antiarrhythmics, antihypertensives, hormones and nutrients, antiarthritis drugs, antiasthmatics, anticonvulsants, antihistamines, analgesics, antitumor drugs, antipruritics, antipyretics, antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrhythmics), antihypertensives, diuretics, vasodilators, This includes, but is not limited to, central nervous system stimulants, cough suppressants and cold medicines, decongestants, diagnostic reagents, hormones, bone growth promoters and bone resorption inhibitors, immunosuppressants, muscle relaxants, psychostimulants, sedatives, sedatives, proteins, peptides and their fragments (naturally derived, chemically synthesized, or recombinantly produced), and nucleic acid molecules (ribonucleotides (RNA) or deoxyribonucleotides (DNA) including polymer forms of two or more nucleotides, double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (such as doxorubicin), and other bioactive polymers such as proteins and enzymes, encompassing all major compounds or compositions used in the therapeutic field. The therapeutic agents may also be bioactive agents used in medicine, including veterinary and agricultural (e.g., plant) applications, as well as bioactive agents used in other fields.The term therapeutic agent includes, but is not limited to, drugs, vitamins, mineral supplements, substances used to treat, prevent, diagnose, cure or alleviate a disease or condition, substances that affect the structure or function of the body, or prodrugs that become biologically active or more active when placed in a particular physiological environment.

[0042] As used herein, "kit" refers to a packaging box consisting of at least two components. These components together constitute a functional unit for a particular purpose. Individual components may be physically packaged together or separately. For example, the instruction manual included in the kit may or may not be physically packaged together with the other individual components. The instruction manual may be provided independently of the other components in paper or electronic format, stored on a computer storage device, downloaded from an internet website, or as a recording of a presentation.

[0043] The “Instruction Manual” mentioned above refers to a document describing the materials or methods associated with the kit. These materials may include any combination of background information, a list of components and their availability (such as purchase information), simple or detailed protocols for using the kit, troubleshooting, references, technical support, and other relevant documentation. The Instruction Manual may be provided with the kit or as a separate member component, and may be available in paper or electronic format, on a computer-readable storage device, downloaded from an internet website, or as a recorded presentation. The Instruction Manual may consist of one or more documents and may include future updates.

[0044] The terms “subject,” “individual,” or “patient,” as used interchangeably herein, may refer to vertebrates such as mammals (e.g., humans). “Subject” refers to cells, cell populations, tissues, organs, or organisms, preferably humans and their components.

[0045] As used herein, the terms “treatment” and “therapeutic” may generally refer to obtaining a desired pharmacological and / or physiological effect. This effect may be (but not necessarily) a prophylactic treatment to prevent or partially prevent a disease, symptom, or related condition. This effect may also be therapeutic, i.e., it may partially or completely cure a disease, symptom, or related condition. As used herein, the term “treatment” includes any treatment of inflammation associated with any disease in a subject, particularly in humans, and includes one or more of the following: (a) preventing the onset of the disease in a subject who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its progression; and (c) alleviating the disease, i.e., reducing or improving the disease and / or its symptoms or condition. As used herein, the term “treatment” may refer to treatment only, prophylactic treatment only, or both treatment and prophylactic treatment. Persons requiring treatment (subjects requiring treatment) include those who already have the disease and / or those who need to prevent the disease, etc. As used herein, the term “treatment” includes inhibiting a disease, discomfort, or medical condition. For example, it includes alleviating a disease, discomfort, or medical condition by preventing its progression and causing regression of the disease, discomfort, and / or medical condition. Treatment of a disease, discomfort, or medical condition includes improving at least one symptom of a particular disease, discomfort, or medical condition, even if it does not affect the underlying pathophysiology. For example, administering an analgesic may treat a subject’s pain even if the drug does not treat the cause of the pain.

[0046] As used herein, the term “therapeutic dose” refers to an amount sufficient to achieve the desired therapeutic outcome or to affect an undesirable symptom, but not usually sufficient to cause adverse side effects. Specific therapeutically effective dose levels vary from patient to patient depending on a variety of factors, including the disease being treated and its severity, the specific composition used, the patient’s age, weight, overall health, sex and diet, time of administration, route of administration, excretion rate of the specific compound used, duration of treatment, drugs used in combination with or concurrently with the specific compound used, and similar factors within the scope of the general practitioner’s knowledge and expertise, although these factors may be well known in the medical field. When treating a particular disease or condition, the desired response may, in some cases, be the inhibition of disease or condition progression. This may only be a temporary slowing of disease progression. However, in other cases, it may be necessary to permanently halt disease progression. This can be monitored by conventional diagnostic methods for any particular disease, known to those skilled in the art. Expected responses to treatment of a disease or condition may include delaying the onset of the disease or condition, or even preventing its onset.

[0047] For example, it is within the skill of a person skilled in the art to schedule the dosage of the drug required to obtain a therapeutic effect, starting at a low level and gradually increasing until the desired effect is achieved. If necessary, the therapeutically effective daily dose can be administered in multiple doses. Therefore, a single-dose composition may contain these amounts or multiples thereof to constitute a daily dose. If there are contraindications, a specialist can adjust the dose. In general, it is preferable to use the maximum dose of the pharmacological agent of the present invention (alone or in combination with other therapeutic agents), i.e., the safest dose based on reasonable medical judgment. However, a person skilled in the art will understand that a patient may request a lower dose or tolerable dose for medical, psychological, or almost any other reason.

[0048] For example, the response to a therapeutically effective dose of a disclosed compound and / or pharmaceutical composition can be measured by determining the physiological effect of the treatment or drug, such as the reduction or disappearance of disease symptoms after administration of the treatment or pharmacological agent. Measuring the response level is within the scope of the art for those skilled in the art. The therapeutic dose can be varied, for example, by increasing or decreasing the amount of the disclosed compound and / or pharmaceutical composition, changing the disclosed administered compound and / or pharmaceutical composition, changing the route of administration, or changing the administration schedule. The dose may vary and may be administered once or multiple times a day, for one day or several days. Guidelines on appropriate doses for specific types of pharmaceuticals can be found in the literature.

[0049] As used herein, the term “preventive effective dose” refers to the amount that effectively prevents the onset or commencement of a disease or condition.

[0050] As used herein, the terms “prevention” or “prevention” refer to eliminating, avoiding, erasing, preventing, blocking, or suppressing the occurrence of something through prior action. Unless otherwise specified, when using “mitigation,” “suppression,” or “prevention” herein, it should be understood that the use of the other two words is also expressly disclosed.

[0051] As used herein, the term “pharmaceutically acceptable” means a substance that does not cause biological or other adverse effects, i.e., a substance that does not cause unacceptable levels of harmful biological effects or interact in a harmful manner.

[0052] As used herein, the term “pharmaceutically acceptable salt” refers to a salt prepared from an acid or base and an activator that is tolerable by a living organism, or a salt prepared from an activator and an acid or base that is tolerable by a subject or living organism when used within an effective therapeutic dose range. When the compounds described in the present invention contain relatively acidic functional groups, base addition salts can be obtained in their pure form or in a suitable inert solvent by contacting these compounds in a neutral form or with a sufficient amount of base. 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 in their pure form or in a suitable inert solvent by contacting the neutral form of these compounds with a sufficient amount of the desired acid. 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, bicarbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, and phosphoric acid, as well as salts 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, and methanesulfonic acid. They also include salts of amino acids such as arginates, and salts of organic acids such as glucuronates or galacturonates.

[0053] As used herein, the term “pharmaceutically acceptable ester” refers to esters of the compounds disclosed herein, including those that are hydrolyzed in vivo and readily decomposed in the human body, leaving the parent compound or a salt thereof. Examples of pharmaceutically acceptable non-toxic esters disclosed herein include C1-C6 alkyl esters and C5-C7 cycloalkyl esters, but C1-C4 alkyl esters are preferred. Esters of the compounds disclosed herein can be produced according to conventional methods. Pharmaceutically acceptable esters can be produced by attaching a hydroxyl group to a compound containing a hydroxyl group by reaction with an acid and an alkyl carboxylic acid such as acetic acid or an acid and an aryl carboxylic acid such as benzoic acid. In the case of compounds containing a carboxylic acid group, pharmaceutically acceptable esters are produced from compounds containing a carboxyl group by reaction with a base such as triethylamine and an alkyl halide such as methyl iodide, benzyl iodide, or cyclopentyl iodide, or an alkyl ester of trifluoromethanesulfonate. They can also be produced by reaction with an acid such as hydrochloric acid and an alcohol such as ethanol or methanol.

[0054] The term "pharmaceutically acceptable amide" refers to the non-toxic amides disclosed in this invention, derived from ammonia, primary C1-C6 alkylamines, and secondary C1-C6 dialkylamines. In the case of secondary amines, the amine may be in the form of a five- or six-membered heterocyclic ring containing one nitrogen atom. Amides derived from ammonia, C1-C3 alkyl primary amides, and C1-C2 dialkyl secondary amides are preferred. Amides of the compounds disclosed in this invention can be produced according to conventional methods. pharmaceutically acceptable amides can be produced from compounds containing a primary or secondary amine group by reacting the amino group-containing compound with an alkyl anhydride, aryl anhydride, acyl halide, or alloyl halide. In the case of compounds containing a carboxylic acid group, pharmaceutically acceptable amides are produced from compounds 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 or dialkylamine such as methylamine, diethylamine, or piperidine. These can also be produced under dehydrating conditions, such as the addition of molecular sieves, by reacting the compound with an acid such as sulfuric acid and an alkylcarboxylic acid such as acetic acid, or with an acid and an arylcarboxylic acid such as benzoic acid. This composition may contain the compounds disclosed in this invention in a pharmaceutically acceptable prodrug form.

[0055] The terms “pharmaceutically acceptable prodrug” or “prodrug” mean a prodrug of the compounds disclosed in this invention, which, within reasonable medical judgment, are suitable for use in contact with human and lower animal tissues, do not exhibit excessive toxicity, irritation, or allergic reactions, and are effective for their intended use in proportion to reasonable benefits / risks. The prodrugs disclosed in this invention can be rapidly converted in vivo, for example, by hydrolysis in the blood, to parent compounds having the disclosed compound structure. T. Higuchi and V. Stella discuss this in detail in ACS Symposium Series Vol. 14, “Prodrugs as Novel Delivery Systems,” and Edward B. Roche (ed.), “Bioreversible Carriers in Drug Design,” American Pharmaceutical Society and Pergamon Press (1987).

[0056] As used herein, the term “derivative” refers to a compound having a structure derived from a parent compound structure (e.g., a compound disclosed herein) whose structure is sufficiently similar to the structure disclosed herein, and based on this similarity, it is expected that, within the scope of the art of those skilled in the art, it may be possible to produce compounds having the same or similar activity and uses as the compound in question, or compounds that induce the same or similar activity and uses as precursors. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound. These should also be included within the scope of protection of this patent.

[0057] As used herein, the nomenclature of compounds, including organic compounds, may be specified using the common names IUPAC, IUBMB, or CAS nomenclature recommendations. If one or more stereochemical features are present, the Cahn-Ingold-Prelog stereochemical rules may be adopted to specify stereochemical priority, E / Z rules, etc. When defining the name of a compound, a person skilled in the art can systematically reduce the compound's structure using the nomenclature rules or CHEMDRAW. TMThe structure of a compound can be easily determined using commercially available software such as that from Cambridgesoft Corporation (USA).

[0058] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., 1 atmosphere). Treatment and / or prevention methods

[0059] The present invention discloses a method for treating and / or preventing various diseases and / or related conditions caused by Porphyromonas gingivalis infection in patients in need, comprising administering to the patient a therapeutically effective dose of one or more monoclonal and / or bispecific antibodies disclosed herein. In some 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 infection level of 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 about 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%. Monoclonal antibodies and bispecific antibodies

[0060] As disclosed in the present invention, in some embodiments, the nucleotide and amino acid sequences of some specific antibodies are provided as follows:

[0061] 1. The RagB-2-1A4-3-7-7 monoclonal antibody contains a heavy chain (mouse IgG1) variable region (VH) having the nucleotide sequence (357 bases) shown in SEQ ID NO:128 and the encoded amino acid sequence (119 amino acids) shown in SEQ ID NO:129, and a light chain (mouse κ type) variable region (VL) having the nucleotide sequence (282 bases) shown in SEQ ID NO:133 and the encoded amino acid sequence (94 amino acids) shown in SEQ ID NO:134. The nucleotide sequence of the heavy chain of the RagB-2-1A4-3-7-7 monoclonal antibody is shown in SEQ ID NO.175, the encoded amino acid sequence in SEQ ID NO.176, the nucleotide sequence of the light chain in SEQ ID NO.177, and the encoded amino acid sequence in SEQ ID NO.178.

[0062] 2. The RagB-3-1D2-2-1-3 monoclonal antibody contains a heavy chain (mouse IgG2a) variable region (VH) having the nucleotide sequence (360 bases) shown in SEQ ID NO:138 and the encoded amino acid sequence (120 amino acids) shown in SEQ ID NO:139, and a light chain (mouse κ type) variable region (VL) having the nucleotide sequence (336 bases) shown in SEQ ID NO:143 and the encoded amino acid sequence (112 amino acids) shown in SEQ ID NO:144. The nucleotide sequence of the heavy chain of the RagB-3-1D2-2-1-3 monoclonal antibody is shown in SEQ ID NO.179, the encoded amino acid sequence in SEQ ID NO.180, the nucleotide sequence of the light chain in SEQ ID NO.181, and the encoded amino acid sequence in SEQ ID NO.182.

[0063] 3. The RagB-4-1B11-4-4 monoclonal antibody contains a heavy chain (mouse IgG1) variable region (VH) having the nucleotide sequence (375 bases) shown in SEQ ID NO:148 and the encoded amino acid sequence (125 amino acids) shown in SEQ ID NO:149, and a light chain (mouse κ type) variable region (VL) having the nucleotide sequence (336 bases) shown in SEQ ID NO:153 and the encoded amino acid sequence (112 amino acids) shown in SEQ ID NO:154. The nucleotide sequence of the heavy chain of the RagB-4-1B11-4-4 monoclonal antibody is shown in SEQ ID NO.183, the encoded amino acid sequence in SEQ ID NO.184, the nucleotide sequence of the light chain in SEQ ID NO.185, and the encoded amino acid sequence in SEQ ID NO.186.

[0064] 4. The RagB-4-1C3-7-8 monoclonal antibody contains a heavy chain (mouse IgG1) variable region (VH) having the nucleotide sequence (357 bases) shown in SEQ ID NO:158 and the encoded amino acid sequence (119 amino acids) shown in SEQ ID NO:159, and a light chain (mouse κ type) variable region (VL) having the nucleotide sequence (321 bases) shown in SEQ ID NO:163 and the encoded amino acid sequence (107 amino acids) shown in SEQ ID NO:164. The nucleotide sequence of the RagB-4-1C3-7-8 monoclonal antibody heavy chain is shown in SEQ ID NO.187, the encoded amino acid sequence in SEQ ID NO.188, the nucleotide sequence of the light chain in SEQ ID NO.189, and the encoded amino acid sequence in SEQ ID NO.190.

[0065] 5. The 1B11-1C3 bispecific antibody comprises a heavy chain (mouse IgG1) having the nucleotide sequence (2232 bases) shown in SEQ ID NO:168 and the encoded amino acid sequence (728 amino acids) shown in SEQ ID NO:172, and a light chain (mouse κ type) having the nucleotide sequence (744 bases) shown in SEQ ID NO:173 and the encoded amino acid sequence (232 amino acids) shown in SEQ ID NO:174. Administration and medication

[0066] While active ingredients may be administered alone, it may be more preferable to administer them as a pharmaceutical formulation or pharmaceutical composition. As described below, the disclosed formulations, whether for veterinary or human use, include at least one active ingredient, as well as one or more acceptable carriers and other optional therapeutic ingredients. The carriers must be “acceptable,” meaning they are compatible with the other ingredients in the formulation and not physiologically harmful to the receptor.

[0067] Each active ingredient can be prepared using conventional carriers and formulations selected according to conventional methods. Tablets may contain formulations, flow aids, fillers, binders, etc. Aqueous formulations are prepared under sterile conditions and are usually isotonic when delivered by methods other than oral administration. All formulations should be used in reference to the "Handbook of Pharmaceutical Additives" (1986). Formulations may contain ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, and stearic acid. The pH range of the formulations is approximately 3 to approximately 11, but is usually approximately 7 to 10. Experienced clinicians can easily determine the therapeutically effective dose of the active ingredient using conventional dose escalation studies. Typically, the dose of the active ingredient is 0.01 mg to 2 g. In one embodiment, the dose is approximately 10 mg to 450 mg. In another embodiment, the dose is approximately 25 mg to approximately 250 mg. In yet another embodiment, the dose may be approximately 50 or 100 mg. For example, in one example, the dose is approximately 100 mg, and it is expected that the active ingredient can be administered once, twice, or three times a day. Furthermore, 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.

[0068] The composition of the active ingredient of a drug includes an appropriate route of administration. The formulation can be simplified to present in unit dosage form and prepared by any method known in the field of pharmacy. For techniques and formulations, see "Remington Pharmaceutical Sciences" (Mack Publishing Co., Easton, Pa.). These methods include the step of combining the active ingredient with a carrier constituting one or more auxiliary components. Typically, the formulation is finally formed by homogeneously and closely mixing the active ingredient with a liquid carrier, a fragmented solid carrier, or both.

[0069] Formulations suitable for oral administration are provided as individual packages such as capsules, coated tablets, or granules containing a predetermined amount of the active pharmaceutical ingredient, and may also be in the form of powders or granules, aqueous or non-aqueous liquids or suspensions, or water-in-oil or oil-in-water liquid emulsions. The active ingredient may also be administered as pills, ointments, or pastes. In some embodiments, the active ingredient is administered by subcutaneous injection.

[0070] Tablets can be manufactured by compression or molding, with optionally one or more auxiliary components. Compressed tablets can be manufactured by compressing a free-flowing active ingredient (such as a powder or granules) mixed with optionally a binder, lubricant, inert diluent, preservative, or surfactant using appropriate machinery. Molded tablets can be manufactured by molding a mixture of an inert liquid-containing diluent and a powdered active pharmaceutical ingredient using appropriate machinery. Tablets can optionally be coated or scored, and can be formulated as sustained-release or controlled-release tablets to ensure the slow release of the active ingredient.

[0071] The active ingredient can be administered via a route appropriate to the condition being treated. Suitable routes include oral, rectal, intranasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). It should be understood that the preferred route of administration may vary depending on the recipient's condition. In some embodiments, the active ingredient can be administered orally due to its high bioavailability. In the embodiments, the patient is human. Active ingredients in pharmaceuticals

[0072] The pharmaceutical active ingredient disclosed in this invention comprises one or more effective amounts of antibodies.

[0073] For oral administration, for example, tablets, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs can be manufactured. Oral administration compositions can be manufactured according to any method known in the pharmaceutical art, and such compositions may contain one or more excipients, including sweeteners, flavorings, colorants, and preservatives, to provide a palatable formulation. Tablets containing an active ingredient mixed with non-toxic and pharmaceutically acceptable excipients are acceptable. These excipients may include, 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; binders such as cellulose, crystalline cellulose, starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. The tablets do not need to be coated, but may be coated using known techniques (such as microencapsulation) to prolong their effect by slowing down their breakdown and absorption in the gastrointestinal tract. For example, time-delaying materials such as glyceryl monostearate or glyceryl distearate can be used alone or in combination with wax.

[0074] Oral formulations may be provided 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).

[0075] The aqueous suspensions disclosed in this invention comprise an active substance mixed with excipients suitable for the preparation of aqueous suspensions. These excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum arabic, and gum tragacanth, as well as dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of fatty acids and alkylene oxides (e.g., polyoxyethylene stearate), condensation products of ethylene glycol and long-chain fatty alcohols (e.g., heptadecaoxyethane), and condensation products of fatty acids and partial esters of hexitol (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives (e.g., ethyl or propyl p-hydroxybenzoic acid) and one or more colorants, flavorings, and sweeteners (e.g., sucrose or saccharin).

[0076] Oily suspensions can be prepared by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, coconut oil) or mineral oil (e.g., liquid paraffin). Thickeners such as beeswax, hard paraffin, or cetyl alcohol may be added to the oily suspension. Sweeteners and flavorings may be added to provide a palatable oral formulation. These compositions can be preserved by adding antioxidants (such as ascorbic acid).

[0077] The dispersible powders and granules disclosed in the present invention are suitable for preparing aqueous suspensions by adding water and provide compositions of active ingredients mixed with dispersants, wetting agents, suspending agents, and one or more preservatives. Suitable dispersants and suspending agents are exemplified above. Furthermore, excipients such as sweeteners, flavorings, and colorants may also be present.

[0078] The pharmaceutical active ingredients disclosed in this invention may be in the form of a water-in-oil emulsion. The oil phase may be a vegetable oil (such as olive oil or peanut oil), a mineral oil (such as liquid paraffin), or a mixture thereof. Suitable emulsifiers include natural gums (such as gum arabic and tragacanth), naturally occurring phospholipids (such as soy 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). The emulsion may contain sweeteners and flavorings. The syrup and elixir formulations can be prepared using sweeteners such as glycerol, sorbitol, or sucrose. Furthermore, these formulations may contain mucosal protectants, preservatives, flavorings, or colorings.

[0079] The pharmaceutical active ingredients disclosed in this invention may be sterile injection formulations, such as sterile aqueous suspensions or oily suspensions. Such suspensions can be prepared according to known techniques using the appropriate dispersants or wetting agents and suspending agents described above. The sterile injection formulation may also be a sterile injection solution or suspension prepared using a non-toxic and acceptable diluent or solvent (such as a 1,3-butanediol solution), or it may be prepared as a lyophilized powder. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixative oils are usually used as solvents or suspension media. For this purpose, any non-irritating fixative oil, including synthetic monoglycerides or diglycerides, can be used. In addition, fatty acids such as oleic acid can also be used in the injection formulation.

[0080] The amounts of active ingredient and carrier material included in a single dosage form vary depending on the host being treated and the specific method of administration (e.g., oral administration or subcutaneous injection). For example, a sustained-release formulation for oral administration in humans may contain approximately 1 to 1000 mg of the active ingredient mixed with a suitable and convenient carrier material, accounting for approximately 5% to 95% (by weight) of the total composition. Pharmaceutical compositions can be prepared in easily measurable doses. For example, an aqueous solution suitable for intravenous infusion may contain approximately 3 to 500 μg of the active ingredient per mL of solution to allow for the injection of an appropriate amount at a rate of approximately 30 mL / hour. Subcutaneous formulations are typically administered once every 2 to 4 weeks for 2 to 4 months.

[0081] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bactericides, and solutes that make the formulation isotonic with the recipient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners.

[0082] These formulations are provided in single-dose or multi-dose containers such as sealed ampoules or vials and can be stored lyophilized simply by adding a sterile liquid carrier such as water for injection before use. Immediately prepared injectable solutions and suspensions are prepared from the above-mentioned sterile powders, granules, and tablets. Preferred unit dosage forms contain the daily dose or a daily unit subdose (or an appropriate portion thereof) of the active ingredient, as described above.

[0083] In some embodiments, the antibodies disclosed herein can be prepared in any suitable dosage form for a suitable method of administration. In some embodiments, the provided method involves administering a pharmaceutical composition comprising one or more antibodies disclosed herein and a pharmaceutically acceptable carrier or excipient. The combination formulations and / or therapeutic regimens disclosed herein comprise the antibodies disclosed herein together with one or more pharmaceutically acceptable carriers or excipients and other therapeutic agents for the treatment and / or prevention of an optional infection, whether currently known or to be developed in the future. The combination formulations containing the active ingredients may exist in any form suitable for the intended method of administration.

[0084] In some embodiments, the vaccines disclosed herein can be prepared in any suitable dosage form for a suitable route of administration. In some embodiments, the methods provided involve administering a pharmaceutical composition comprising one or more vaccines disclosed herein and a pharmaceutically acceptable carrier or excipient. The combination formulations and / or therapeutic regimens disclosed herein comprise the vaccines disclosed herein together with one or more pharmaceutically acceptable carriers or excipients and other therapeutic agents for the treatment and / or prevention of an optional infection, whether currently known or to be developed in the future. The combination formulations containing the active ingredient may exist in any form suitable for the intended method of administration.

[0085] According to the techniques disclosed in PCT / GB2005 / 001976 and PCT / CN2019 / 124433, the rag locus encoding the outer membrane protein of Porphyromonas gingivalis consists of two co-transcribed and independent genes, ragA and ragB. Data showed that there are four major distinct subtypes of the outer membrane protein encoded by the ragB gene. These four subtypes account for over 95% of clinically collected samples from 197 periodontal disease patients obtained from a global collaborative study. The four subtypes of Porphyromonas gingivalis were named W50ragB protein, HairagB protein, QMLragB protein, and 381ragB protein, respectively, based on their RagB outer membrane proteins.

[0086] These four allotype proteins differ significantly in sequence, with similarities of only 42–59% in pairwise comparisons (Figure 6). RagA, like RagB, was found to have an allotype protein. However, since Cra4S1 is a polypeptide fragment near the N-terminus of the RagA protein encoded by the ragA gene and contains a conserved domain, this means that the Cra4S1 polypeptide is present in all allotype strains of Porphyromonas gingivalis.

[0087] In some embodiments, the present invention discloses gene modification sequences of the Porphyromonas gingivalis outer membrane protein RagB and successfully completes pilot-scale production of untagged RagB and Cra4S1 recombinant proteins (Example 1, Figures 1-5 and 7-11).

[0088] Different RagB outer membrane protein subtypes each possess unique antigenic properties. However, studies have shown that polyclonal antibody serums obtained by immunizing animals with a single recombinant RagB protein exhibit significant cross-reactivity with other RagB protein isoforms under specific dilution conditions (Example 2).

[0089] Mouse monoclonal antibodies against four different RagB and Cra4S1 proteins have been produced, some of which not only react with homologous proteins but also cross-react with other subtypes of proteins (Example 3).

[0090] Despite the sequence similarity of RagB subtypes being only 42–59%, cross-reactivity between different RagB proteins suggests the possibility of a common antigen or compatible epitope. Are these cross-reactivity triggered by linear protein sequences, or by two-dimensional planar and / or multidimensional structures? What is the molecular basis for these cross-reactivity? Where are the target antigens located? Do monoclonal antibodies exhibit cross-immunoprotection?

[0091] In embodiments of the present invention, a method for constructing an artificially synthesized peptide library is provided. These peptide libraries contain five different subtypes of the Porphyromonas gingivalis outer membrane protein RagB and a peptide fragment of Cra4S1, and are used to screen for target antigens of monoclonal antibodies. Antigen targets are identified by utilizing the immunoreaction between the monoclonal antibody and peptides located at different positions in the corresponding protein. In the embodiments, several monoclonal antibodies with cross-reactivity properties all found corresponding antigen targets in the peptide library. This significant finding has profound implications for manufacturing processes and costs in future product development. In previous concepts, homologous bacteria were targeted using specific monoclonal antibodies based on differences in Porphyromonas gingivalis strains, but the new method uses an antibody product that can simultaneously target four Porphyromonas gingivalis subtypes (Example 4).

[0092] More specifically, the present invention elucidates the pathogenesis of Porphyromonas gingivalis based on research methods and results regarding the pathogenesis of Porphyromonas gingivalis. In the examples, the heterophilic antigenic characteristics of Porphyromonas gingivalis and the heterophilic antibodies of the individual are considered to be the cause of the chronic persistence of the disease and multi-organ damage.

[0093] In the examples, serological test results of clinical specimens showed that the outer membrane proteins (including RagB and Cra4S1) of Porphyromonas gingivalis possess heterophilic antigenic properties. The test results indicate that local lesions and systemic disorders caused by Porphyromonas gingivalis infection are closely related to heterophilic antigens and / or heterophilic antibodies (Example 5).

[0094] In the examples, the present invention also discloses a method for establishing an animal model of Porphyromonas gingivalis infection. This animal model is used to evaluate systemic disorders caused by pathogenic infection and methods for treating Porphyromonas gingivalis infection.

[0095] Experimental results of compound antibodies in a mouse reproductive model demonstrated far-reaching significance, facilitating the development of preventive and therapeutic products for Porphyromonas gingivalis and related chronic systemic diseases. Of particular importance was the discovery of two monoclonal antibodies with adjacent antigenic targets, each targeting a different subtype of bacteria, with the location and sequence of the antigenic targets in each outer membrane protein identified. As research in this field continues to expand, further therapeutic applications will be established, and the antibodies and methods of use disclosed herein open up broad market prospects for preventive and / or therapeutic products for various periodontal disease-related conditions. Therefore, the present invention provides stable and efficient biological formulations, solves the problem of treating different subtypes of pathogens, simplifies the manufacturing process, reduces manufacturing costs, and ultimately brings overall benefits to public health (Example 6).

[0096] In the examples, the present invention discloses the nucleotide and protein sequences of mouse monoclonal antibody genes produced by immunization of the Porphyromonas gingivalis outer membrane protein RagB. Recombinant antibody plasmids constructed using these antibody sequences were successfully expressed in eukaryotic cells. These plasmids retain the ability of the original monoclonal antibodies to bind to antigens and provide a molecular basis for the development of therapeutic agents and the industrial production of products.

[0097] The present invention further provides a humanization mutation scheme for one or more monoclonal antibodies and a large-scale manufacturing process for these antibodies (Example 7).

[0098] In the examples provided, the present invention offers two specific monoclonal antibodies belonging to the same subtype of Porphyromonas gingivalis, whose antigen targets are closely related. Importantly, these two antibodies exhibited cross-reactivity with different isoforms of outer membrane proteins, and these cross-antigens differed in polypeptide sequence and position on the outer membrane protein. This discovery significantly simplified the manufacturing process and effectively improved the production efficiency of the product.

[0099] In the examples, the present invention discloses the design and manipulation of bispecific antibodies targeting different subtypes of Porphyromonas gingivalis, and successfully constructs recombinant antibody plasmids using the designed antibody sequences, which are then successfully expressed in eukaryotic cells. The data presented by the present invention demonstrate that recombinant bispecific antibodies not only retain the antigen-binding ability of the original different monoclonal antibodies but also compensate for the shortcomings of the original monoclonal antibodies. These data demonstrate the feasibility and potential of developing bispecific antibody products (Example 8).

[0100] In the examples, the present invention also discloses diagnostic kits for the diagnosis, treatment, and prediction of diseases associated with Porphyromonas gingivalis infection, including but not limited to periodontal disease, cardiovascular disease, rheumatoid arthritis, oral gastrointestinal cancer, ulcerative colitis, neurological disorders, autoimmune encephalomyelitis, lung cancer, abnormal pregnancy, pancreatic cancer, diabetes mellitus, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease.

[0101] This invention discloses a diagnostic kit reagent for the diagnosis, treatment, and prediction of diseases associated with Porphyromonas gingivalis infection. In some examples, the amino acid sequences of the diagnostic reagent are described in SEQ ID NO. 2, 4, 6, 8, and 10. The diagnostic kit also provides instructions for performing a reaction using the reagent to test for specific antibodies in biological samples, including but not limited to blood, gingival crevicular exudate, urine, saliva, cerebrospinal fluid, pleural fluid, ascites, and amniotic fluid. Details of the preparation, use, and analysis of the results of the diagnostic kit are described in Example 9 of this invention.

[0102] The present invention also discloses polypeptide vaccines capable of mobilizing the active immune function of an individual. In the examples, the sequence of the polypeptide vaccine was determined by the target of a neutralizing antibody, while retaining the antigenic properties of the polypeptide vaccine and removing irrelevant and harmful components. The polypeptide vaccines disclosed in the present invention reduce the side effects of commonly used vaccines, can be administered repeatedly, and provide efficient and sustained protection against Porphyromonas gingivalis infection.

[0103] The present invention further provides polypeptide vaccines and compound antibodies for active and passive immunization of infected animals. Details of the preparation, use, and results of these polypeptide vaccines and / or compound antibodies refer to Example 10 of the present invention.

[0104] The above provides a general overview of various aspects of the present invention, and the following examples illustrate some additional and / or more detailed aspects of the present invention. While various aspects of the present invention are expressed in conjunction with the following examples and corresponding textual descriptions and illustrations, the present invention is not limited to these descriptions. Rather, the meaning of "aspects of the present invention" is to encompass all alternatives, modifications, and equivalents that fall within the spirit and scope of the invention.

[0105] The following examples are intended to provide those skilled in the art with a complete schematic diagram and description of how to prepare and evaluate the compounds, compositions, materials, settings and / or methods claimed herein, and the disclosure of these examples is not intended to limit the scope of the inventors' disclosure. While efforts have been made to ensure the accuracy of the figures (e.g., quantities, temperatures, etc.), some errors and deviations should be taken into account. Unless otherwise stated, components are expressed by weight, temperatures are given in degrees Celsius (°C) or as ambient temperature, and pressures are atmospheric pressure or close to atmospheric pressure. [Examples]

[0106] Example 1: Expression of RagB and Cra4S1 in Escherichia coli The outer membrane protein RagB of Porphyromonas gingivalis is primarily present in four isoforms in clinical periodontal disease samples. These four isoforms of the outer membrane protein have been named W50RagB, ThaiRagB, QMLRagB, and 381RagB.

[0107] In this invention, the names of the four RagB subtypes have been simplified, with W50RagB designated as RagB-1, ThaiRagB as RagB-2, QMLRagB as RagB-3, and 381RagB as RagB-4. As shown in Figure 6 and Table 1, the similarity of protein sequences between RagB isoforms is only 42% to 57%, with RagB-1 and RagB-2 having a similarity of 47%, RagB-1 and RagB-3 a similarity of 57%, RagB-1 and RagB-4 a similarity of 49%, RagB-2 and RagB-3 a similarity of 45%, RagB-2 and RagB-4 a similarity of 42%, and RagB-3 and RagB-4 a similarity of 49%.

[0108] The recombinant plasmids constructed using the four different genes encoding Porphyromonas gingivalis outer membrane proteins (RagBs) and Cra4S1 proteins, as well as the vectors, were all transfected into host Escherichia coli (E. coli), and after induction, the recombinant proteins were successfully expressed.

[0109] Table 1. RagB multiplex sequence alignment (percentage of identical sequences) JPEG0007917864000001.jpg27156

[0110] The original nucleotide sequence of the target protein was modified based on the codon bias of the host E. coli. After nucleotide sequence optimization, the encoded protein sequence matches the original target protein, thereby enabling accurate expression of the target protein.

[0111] 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 Figures 1A-1B (particularly for RagB-1, SEQ ID NO: 1-2), 2A-2B (particularly for RagB-2, SEQ ID NO: 3-4), 3A-3B (particularly for RagB-3, SEQ ID NO: 5-6), 4A-4B (particularly for RagB-4, SEQ ID NO: 7-8), and 5A-5B (particularly for Cra4S1, SEQ ID NO: 9-10), respectively, with SEQ ID NO: 1-10.

[0112] Table 2 Expression of recombinant outer membrane proteins in Escherichia coli JPEG0007917864000002.jpg26161

[0113] All five Porphyromonas gingivalis outer membrane proteins, including RagB-1 (W50 RagB), RagB-2 (ThaiRagB), RagB-3 (QMLRagB), RagB-4 (381RagB), and Cra4S1, were successfully expressed in host E. coli. Specifically, after successful plasmid construction, the cloned genes (including optimized nucleotide sequences) were sequenced to ensure the accuracy of the protein sequences. After transforming E. coli with the plasmids, recombinant protein expression was promoted by induction. The precipitate from the bacteria expressing the recombinant proteins was collected by centrifugation, the resulting pellet was mechanically disrupted, and the supernatant was purified by ion-exchange chromatography to obtain the target proteins. All recombinant proteins are tagless and soluble (see Table 2).

[0114] The pilot-scale production of the five untagged recombinant proteins described above was completed by a third-party contract research organization (CRO) (GenScript, USA, contract number C9458FG140). Specifically, recombinant plasmids and / or seed bacteria were sent to the CRO for verification and identification before fermentation production. During the high-density fermentation process, the culture medium did not contain animal-derived proteins or antibiotics, and after fermentation was complete, the proteins were purified by a five-step chromatography method. All five recombinant protein products are accompanied by a certificate of analysis. Figures 7A-7B (specifically referring to RagB-1), 8A-8B (specifically referring to RagB-2), 9A-9B (specifically referring to RagB-3), 10A-10B (specifically referring to RagB-4), and 11A-11B (specifically referring to Cra4S1) show the induced expression and purification results of the five untagged target proteins.

[0115] Table 3 Small-scale prototypes of recombinant proteins JPEG0007917864000003.jpg16167

[0116] The successful pilot-scale production of five different outer membrane proteins of Porphyromonas gingivalis demonstrates the feasibility of industrializing the product.

[0117] Example 2: Cross-reactivity of polyclonal antibodies against Porphyromonas gingivalis outer membrane protein Although the protein sequences of different subtypes of Porphyromonas gingivalis RagB differ, animal serological studies have revealed that antibodies cross-react to different subtypes of RagB protein. Therefore, a step to account for cross-reactivity of antibodies to different RagB subtypes was added to the design of detection experiments, and in this study, enzyme-linked immunosorbent assay (ELISA) was primarily used to detect the different affinities of antibodies to antigens.

[0118] Specifically, the method for preparing mouse and rabbit antibody serum is briefly described as follows: Mice and rabbits are immunized 3-4 times with the antigen protein (RagB / Cra4S1), and the titer of specific antibodies in the serum is measured by indirect ELISA. After the antibody level reaches a predetermined standard, whole blood samples are collected from the animals, serum is obtained by incubation and centrifugation, and then frozen for storage.

[0119] ELISA is an enzyme-linked immunosorbent assay (ELISA) method. The basic principle involves immobilizing a certain concentration of antigen on the surface of a polystyrene microplate by physical adsorption, adding diluted serum (host antibody) to the reaction plate, allowing sufficient time for the antigen and antibody to bind, and then washing away any unbound free antibody molecules. Next, an enzyme-labeled secondary antibody (anti-host antibody) is added, and the free molecules are washed away again. Finally, an enzyme substrate solution is added, and the enzymatic reaction is allowed to proceed for a certain period. By measuring the color reaction of the substrate, the qualitative and semi-quantitative characteristics of the substrate are indirectly reflected. The specific steps are as follows:

[0120] 1. Antigen coating: Prepare the protein (antigen) solution to a concentration of 4 μg / mL in PBS, add 100 μL of antigen solution to each well, seal the plate, and incubate overnight at room temperature (approximately 20°C). Wash the plate four times with PBS buffer, adding 300 μL / well, and tap off any remaining buffer from the wells with absorbent paper. Add 100 μL of blocking buffer (1% BSA in PBS) to each well and incubate for 2 hours at room temperature (20°C). Wash the plate four times with PBS buffer, adding 300 μL / well, and tap off any remaining buffer from the wells with absorbent paper. The coated ELISA plate can be stored in a -20°C freezer for 4-6 weeks.

[0121] 2. Addition of primary antibody: Using sample dilution buffer (1% BSA in PBS), adjust the cell culture supernatant or serum antibody to the desired working concentration, add 100 μL of primary antibody solution per well, seal the plate, incubate at 37°C for 60-90 minutes, wash the plate four times with PBS buffer to 300 μL per well, and tap the remaining buffer in the wells on absorbent paper to dry.

[0122] 3. Addition of enzyme-labeled secondary antibody: Dilute the secondary antibody with sample dilution buffer according to the manufacturer's instructions, add 100 μL of enzyme-labeled secondary antibody to each well, incubate at 37°C for 40-60 minutes, wash the plate three times with PBS buffer, tap the remaining buffer in the wells with absorbent paper (300 μL / well), and finally wash the plate with deionized water, tap the remaining liquid in the wells with absorbent paper (300 μL / well).

[0123] 4. Reaction color development: Prepare the substrate solution, dissolve 4-nitrophenyl phosphate disodium hexahydrate in 1M Tris-MgCl2 to a concentration of 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 a dual-wavelength program (405 nm and 630 nm) on the microplate reader, place the plate on the preheated microplate reader, read the data, save it, and perform the analysis.

[0124] Conventional ELISA methods offer many different detection systems to choose from. In this example, an alkaline phosphatase-conjugated secondary antibody was selected for enzymatic labeling. This was due to its long stability and low false-positive error. Specifically, for ELISA plate number 17003 shown in Table 4, the antigen layout design described here was as follows: columns 1 and 7 were coated with RagB-1 recombinant protein, columns 2 and 8 with RagB-2, columns 3 and 9 with RagB-3, columns 4 and 10 with RagB-4, columns 5 and 11 with Cra4S1 recombinant protein, and columns 6 and 12 with GST recombinant protein as negative, irrelevant protein controls. All serum from the test and control groups was diluted to 1:2000, and each serum sample had the opportunity to detect 6 wells of antigens containing RagB-1, RagB-2, RagB-3, RagB-4, Cra4S1, and GST. The optical density (OD) values ​​of ELISA plate number 17003 are shown in Table 4, with PBS and normal serum used as negative controls.

[0125] Table 4 Detection of polyclonal antibodies in animals infected with Porphyromonas gingivalis JPEG0007917864000004.jpg77161

[0126] ELISA results show that antibodies to the corresponding outer membrane protein are detectable in the serum of animals infected with homologous Porphyromonas gingivalis. For example, in Table 5, a total of eight mice (G6-1, G6-2, G7-1, G7-2, G8-1, G8-2, G8-3, and G8-4) were infected with the Porphyromonas gingivalis RagB-2 subtype. Their serum reacted with the RagB-2 recombinant protein, confirming that the RagB-2 protein is the major antigen on the bacterial surface. The results also show that the more infections there are, the higher the corresponding antibody levels; for example, the four mice in group G8 (G8-1, G8-2, G8-3, G8-4) were infected with Porphyromonas gingivalis only once, while the other four mice (G6-1, G6-2, G7-1, G7-2) were infected with Porphyromonas gingivalis three times. The antibody detection level (OD) in mice infected multiple times was higher than in mice infected only once. Furthermore, inoculation of animals with a history of Porphyromonas gingivalis infection with RagB-2 protein resulted in the production of high-titer specific antibodies (Tables 4 and 5, G6-3, G6-4, G7-3, and G7-4). Compared to animals in a vaccination-only protocol, post-vaccination antibody OD levels were significantly higher in animals with a history of bacterial infection. This phenomenon indicates that bacterial infection sensitized the animals, suggesting that the vaccine product has an immunostimulatory function in patients with a history of infection.

[0127] Table 5 Information on experimental animals JPEG0007917864000005.jpg68161

[0128] The results showed that when animals pre-infected with Porphyromonas gingivalis RagB-2 were inoculated with RagB-2 protein (G6-3, G6-4, G7-3, and G7-4), their serum antibodies not only showed a strong immune response to RagB-2, but also cross-reacted to different subtypes of RagB protein (including RagB-1, RagB-3, and RagB-4), and showed a weak but visible cross-reaction to Cra4S1 protein, while showing no reaction to the negative control GST protein.

[0129] Table 6 Detection of polyclonal antibodies in animals infected with Porphyromonas gingivalis JPEG0007917864000006.jpg77161

[0130] Table 7 Information on experimental animals JPEG0007917864000007.jpg65153

[0131] Similarly, animals previously infected produced high-titer specific antibodies after inoculation with RagB-3 protein, and serum antibodies showed cross-reactivity with RagB-1, RagB-2, RagB-4, and Cra4S1 (Tables 6 and 7).

[0132] This example demonstrated that recombinant outer membrane proteins of Porphyromonas gingivalis, including RagB and Cra4S1, possess good antigenicity and can stimulate animals to produce high-titer antibodies. Furthermore, it was observed that polyclonal serum antibodies cross-reactive with other subtypes of RagB protein. Although there are significant differences in the gene and protein sequences of the Porphyromonas gingivalis outer membrane protein RagB between different subtypes, these are homologous alleles. These results suggest that there may be common antigens or compatible antigenic determinants among the subtypes of Porphyromonas gingivalis. In particular, the cross-reactivity of antibodies produced by immunization with the RagB protein with the Cra4S1 protein was surprising and unexpected.

[0133] Example 3: Cross-reactivity of RagB monoclonal antibody Monoclonal antibodies against Porphyromonas gingivalis outer membrane proteins, including RagB and Cra4S1 proteins, were prepared by third-party CRO (Contract Research Organization) companies. Specifically, the general process for preparing mouse hybridoma cells and obtaining monoclonal antibodies is briefly described below: First, animals are immunized five times with antigen proteins, with intervals of 1-2 weeks between each immunization. Mouse spleen cells are fused with well-growthed SP2 / 0 myeloma cells, and the fused cells are grown in culture medium for 7 days. Then, positive clones are screened using immunoantigens, and three further subclonings are performed to confirm that the positive cell lines produce antibodies sustainably and stably. The final hybridoma cell lines were stored in liquid nitrogen.

[0134] Table 8 RagB hybridoma cell lines JPEG0007917864000008.jpg165138

[0135] Table 9 Cra4S1 Hybridoma Cell Lines JPEG0007917864000009.jpg20161

[0136] Each antigen item provides 5 to 10 monoclonal antibody-positive hybridoma cell lines (Tables 8 and 9). Monoclonal antibodies are mainly extracted and purified from cell culture supernatant and ascites fluid produced after implantation of mouse hybridoma cells into the peritoneal cavity. Immunoglobulin subclasses are detected from the purified monoclonal antibodies, and positive results are indicated with "P" in the table.

[0137] Table 10 Detection of cross-reactivity of monoclonal antibodies JPEG0007917864000010.jpg78161

[0138] Table 11 Detection of cross-reactivity antigens of monoclonal antibodies JPEG0007917864000011.jpg76161

[0139] Tables 10 and 11 show the original results of cross-reactivity between monoclonal antibodies and RagB recombinant proteins. The ELISA procedure is as described in Example 2. The antigen adsorption plate design is as follows, with RagB-1 recombinant protein adsorbed in columns 1, 5, and 9; RagB-2 adsorbed in columns 2, 6, and 10; RagB-3 adsorbed in columns 3, 7, and 11; and RagB-4 adsorbed in columns 4, 8, and 12. The antibodies were from hybridoma culture supernatant or purified antibodies, with dilution ratios of 1:1000 to 1:4000. Each antibody sample can be reacted with the antigen proteins RagB-1, RagB-2, RagB-3, and RagB-4 in 4 wells.

[0140] A positive result was determined when the OD measurement was more than twice that of the normal sero-negative control group. From the data presented in this example, it was found that all monoclonal antibodies tested showed a positive immune response to the antigen, with the exception of one hybridoma (wells B5-8 listed in Table 11, cell name RagB-4-1C11-1-5, which showed a negative reaction). Furthermore, several hybridoma antibodies were identified, which not only react with the corresponding RagB protein but also cross-react with other subtypes of RagB protein.

[0141] For example, in Table 10, RagB-2-1A4-3-7-7 in plate wells G1-G4 is a specific monoclonal antibody against the RagB-2 protein, but it also cross-reacted with the RagB-1 protein. RagB-3-1D2-2-1-3 in plate wells E5-E8 is a specific monoclonal antibody against the RagB-3 protein, but it also cross-reacted with the RagB-1 and RagB-2 proteins. In Table 11, the 1B11-4-4 antibody and the 1C3-7-8 antibody in wells H1-H4 and A5-A8 are specific monoclonal antibodies against the RagB-4 protein, but they cross-reacted with the RagB-1, RagB-2, and RagB-3 proteins, respectively.

[0142] Due to significant differences in nucleic acid and protein sequences between RagB subtypes of Porphyromonas gingivalis, and the lack of clear contiguous common sequences in pairwise alignment, previous bacterial immunotherapy prioritized precision, first identifying the subtype and then selecting a corresponding specific monoclonal antibody. However, this invention discloses several monoclonal antibodies that can cross-react with different subtypes of RagB protein, demonstrating that different subtypes of Porphyromonas gingivalis share common targets or compatible epitopes. So, what exactly are these common targets and / or compatible epitopes? Where are they located?

[0143] Example 4 Target discovery of monoclonal antibodies in a peptide library To identify the antigenicity of the Porphyromonas gingivalis RagB outer membrane protein and the antigenic targets that bind to monoclonal antibodies, five peptide libraries targeting RagB-1, RagB-2, RagB-3, RagB-4, and Cra4S1 were constructed. The synthetic peptides were manufactured by a third-party CRO company (GenScript, USA). Specifically, based on the amino acid sequence of each protein, peptide chains consisting of 18 to 25 amino acids were artificially synthesized, starting from the N-terminus. The next peptide chain would then form a sequence duplication by including five amino acids derived from the C-terminus of the previous peptide chain, and so on. Except for the peptides at both ends of each protein (each peptide chain has only a single duplicate sequence), all other peptide chains have a 5-amino acid duplication with the upstream and downstream peptide chains on both sides, avoiding the omission of any antigenic determinants. The peptide sequences are shown in Tables 12-16. Each RagB subtype protein has 24 peptide chains. RagB-1 peptides are named with a serial number in RA, e.g., RA-1, RA-2, etc. RagB-2 peptides are named with a serial number in RB, RagB-3 peptides with a serial number in RC, and RagB-4 peptides with a serial number in RD. Cra4S1 peptides are named with serial numbers in Cra and NewCra. Most synthetic peptide chain fragments dissolve in ultrapure water, PBS, and / or DMSO. The peptide chain sequences and physicochemical properties of the peptide chains are shown in Tables 12-16.

[0144] After constructing a peptide library of Porphyromonas gingivalis outer membrane proteins, all monoclonal antibodies were reacted with fragments in the peptide library to search for antigen-antibody binding targets and important sequence fragments. Specifically, 1 mg / ml soluble solutions were prepared according to the peptide solubility, and then the polypeptide solutions were further diluted to 20 μg / ml and adsorbed onto ELISA plates. The subsequent antibody testing steps are as described in Example 2. Each monoclonal antibody reacts with its corresponding RagB subtype outer membrane protein polypeptide fragment. To avoid missing antigens and to determine binding sites, each synthetic peptide chain has overlapping portions with upstream and downstream polypeptides, as described above.

[0145] The construction of four RagB subtype peptide libraries aims to capture antigen epitopes, as well as antigen targets that cross-react with monoclonal antibodies. As described in Example 3, some monoclonal antibodies targeting specific RagB subtypes exhibited cross-reactivity to other RagB subtypes. These antibodies were screened in the peptide libraries to identify antibody-antigen targets.

[0146] Table 12 RagB-1 (SEQ ID NO: 2) peptide library (GenScript, USA, C4965GB230) JPEG0007917864000012.jpg90153

[0147] Table 13 RagB-2 (SEQ ID NO: 4) peptide library (GenScript, USA, C4965GB230) JPEG0007917864000013.jpg82161

[0148] Table 14 RagB-3 (SEQ ID NO: 6) peptide library (GenScript, USA, C4965GB230) JPEG0007917864000014.jpg81155

[0149] Table 15 RagB-4 (SEQ ID NO: 8) peptide library (GenScript, USA, C4965GB230) JPEG0007917864000015.jpg94161

[0150] Table 16 RagB-4 (SEQ ID NO: 10) Peptide Library JPEG0007917864000016.jpg16696

[0151] Antigen-antibody binding was determined by the OD value in an indirect ELISA experiment. A positive result was considered to be when the OD value was twice that of the negative serum control.

[0152] Table 17 shows the capture of antigen targets by monoclonal antibodies, including a peptide library of RagB-1 (SEQ ID NO: 2) and monoclonal antibodies against RagB-1 and other RagB subtypes that were recognized as reacting with these antibodies. ELISA results showed that three strains of RagB-1 hybridoma antibodies detected the peptide antigen target; 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). Mouse serum of polyclonal antibodies produced by inoculation with RagB-1 protein was named 2019-G85-1 serum (P), and this 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 to RagB-2 protein) bound to the RA-19 peptide chain, 1D2-2-1-3 (specific to RagB-3 protein) bound to the RA-17 peptide chain, 2C1-4-6-2 (specific to RagB-3 protein) bound to RA-9 and RA-17, 2B7-4-6-7 (specific to RagB-3 protein) bound to RA-9, while 1B11-4-4 (specific to RagB-4 protein) bound to the RA-21 peptide chain.

[0153] Table 17 Screening of target antigens using a RagB-1 (SEQ ID NO: 2) peptide library JPEG0007917864000017.jpg179104

[0154] Table 18 shows the capture of antigen targets by monoclonal antibodies, including a peptide library of RagB-2 (SEQ ID NO: 4) and monoclonal antibodies recognized as reacting with RagB-2 and other RagB subtypes. ELISA results showed that three strains of RagB-2 hybridoma antibodies detected the peptide antigen targets; for example, monoclonal antibodies 1A4-3-7-7, 1A6-3-3, and 1B3-1-5 reacted with the RB-18, RB-21, and RB-7 peptide chains, respectively (Table 18). Mouse serum polyclonal antibodies generated by inoculation with RagB-2 protein were named 2019-G65-1 and reacted with the RB-17 peptide chain.

[0155] Peptide targets were also found in the RagB-2 peptide library for monoclonal antibodies that cross-react with the RagB-2 protein. For example, 1D2-2-1-3 (specific to the RagB-3 protein) binds to the RB-17 peptide chain, while 1C3-7-8 (specific to the RagB-4 protein) binds to the RB-21 peptide chain.

[0156] Table 18 Screening of target antigens using a RagB-2 (SEQ ID NO: 4) peptide library JPEG0007917864000018.jpg163111

[0157] Table 19 Screening of target antigens using a RagB-3 (SEQ ID NO: 6) peptide library JPEG0007917864000019.jpg164117

[0158] Table 20 Screening of target antigens using a RagB-3 (SEQ ID NO: 6) peptide library JPEG0007917864000020.jpg156120

[0159] Tables 19 and 20 show the capture of the antigen target by monoclonal antibodies, including the RagB-3 (SEQ ID NO: 6) peptide library and monoclonal antibodies identified to react with other RagB subtypes. ELISA results showed that almost all of the tested RagB-3 hybridoma antibodies detected the peptide antigen target, and 15 of the 16 hybridoma antibody strains reacted with the peptide chain of the RagB-3 peptide library.

[0160] Surprisingly, the data from the RagB-3 group also showed that some monoclonal antibodies reacted with multiple peptide chains at different positions. Furthermore, four different mouse polyclonal antibodies produced by single-protein vaccines (Table 20, 2019-G75-2) and multivalent RagB vaccine immunization (Table 20, 2019-G160-2, 2019-G160-4, and 2019-G250-3) all failed to detect clear polypeptide targets in peptide libraries. The 1D2-2-1-3 recombinant plasmid antibody showed a highly specific reaction to the RC-17 peptide chain, but no reaction was observed with other polypeptide fragments.

[0161] In the RagB-3 peptide library, specific polypeptide targets also interacted with the monoclonal antibodies tested. For example, the 1B11-4-4 antibody, which is specific to the RagB-4 protein, was found to react with the RC-21 peptide, and the 1C3-7-8 antibody, which is specific to the RagB-4 protein, was found to react with the RC-22 peptide chain.

[0162] Table 21 Screening of target antigens using a RagB-4 (SEQ ID NO: 8) peptide library. JPEG0007917864000021.jpg161119

[0163] Table 21 shows the peptide library of RagB-4 (SEQ ID NO: 8), as well as the capture of antigen targets by monoclonal antibodies, including monoclonal antibodies against RagB-4 and monoclonal antibodies identified to react with other RagB subtypes. ELISA results showed that hybridoma antibodies of RagB-4 detected polypeptide antigen targets, and 6 out of 7 hybridoma antibody strains reacted with 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-reactions were found between these antibodies and RagB-4 polypeptide fragments. Mouse serum containing polyclonal antibodies produced by inoculation with RagB-4 protein was named G90 serum (P) and reacts with the RD-1 peptide chain.

[0164] In antigen target capture studies of Cra4S1 monoclonal antibodies, culture supernatants from over 40 positive hybridoma cell lines were tested to screen for antigen targets. Although these antibodies reacted with high titers to recombinant proteins, they did not react with artificially synthesized peptide antigens created from Cra4S1 peptide libraries. Only a few clones showed weak antigen-antibody reactions. Table 16 provides two peptide libraries of Cra4S1. Antigen-antibody binding reactions were still not detected after adjustments to peptide chain length and position.

[0165] As described above, several monoclonal antibody strains showed cross-reactivity to other RagB proteins and their peptide libraries. Key cross-antigen targets are as follows:

[0166] 1. Binding site of monoclonal antibody RagB-2-1A4-3-7-7 to polypeptide:

[0167] RA-19: V AE V YLIL V E SALQTGDTPTAEKYL (SEQ ID NO: 29), and / or

[0168] RB-18: PKKENFKTGCRFFSL AE A YLIL A E A (SEQ ID NO: 52)

[0169] The monoclonal antibody 1A4-3-7-7 specifically reacts with the RagB-2 protein and also with the RA-19 and RB-18 peptide chains. The underlined amino acids are identical in both sequences, indicating that the specific antigen-binding site of the RagB-2-1A4-3-7-7 monoclonal antibody is located at these highlighted epitopes in RA-19 (SEQ ID NO: 29) and / or RB-18 (SEQ ID NO: 52).

[0170] 2. Binding site of monoclonal antibody RagB-3-1D2-2-1-3 to polypeptide (1):

[0171] RA-17&18: YIAKVVKKD KGYLVNKFLED K AYR DVQDKPNLKVGARYFSVAEVY (SEQ ID NO: 123), and / or

[0172] RC-17&18: KSVYIDKTVSNGSE KGYLVNKFLED P AYR ETADIPILKIGVRMFS (SEQ ID NO: 124)

[0173] Binding site of monoclonal antibody RagB-3-1D2-2-1-3 to polypeptide (2):

[0174] RB-17: DGG KGY V VNKFL GDPELREDPKKEN (SEQ ID NO: 51), and / or

[0175] RC-17&18: KSVYIDKTVSNGSE KGY L VNKFL EDPAYRETADIPILKIGVRMFS (SEQ ID NO: 124)

[0176] The specific antigen-binding sites of the RagB-3-1D2-2-1-3 monoclonal antibody are located at 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 completely identical in both sequences, indicating that the monoclonal antibody RagB-3-1D2-2-1-3 is cross-reactive to the other two RagB subtypes, but that the epitopes and lengths of the antibody-antigen-binding sites are not the same.

[0177] 3. Binding site of monoclonal antibody RagB-4-1B11-4-4 to polypeptide (1):

[0178] RA-21: MEALQ AERTRELIGEGSRL R DMVRW (SEQ ID NO: 31), and / or

[0179] RD-21: VM AERTRELIGEGSRL NDMI RWNLP (SEQ ID NO: 103)

[0180] Binding site of monoclonal antibody RagB-4-1B11-4-4 to polypeptide (2):

[0181] RB-21: D ERTRE M IGEGSRLN DMIRWNMDLV (SEQ ID NO: 55), and / or

[0182] RD-21: VMA ERTRE L IGEGSRLN DMIRWNLP (SEQ ID NO: 103)

[0183] Binding site of monoclonal antibody RagB-4-1B11-4-4 to polypeptide (3):

[0184] RC-21: IDTGDVMKAIQE ERTRELIGEG ARL (SEQ ID NO: 79), and / or

[0185] RD-21: VMA ERTRELIGEG SRLNDMIRWNLP (SEQ ID NO: 103)

[0186] The specific antigen-binding sites of the RagB-4-1B11-4-4 monoclonal antibody are located at 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 regions where two sequences are completely identical, indicating that the monoclonal antibody RagB-4-1B11-4-4 cross-reactive to the other three RagB subtypes, but the epitopes and lengths of the antibody-antigen-binding sites are not the same.

[0187] 4. Binding site of monoclonal antibody RagB-4-1C3-7-8 to polypeptide (1):

[0188] RA-21&22: MEALQAERTRELIGEGSRLR DM V RW SI PNNHD AF E T QP GLEGFAN (SEQ ID NO: 125), and / or

[0189] RD-21&22: VMAERTRELIGEGSRLN DM I RW NL PNNHD DM E N QP VFLQIGLA (SEQ ID NO: 126)

[0190] Binding site of the P monoclonal antibody RagB-4-1C3-7-8 to polypeptide (2)

[0191] RB-21: DERTREMIGEGSRLN DMIRWN MDLV (SEQ ID NO: 55), and / or

[0192] RD-21: VMAERTRELIGEGSRLN DMIRWN LP (SEQ ID NO: 103)

[0193] Binding site of monoclonal antibody RagB-4-1C3-7-8 to polypeptide (3)

[0194] RC-22: EGARLR DMIRWNLPN IDKTEIQPAL (SEQ ID NO: 80), and / or

[0195] RD-21&22: VMAERTRELIGEGSRLN DMIRWNLPN NHDDMENQPVFLQIGLAKA (SEQ ID NO: 126)

[0196] The specific antigen-binding sites of the RagB-4-1C3-7-8 monoclonal antibody are located at 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 regions where the two sequences are completely identical, indicating that the monoclonal antibody RagB-4-1C3-7-8 cross-reactive with the other three RagB subtypes, but the epitopes and lengths of the antibody-antigen-binding sites are not the same.

[0197] The discovery of polyclonal antibodies that cross-react with different subtypes of RagB protein, as well as monoclonal antibodies with multiple antigen targets, was surprising and unexpected. Data from two monoclonal antibody strains with multiple cross-reactivity obtained from RagB-4 immunization, 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), confirmed 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 these antigen targets are close to each other, each antigen target has its own unique position and sequence. Importantly, they share common antigens with antigenic determinants located at different positions in the RagB-1, RagB-2, and RagB-3 proteins.

[0198] Based on the above, antigen-antibody reactions between monoclonal antibodies and peptide chains in the RagB peptide library confirm the antigenicity of RagB polypeptide fragments, provide a molecular basis for the development of antibodies and vaccines that can react with these antigenic fragments, and contribute to the development of therapeutic agents to control diseases caused by Porphyromonas gingivalis infection. 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) each 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 all of them can recognize antigens from four different RagB subtypes. The adjacent antigen targets provide ideal conditions for the preparation of mixed monoclonal antibodies and bispecific antibodies.

[0199] Example 5: Mechanism of development of heterophilic antigens and Porphyromonas gingivalis Recent clinical studies and statistical surveys have shown that periodontal disease is associated with various systemic diseases, particularly chronic diseases and tumors. Despite numerous detailed studies, the direct link between Porphyromonas gingivalis and chronic diseases, or the pathogenesis, remains unclear.

[0200] In Example 4, when monoclonal antibodies against the Cra4S1 protein were screened, the antibodies showed a strong reaction with the full-length recombinant Cra4S1 protein, but they could not identify specific antigenic target fragments of this protein. Cra4S1 polyclonal antibody serum also failed to identify the major polypeptide target in the Cra4S1 peptide library. These data suggest that there is no prominent dominant antigenic fragment in Cra4S1, or that the antigenicity of Cra4S1 is diffused and presented only by its three-dimensional spatial structure. Furthermore, in Example 4, monoclonal antibodies against the RagB-3 protein identified multiple target antigen polypeptide sequence fragments in the RagB-3 peptide library. However, tests using polyclonal antibody serum failed to identify the major peptide fragment and / or epitope of RagB-3 in the peptide library. Nevertheless, animals immunized with RagB-1 and RagB-4 proteins, and the resulting polyclonal antibodies, were able to recognize the 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 peptide RB-17. Overall, these results indicate that the antigenic properties of different outer membrane protein subtypes of Porphyromonas gingivalis are not the same.

[0201] Looking back at the research on the recombinant Cra4S1 protein, Example 2 showed that animal serum after Porphyromonas gingivalis infection did not react with Cra4S1 but reacted with RagB, suggesting that the antigenicity of the Cra4S1 protein may not be dominant. The data from Example 2 also showed that when RagB protein was used to immunize animals with a history of bacterial infection, serum antibodies showed an immune response with Cra4S1 but not with an unrelated protein GST-negative control, suggesting that RagB antibodies cross-react with the Cra4S1 protein. In several examples, it was found that RagB-4-1B11-4-4 monoclonal antibodies did indeed cross-react with Cra4S1.

[0202] Surprisingly, when antibodies against Porphyromonas gingivalis were detected using human serum samples, antibodies against RagB and Cra4S1 were detected in a considerably significant proportion in the samples. Specifically, the human serum samples collected in this example included those from healthy individuals, patients with cardiovascular disease, and geriatric patients. Tables 22, 26, and 29 show the ELISA results for these human serum samples, in which antibodies against Porphyromonas gingivalis outer membrane proteins were detected. The design and implementation of antigen adsorption on the ELISA plate were the same as described in Example 2, and the antigen layout design was briefly described as follows: RagB-1 recombinant protein was adsorbed in columns 1 and 7, RagB-2 in columns 2 and 8, RagB-3 in columns 3 and 9, RagB-4 in columns 4 and 10, Cra4S1 in columns 5 and 11, and GST recombinant protein as an unrelated protein-negative control in columns 6 and 12.

[0203] The dilution of the first group of serum samples (healthy subjects) was 1:300, and each serum sample was tested with a 6-well antigen protein test containing 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 (indicated as M-PC) was obtained from immunized mouse serum immunized with pentavalent protein containing RagB-1, RagB-2, RagB-3, RagB-4, and Cra4S1 as vaccine components.

[0204] This is the first time that Porphyromonas gingivalis outer membrane protein antibodies have been detected using human serum. Therefore, the mean OD value of at least one-fifth (at least 20%) of all samples was pre-defined as negative, and the cutoff value was provisionally set at 2.5 times that value. If the OD value exceeded 2.5 times the negative value, it was considered positive. Table 22 shows the ELISA results for serum samples from 51 healthy subjects. Table 23 shows the minimum OD values ​​(20% of the 51 samples) for each protein group detected from 10 subjects. Table 23 also shows the number of samples with an OD value higher than 2.5 times the negative OD value (considered a positive result), and data for suspected positive subjects with an OD value between 1.8 and 2.5 times the negative value. OD values ​​exceeding 4 times the negative value (4 times) were considered strongly positive and quantitatively reflected higher protein titers.

[0205] The human serum samples in Group 1 consisted of 51 healthy individuals (blood tests of young adults, numbers H1-H51). The positive rate for the RagB-1 antibody test was 11.8% (6 / 51), of which the strongly positive rate was 2% (1 / 51), with the OD value exceeding 4 times the negative value. The positive rate for the RagB-2 antibody test was 0% (0 / 51), the positive rate for the RagB-3 antibody test was 2% (1 / 51), and the positive rate for the RagB-4 antibody test was 31.3% (16 / 51), of which the strongly positive rate was 13.7% (7 / 51). Weakly positive results (1.8-2.5 times the negative serum OD value) accounted for 29.4% (15 / 51), indicating that RagB-4 antibody-positive samples comprised a relatively large portion of all samples (15 / 51 weakly positive, 9 / 51 positive, 7 / 51 strongly positive). The positive rate for Cra4S1 antibodies was 9.8% (5 / 51), while the positive rate for the unrelated protein GST antibodies was 0% (0 / 51).

[0206] The detection rates of RagB-1 and Cra4S1 antibodies were lower than those of RagB-4, but some RagB-1 antibodies showed a positive correlation with RagB-4 antibodies; that is, when RagB-4 antibodies were detected, the higher the OD value, the higher the OD value of the RagB-1 antibody. The detection rates of RagB-2, RagB-3, and GST antibodies were very low (Figures 12A-12B, Tables 22, 23, and 24). In this group of human serum samples, is it possible that the low antigenicity of the RagB-2 and RagB-3 subtypes prevented them from eliciting a significant antibody response?

[0207] From 2002 to 2004, an etiological study was conducted on periodontal disease patients who visited the dental outpatient clinic of the Royal London Hospital. A total of 107 samples were examined, and Porphyromonas gingivalis and its subtypes were detected in gingival crevicular exudate samples from periodontal disease patients using PCR. PCR nucleic acid testing revealed that the Porphyromonas gingivalis ragB-2 and ragB-3 alleles were the most common group in the periodontal disease patient population. The detailed method is described in patent application PCT / GB2005 / 001976, which is cited here. Briefly, total DNA was extracted from gingival crevicular exudate samples, and the 16S RNA of Porphyromonas gingivalis was identified by PCR. The ragB allele was detected using ragB primers with four pairs of probes 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 samples combined (Figure 13, Table 25). These clinical results indicate that the RagB-2 and RagB-3 Porphyromonas gingivalis subtypes are relatively common in the local East London periodontal disease population.

[0208] Toxicity studies of different bacterial strains using soft tissue injury animal models did not reveal any single outer membrane protein subtype exhibiting significantly stronger or weaker toxicity than others. The mechanisms by which bacterial toxicity and immune responses cause local and systemic damage in the body remain a mystery and an urgently needed milestone.

[0209] A similar ELISA method was also used to detect human serum antibodies in patients with cardiovascular disease and geriatric diseases. Table 26 shows the serum test results for geriatric patients. In summary, the antigen adsorption and detection procedures were the same as in the above examples, with the primary antibody (patient serum) dilution being 1:1000 for plates 20-05-P5, 20-05-P6, and 20-05-P7, and 1:500 for plate 20-05-P8. To avoid false positive results, the sample dilution was increased from 1:300 to 1:500 and 1:1000.

[0210] Table 22: Antibody levels of Porphyromonas gingivalis in the serum of healthy individuals. JPEG0007917864000022.jpg163106

[0211] Table 23 Analysis of Serum Survey Data from Healthy Individuals JPEG0007917864000023.jpg149155

[0212] Table 24 Analysis of antibody distribution of outer membrane proteins JPEG0007917864000024.jpg68161

[0213] Table 25 Distribution analysis of RagB subtypes in periodontal disease patients JPEG0007917864000025.jpg17145

[0214] Table 26: Antibody Survey of Porphyromonas gingivalis in Serum of Elderly Patients JPEG0007917864000026.jpg95163

[0215] Table 27 shows the data analysis of serum sample surveys from elderly patients (samples 201-255 in Table 26 and samples 256-262 in Table 29), totaling 62 samples. Based on pre-defined criteria, Table 28 shows the data analysis of serum sample surveys from elderly patients. The positive rate for RagB-1 antibody detection was 48.4% (30 / 62), of which the strongly positive rate was 22.6% (14 / 62). The positive rate for RagB-2 was 48.4% (30 / 62), of which the strongly positive rate was 24.2% (15 / 62). The positive rate for RagB-3 antibody was 46.8% (29 / 62), of which the strongly positive rate was 24.2% (15 / 62). The positive rate for RagB-4 antibody was 38.7% (24 / 62), of which the strongly positive rate was 22.6% (14 / 62). The positive rate for Cra4S1 antibody was 43.5% (27 / 62), of which 21% (13 / 62) were strongly positive. On the other hand, the positive rate for the unrelated protein GST antibody was 40.3% (25 / 62), of which 21% (13 / 62) were strongly positive, as shown in Figures 14A-14B.

[0216] Table 27 Data analysis of survey results in elderly patients JPEG0007917864000027.jpg138160

[0217] Table 28 Distribution analysis of outer membrane protein antibodies JPEG0007917864000028.jpg67140

[0218] Table 29: Antibody Survey of Porphyromonas gingivalis in Serum of Cardiovascular Patients JPEG0007917864000029.jpg96161

[0219] Table 30 Data Analysis of Survey Results in Cardiovascular Patients JPEG0007917864000030.jpg132160

[0220] Table 29 shows the original ELISA results for serological testing in cardiovascular patients (and some elderly patients; see above for details). In summary, the antigen adsorption and detection procedures were the same as described above, and the dilution ratio of patient serum in the reaction plate was 1:500.

[0221] Table 30 shows the data analysis of serum samples from cardiovascular patients (numbers 101-149), totaling 49 samples. Based on pre-defined criteria, the positive rate for RagB-1 antibody detection was 44.9% (22 / 49), of which the strongly positive rate was 8.2% (4 / 49). The positive rate for RagB-2 was 40.8% (20 / 49), of which the strongly positive rate was 8.2% (4 / 49). The positive rate for RagB-3 antibody was 46.9% (23 / 49), of which the strongly positive rate was 8.2% (4 / 49). The positive rate for RagB-4 antibody was 57.1% (28 / 49), of which the strongly positive rate was 10.2% (5 / 49). The positive rate for Cra4S1 antibody was 40.8% (20 / 49), of which the strongly positive rate was 4.1% (2 / 49). On the other hand, the positive rate for the unrelated protein GST antibody was 53.1% (26 / 49), of which the strongly positive rate was 4.1% (2 / 49), as shown in Figures 15A-15B and Table 31.

[0222] Compared to healthy individuals, serological surveys of patients with cardiovascular disease and geriatric illness exhibit three notable characteristics. First, the detection rates of RagB-2 and RagB-3 antibodies are significantly increased. Second, the detection rate of GST protein antibodies (i.e., antibodies against unrelated antigens) is substantially increased. Third, the proportion of patients with high-titer antibodies in samples from patients with cardiovascular disease and geriatric illness is far higher than in the healthy control group.

[0223] Table 31 Distribution analysis of outer membrane protein antibodies JPEG0007917864000031.jpg73153

[0224] Antibodies against the unrelated antigen GST are not detected in healthy individuals, but the detection rate of GST antibodies increases significantly in patients with cardiovascular disease and geriatric diseases. Furthermore, the detection of GST antibodies is directly related to the levels of RagB and Cra4S1 antibodies, and is only found in patients who are positive for these antibodies. Individuals with high OD values ​​for anti-RagB and anti-Cra4S1 antibodies also have high OD values ​​for GST antibodies. No cases of independent GST antibody positivity were found.

[0225] Serological antibody studies of Porphyromonas gingivalis outer membrane protein have revealed that a significant portion of cardiovascular disease and geriatric disease patients have antibodies to Porphyromonas gingivalis outer membrane protein in their serum. In particular, serum containing high titers of RagB-2, RagB-3, and Cra4S1 antibodies also shows an increased incidence (and high titer) of antibodies against the unrelated protein GST. These data provide scientific evidence suggesting that Porphyromonas gingivalis may cause disease through its heterophilic properties.

[0226] Evidence from multiple sources indicates that Porphyromonas gingivalis outer membrane proteins, including RagB and Cra4S1, possess heterophilic antigenic characteristics. Heterophilic antigens are general antigens that are structurally similar to but completely unrelated to antigens present in host tissues. Numerous scientific studies have reported diseases caused by heterophilic antigens. For example, the cell membrane of hemolytic streptococci shares antigens with the glomerular basement membrane and myocardial tissue, which can lead to tubulitis or myocarditis after streptococcal infection. Similarly, the lipopolysaccharide of Escherichia coli (E. coli) shares antigens with the human colon mucosa, and infection with the former is associated with the development of ulcerative colitis.

[0227] Heterophilic antibodies are polyspecific immunoglobulins with a certain titer that are induced by known or unknown antigenic substances (heterophilic antigens). They can bind to various proteins, but their affinity may not be strong. These antibodies are endogenous and spontaneously occurring, and can react with molecules unrelated to the original antigen. Immunoglobulins with these different chemical structures but multiple binding affinities to test substances are called heterophilic antibodies.

[0228] Heterophilic antibodies can be understood as polyspecific or nonspecific immune enhancements and may possess immunoprotective functions. However, the multiple immune responses of heterophilic antibodies against heterophilic antigens and antigenic components shared across different tissues can lead to immunopathology.

[0229] Serological studies revealed that in patients with cardiovascular disease and geriatric diseases, the majority of the total sample was positive for RagB, Cra4S1, and GST antibodies. Based on epidemiological data, Porphyromonas gingivalis outer membrane proteins, including RagB and Cra4S1, exhibit heterophilic antigenic characteristics.

[0230] Since the RagB-4 (381RagB) DNA / protein sequence (SEQ ID NO: 8) was first disclosed in 2004, scientists have discovered high similarities in protein sequences to RagB-4 in other species. For example, the outer membrane protein (WP_039431148) of Porphyromonas gulae (a major periodontal disease pathogen in dogs and cats) has 93% similarity to RagB-4 (see Figure 16), further supporting the heterophilic antigenic properties of Porphyromonas gingivalis and its broad potential applications. Furthermore, although RagB-4 is unrelated to the Cra4S1 molecular sequence, as described in Example 4 and later Example 7, the RagB-4-1B11-4-4 monoclonal antibody cross-reacts with the Cra4S1 protein. The fact that this cross-reaction is with the full-length Cra4S1 protein and not with a specific polypeptide suggests the possibility of diffusibility or weak binding, a feature commonly observed in heterophile antigens.

[0231] Periodontal disease is a very common disease in humans. Porphyromonas gingivalis is the main pathogen, and the detection of antibodies against Porphyromonas gingivalis outer membrane protein in human serum means that the subject has natural antibodies or a history of Porphyromonas gingivalis infection. Periodontal disease is usually chronic and progressive, and although the degree of the body's immune response to Porphyromonas gingivalis varies, it is usually not possible to completely eliminate the infection.

[0232] This example demonstrates relatively high detection rates of Porphyromonas gingivalis outer membrane protein antibodies in the serum of patients with cardiovascular disease and geriatric disease. Furthermore, the majority of these patients also show reactions between immunoglobulins and proteins unrelated to these antibodies in their serum. This suggests that a significant portion of these patients are immunosensitized, and that these antibodies are likely heterophile antibodies, strongly correlated with chronic disease and pathological damage, and may even exert pathogenicity in the pathogenesis of specific diseases.

[0233] As the experimental results show, Porphyromonas gingivalis appears to be less toxic to younger individuals. It is presumed that heterophilic antibodies do not accumulate to harmful levels in younger individuals. Human serum samples also show that in all three groups (healthy individuals, patients with cardiovascular disease, and geriatric patients), Porphyromonas gingivalis outer membrane protein antibodies were not detected in some individuals (Tables 22, 26, and 29). These individuals either have innate / nonspecific resistance to Porphyromonas gingivalis or have never been infected with Porphyromonas gingivalis. This finding highlights that Porphyromonas gingivalis may be one of the multifactorial contributors to complex systemic diseases, and that differences in individual immune responses play a crucial role in determining whether the infection is cleared or persists.

[0234] Detecting antibodies against RagB and Cra4S1 antigens, as well as GST or other tissue antigens in serum, is a simple and viable method for predicting the onset of Porphyromonas gingivalis infection and associated diseases. This can provide timely attention to disease development, leading to significant benefits in early patient treatment and reducing potential morbidity and mortality.

[0235] Further research can deepen our understanding of the association between Porphyromonas gingivalis infection and chronic disease. The data provided in this embodiment suggest that the heterophilic antigenic properties of Porphyromonas gingivalis, combined with individual characteristics, can explain heterophilic antibodies and / or hypersensitivity reactions, which are important elements in chronic Porphyromonas gingivalis infection and systemic disease. Therefore, by eliminating pathogenic infection, reducing the release of heterophilic antigens, and / or lowering circulating heterophilic antibody levels, it is possible to mitigate tissue damage caused by harmful heterophilic antibodies, actively treat related diseases, and control and prevent further deterioration of multiple tissues.

[0236] Example 6: Application of compound antibodies in animal breeding models Animal study data have shown that a combined vaccine of RagB and Cra4S1 proteins provides superior local tissue protection compared to a single-protein vaccine. However, the combined vaccine did not exhibit cross-protection against other subtypes of Porphyromonas gingivalis. For example, when animals were administered a combined vaccine containing RagB-1, RagB-3, and Cra4S1, they were protected against infection by Porphyromonas gingivalis RagB-1 and RagB-3 subtypes, but not against infection by the RagB-2 subtype. Passive immunization confirmed that antibody combinations, such as a mixture of anti-RagB and anti-Cra4S1, exhibited superior protective effects compared to either single antibody and the control group. Further details of protection are described in PCT / CN2019 / 124433, which is incorporated herein by reference.

[0237] Conventional antibody drug therapy focuses on the use of specific antibodies and provides precise treatment based on the diagnosis of Porphyromonas gingivalis outer membrane protein RagB subtypes. As described in Example 3, monoclonal antibodies against four different RagB proteins and Cra4S1 protein have been identified and described.

[0238] An antibody combination experiment was designed. In this example, the 1D2-2-1-3 monoclonal antibody specific to RagB-3 was combined with Cra4S1 polyclonal antibody derived from mouse serum. Specifically, 5- to 6-week-old male Balb / c mice (body weight 15-18 g) were divided into groups of 6 mice each. Four days and one day before bacterial infection, 100 μl of antibody solution was injected intraperitoneally into the mice (see Table 32). The injection solution contained 50 μg of monoclonal antibody, 200 μg of mouse serum (Pab in Table 32 refers to mouse polyclonal antibody), or a mixture of 50 μg of monoclonal antibody and 200 μg of mouse serum, after which the animals were exposed to bacterial challenge.

[0239] The preparation of the bacterial suspension is briefly described below. Porphyromonas gingivalis comprising various subtypes (RagB-1, RagB-2, RagB-3 and RagB-4) was grown on obligate anaerobic agar medium (FAA) containing 5% defibrinated horse blood, placed in an anaerobic incubator at 37°C, and the culture gas composition was 80% nitrogen, 10% hydrogen and 10% carbon dioxide. Bacterial colonies were transferred to freshly prepared brain heart extract (BHI) medium (containing 5 μg / ml hemin) and cultured for 18-24 hours until the OD600nm reached 1-1.2. Thereafter, the culture was centrifuged and washed twice. After washing, bacterial suspensions of different concentrations (2-8x10 10 CFU / ml) were prepared using BHI medium and used for animal experiments.

[0240] Table 32 Information of animals participating in the passive immunization experiment JPEG0007917864000032.jpg27161

[0241] After bacterial infection, the general condition of the animals and the progression of local injuries were observed, and photographs were taken. The records are shown in Tables 33 and 34, and in Figures 17A-17E and 18A-18B.

[0242] The data shows that animals treated with a combination of RagB-3 monoclonal antibody 1D2-2-1-3 and Cra4S1 polyclonal antibody acquired better local immune protection at the site of bacterial infection, exhibiting less soft tissue damage and faster healing compared to other animal groups that did not receive the antibody combination. The data also indicates that the antibody combination achieved a local effect even while circulating in body fluids. In this example, intraperitoneal injection (systemic administration technique) exerted a local subcutaneous protective effect against subsequent Porphyromonas gingivalis bacterial infection.

[0243] Table 33 Experimental data record of monoclonal antibody immune defense JPEG0007917864000033.jpg124161

[0244] Table 34 Data compilation JPEG0007917864000034.jpg33161

[0245] Periodontal disease caused by Porphyromonas gingivalis can manifest as localized damage visible to the naked eye. Clinicians can assess the severity of periodontal disease by examining the depth of periodontal pockets and susceptibility to gingival bleeding. However, the association between Porphyromonas gingivalis infection and a range of systemic diseases suggests that periodontal disease may be just the tip of the iceberg of the harm caused by Porphyromonas gingivalis infection.

[0246] Currently, conventional animal models of Porphyromonas gingivalis infection primarily focus on soft tissue destruction and alveolar bone resorption. The pathogenicity of different strains of Porphyromonas gingivalis is inconsistent, and local tissue damage caused by Porphyromonas gingivalis infection is observed in almost all animals, with the degree of damage depending on the infectivity dose. Generally, the higher the bacterial concentration infecting the animal, the more severe the local tissue damage. In most cases, infected animals self-heal, although some abscesses may recur after initial wound healing. In cases of bacterial infection alone, animals generally do not exhibit high mortality rates. The pathogenic mechanism of Porphyromonas gingivalis has never been definitively proven, and the scientific community has so far failed to establish an animal model associated with Porphyromonas gingivalis infection and systemic disease.

[0247] Over many years, clinical and epidemiological data have suggested that Porphyromonas gingivalis infection in pregnant women is associated with the birth of premature and low-birth-weight infants. Inspired by these clinical findings, we established an animal model to study the effects of Porphyromonas gingivalis infection on mouse reproduction.

[0248] In the first established animal model, three groups were set up: Group 1 consisted of male and female mice that received both antibody combinations and Porphyromonas gingivalis infection; Group 2 consisted of male mice as a control group and female mice that received both antibody combinations and Porphyromonas gingivalis infection; and Group 3 consisted of male mice that received both antibody combinations and Porphyromonas gingivalis infection and female mice as a control group. The antibodies were derived from rabbit serum, and details of the antibody combinations are shown in Table 35. Specifically, two intraperitoneal antibody injections were administered 4 days apart, after which the mice were infected with Porphyromonas gingivalis. During the recovery period, male and female mice from each group were randomly selected and mated. Each cage contained one male and two females. The PBS control group consisted of one male and one female per cage.

[0249] In the initial experiment, due to the limited number of control female mice (i.e., those not infected with antibodies or Porphyromonas gingivalis), younger female mice (11-12 weeks old instead of 18-20 weeks old) were used; see Table 35 for details. As a control, when control female mice of similar age were mated with control male mice, it was shown that 11-12 week old female mice produced fewer offspring.

[0250] The results showed that when both male and female mice were injected with antibodies and subsequently infected with Porphyromonas gingivalis, the rate of live offspring was low and the rate of stillbirth was high (Table 36). In contrast, when mice that were not injected with antibodies (PBS group) were infected with Porphyromonas gingivalis, they had a higher number of offspring and no stillbirths were observed compared to mice inoculated with the antibody combination.

[0251] Table 37 shows that female mice inoculated with a combination antibody and subsequently infected with Porphyromonas gingivalis were mated with normal control male mice. The results showed an increased number of offspring in this group compared to the previous group where both males and females were inoculated with the antibody combination. The control group consisted of female mice inoculated with PBS and infected with Porphyromonas gingivalis, mated with normal control male mice. In fact, the average number of offspring from female mice inoculated with the antibody combination (51 / 13, an average of 3.92 offspring per female mouse) was slightly higher than that from female mice inoculated with PBS control mice. While the data may not be sufficient to demonstrate statistical significance, these results suggest that antibody administration and Porphyromonas gingivalis infection play an important role in offspring reproduction in both male and female mice.

[0252] Table 38 shows that male mice administered the antibody combination and subsequently infected with Porphyromonas gingivalis were mated with normal control female mice. The results showed improved offspring numbers compared to the previous group, where both males and females received the antibody combination and Porphyromonas gingivalis infection. The control group consisted of male mice inoculated with PBS and infected with Porphyromonas gingivalis, mated with normal control female mice. The average offspring number of male mice administered the antibody combination and infected was similar to that of control mice vaccinated with PBS. Interestingly, the offspring numbers and survival rates in Table 38 were lower and the stillbirth rates higher than those in Table 37. These data suggest that the infection status of males after antibody administration and infection has a greater impact on survival and offspring rates than the infection status of female mice.

[0253] Table 35 Information on animals that participated in the experiment JPEG0007917864000035.jpg107161

[0254] Table 36 Reproductive Experiment (1) - Antibody administration and infection in both male and female animals (with the addition of a control that did not receive antibodies) JPEG0007917864000036.jpg32161

[0255] Table 37 Reproductive Experiment (2) - Mating between female mice that received antibody administration and infection (with the addition of a control that did not receive antibody administration) and male control mice that did not receive antibody administration or infection. JPEG0007917864000037.jpg33161

[0256] Table 38 Reproductive Experiment (3) - Mating between male mice that received antibody administration and infection (with the addition of a control that did not receive antibody administration) and female control mice that did not receive antibody administration or infection. JPEG0007917864000038.jpg32161

[0257] Table 39 Investigation of the immune-protective function of combined monoclonal antibodies JPEG0007917864000039.jpg45161

[0258] Based on the above results, the next step is to evaluate the effect of administering RagB monoclonal antibodies to these mouse models. As described in Example 4, the three monoclonal antibodies have shown potential as vaccines against *Porphyromonas gingivalis* due to their cross-reactivity against 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). As described in PCT / CN2019 / 124433, since the effect of passive immunity provided by a single antibody is limited, the present example aims to evaluate the effect of passive immunity using a combination of two (or more) monoclonal antibodies disclosed in the present invention.

[0259] Animal experiments with administration of antibody combinations and *Porphyromonas gingivalis* infection were performed as described above. Specifically, mice were administered antibodies via intraperitoneal injection twice at an interval of 4 days, after which the animals were infected with *Porphyromonas gingivalis*, and survival rate and local damage were observed and recorded.

[0260] Table 39 shows the results of passive immunity in animals infected with *Porphyromonas gingivalis* after administration of antibody combinations. Male and female mice were inoculated with the combinations of two monoclonal antibodies listed in Table 39, while the control group was administered normal mouse IgG or PBS, and then challenged with *Porphyromonas gingivalis* RagB-3 subtype. One mouse (1 / 8) administered with normal mouse IgG died after bacterial infection, and two mice (2 / 8) died in the G942 group and G944 group respectively. No female mice died.

[0261] During the recovery period, male and female mice were paired. Each cage contained one male and three females. In each breeding group, two female mice underwent an immunization challenge, while one female mouse was not subjected to antibody or infection. The weight of the female mice, as well as the number of live and stillborn offspring, were recorded. If a female mouse's weight decreased rapidly and no offspring were found, a miscarriage may have occurred. Due to a shortage of female mice in each group because fewer female mice were available than male mice, some female mice were used from other studies. These mice had records of antibody injection and Porphyromonas gingivalis infection, and specific details are shown in Table 40.

[0262] Table 40 Immune profiles of female mice added to the reproductive model JPEG0007917864000040.jpg73161

[0263] Table 41 Effects of combined monoclonal antibodies on reproduction (1) - Male and female mice were infected with mouse IgG, and the experimental group included female mice without antibodies and female mice without infection. JPEG0007917864000041.jpg70161

[0264] G941 / G951 (Table 41) and G945 / G955 (Table 45) were control groups, administered with normal mouse IgG and PBS, respectively. Since these animals were not administered any protective antibodies before infection with Porphyromonas gingivalis, these represent the baseline expected reproductive success rates of animals that recovered from infection. The number of offspring observed in both groups was similar, but in the G941 / G951 group, a high percentage of female mice in four cages did not become fully pregnant (4 / 7), while a high percentage of stillbirths were observed in the G945 / G955 group.

[0265] Table 42 Effects of combined monoclonal antibodies on reproduction (2) - Male and female mice were infected with a combination of monoclonal antibodies 1B11 and 1D2, and the experimental group included female mouse controls without antibodies and without infection. JPEG0007917864000042.jpg61161

[0266] In the G942 / G952 group (Table 42), the animals received a combination of 1D2 and 1B11 monoclonal antibodies prior to infection with Porphyromonas gingivalis. As described above, male mice had a 25% mortality rate following bacterial infection (2 / 8). Subsequent reproductive success in this group was also significantly impaired. Five of the six male mice were unable to impregnate female mice (including the control female), and only one male was able to reproduce. This normal female gave birth to four live offspring and one stillbirth in this group, and two other female mice were suspected to have miscarried.

[0267] In the G943 / G953 group (Table 43), the animals received a combination of 1B11 and 1C3 monoclonal antibodies prior to infection with Porphyromonas gingivalis. All animals, including males and females, survived Porphyromonas gingivalis infection. Males and females recovered well, although two males were unable to impregnate females (including the control female). In the remaining six cages, a total of 48 live offspring were observed from 18 females, with a small number of stillbirths (4) recorded.

[0268] In the G944 / G954 group (Table 44), the animals received a combination of 1D2 and 1C3 monoclonal antibodies prior to infection with Porphyromonas gingivalis. As described above, male mice had a 25% mortality rate following bacterial infection (2 / 8). Subsequent reproductive success was similar to that of the G945 / G955 control group, which was given PBS before infection. Of the remaining six male mice, one was unable to impregnate any female mouse (including the control female mouse). Observed live births: A total of 31 pups were born from 15 female mice. There were many stillbirths (10), and miscarriages were suspected in 3 female mice.

[0269] Table 43 Reproductive effects of combined monoclonal antibodies (3) - Male and female mice were infected with a combination of monoclonal antibodies 1B11 and 1C3, and the experimental group included female mice controls without antibodies and without infection. JPEG0007917864000043.jpg78161

[0270] Table 44 Reproductive effects of combined monoclonal antibodies (4) - Male and female mice were infected with a combination of monoclonal antibodies 1C3 and 1D2, and the experimental group included female mice controls without antibodies and without infection. JPEG0007917864000044.jpg61161

[0271] Table 45 Reproductive effects of combined monoclonal antibodies (5) - Male and female mice were infected with monoclonal antibody PBS, and the experimental group included female mouse controls without antibody and without infection. JPEG0007917864000045.jpg78161

[0272] Notably, the Rag-3 1D2 monoclonal antibody showed a local protective effect in animal infection experiments with Porphyromonas gingivalis (as shown in Tables 33 and 34). In multiple ELISA assays, 1D2 showed high affinity for the antigen. However, the reproductive effects of the 1D2 antibody (in different combinations) on animals may raise safety concerns, as a 25% mortality rate was observed in male mice in groups G942 and G944, along with decreased live birth rates, increased miscarriage and stillbirth rates, decreased fertilization success rates, or a combination of these indicators within the groups.

[0273] The results from the animal model disclosed in this embodiment demonstrate that Porphyromonas gingivalis infection causes reproductive disorders. According to the literature, pregnant women with periodontal disease are 3 to 7 times more likely to give birth prematurely and have low birth weight babies than pregnant women without periodontal disease. For many years, researchers have studied infections to genetic and endocrine characteristics, as well as to tissues and organs, focusing on infections to the maternal reproductive system and fetal characteristics.

[0274] This embodiment clarifies that Porphyromonas gingivalis infection causes reproductive disorders. The worst outcomes occur when both males and females are infected, with high rates of fertilization failure, stillbirth, and miscarriage. The data disclosed in this embodiment also show that when infected females mate with uninfected males, their reproduction remains stable, but conversely, when infected males mate with uninfected females, the final outcome is worse. These results clearly demonstrate that Porphyromonas gingivalis infection and immune status in males adversely affect reproductive outcomes, as infected males consistently exhibit poor reproductive outcomes in these animal models. A possible mechanism of action is mediated by heterophilic antigens on the surface of Porphyromonas gingivalis that resemble host tissues, such as sperm or the tissues of the male reproductive system.

[0275] Furthermore, while Porphyromonas gingivalis-specific antibodies can provide immunoprotective function, they can also cause immune-mediated pathological damage. For example, the 1D2 monoclonal antibody showed a strong antigen-antibody reaction to multiple RagB subtypes, with antigen targets including RA-17, RB-17, and RC-17 peptides (Example 4), exhibiting local immunoprotection. However, it was also noted that this monoclonal antibody caused high mortality and decreased reproductive success in this example.

[0276] The data disclosed in this embodiment also show that two other monoclonal antibodies, namely 1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3), are specific to RagB-4, can cross-react with multiple RagB subtypes, are safe, and do not harm the reproductive system of animals. These combined antibodies show that they effectively improve the physical condition of male and female animals and directly or indirectly restore / cure fertility. 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, neurological disorders, autoimmune encephalomyelitis, lung cancer, abnormal pregnancy, pancreatic cancer, diabetes mellitus, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease. One treatment strategy involves eliminating harmful antigens and / or antibodies associated with the onset and development of the disease, and / or increasing the titer or concentration of beneficial antibodies in the body.

[0277] Example 7 Mouse monoclonal antibody and humanized mutation 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 different subtypes of outer membrane proteins of Porphyromonas gingivalis. Nucleic acid sequences of these antibodies have been obtained. Briefly, the total RNA of each cell line is first extracted, and reverse transcription is performed using antisense primers according to conventional RNA extraction and transcription methods. Next, the variable region sequences of the light and heavy chains are amplified, and the PCR fragments are inserted into a pUC19T vector. Then, clones are screened using PCR techniques, and positive clones are selected and sequenced.

[0278] The nucleotide and amino acid sequences of the specific antibody are shown below.

[0279] RagB-2-1A4-3-7-7 (Baiying, China, B467701, also known as "1A4") contains a heavy chain (mouse IgG1) variable region (VH), its nucleotide sequence is shown in SEQ ID NO: 128, and the encoded amino acid sequence is shown in SEQ ID NO: 129. It also contains a light chain (mouse Kappa) variable region (VL), its nucleotide sequence is shown in SEQ ID NO: 133, and the encoded amino acid sequence is shown in SEQ ID NO: 134 (see also Figures 19A-19D). Recombinant antibody expression is not provided.

[0280] RagB-3-1D2-2-1-3 (Baiying, China, B467702, also known as "1D2") contains a heavy chain (mouse IgG2a) variable region (VH), its nucleotide sequence is shown in SEQ ID NO: 138, and the encoded amino acid sequence is shown in SEQ ID NO: 139. It also contains a light chain (mouse Kappa) variable region (VL), its nucleotide sequence is shown in SEQ ID NO: 143, and the encoded amino acid sequence is shown in SEQ ID NO: 144 (see also Figures 20A-20D). Recombinant antibody expression is shown in Figures 20E and 20F.

[0281] RagB-4-1B11-4-4 (Baiying, China, B583901, also known as "1B11") contains a heavy chain (mouse IgG1) variable region (VH), the nucleotide sequence of which is shown in SEQ ID NO: 148, and the encoded amino acid sequence is shown in SEQ ID NO: 149. It also contains a light chain (mouse Kappa) variable region (VL), the nucleotide sequence of which is shown in SEQ ID NO: 153, and the encoded amino acid sequence is shown in SEQ ID NO: 154 (see also Figures 21A-21D). The present invention also provides a bispecific antibody of this monoclonal antibody. Recombinant antibody expression is shown in Figures 21E and 21F.

[0282] RagB-4-1C3-7-8 (Baiying, China, B583902, also known as "1C3") contains a heavy chain (mouse IgG1) variable region (VH), its nucleotide sequence is shown in SEQ ID NO: 158, and the encoded amino acid sequence is shown in SEQ ID NO: 159. It also contains a light chain (mouse Kappa) variable region (VL), its nucleotide sequence is shown in SEQ ID NO: 163, and the encoded amino acid sequence is shown in SEQ ID NO: 164 (see also Figures 22A-22D). This invention provides a detailed description of this monoclonal antibody and its bispecific antibody. Recombinant antibody expression is shown in Figures 22E and 22F.

[0283] Table 46 Physicochemical properties of recombinant antibodies JPEG0007917864000046.jpg14160

[0284] Recombinant plasmids of three RagB monoclonal antibodies, including 1D2, 1B11, and 1C3, were expressed in eukaryotic cells HEK293 by a CRO company (Baiying, China). The purification results of the recombinant antibodies are shown in Figures 20E-20F, 21E-21F, and 22E-22F, and the physical and chemical properties of each recombinant antibody are shown in Table 46. The antigen-binding function of the recombinant antibodies was detected using ELISA (see Table 47).

[0285] Table 47 shows the detection results of recombinant antibody plasmids. The ELISA procedure was as described in Example 2, and the antigen adsorption design was as follows: Column 1: RA-21 peptide, Column 2: RB-21 peptide, Column 3: RC-21 peptide, Column 4: RC-22 peptide, Column 5: RD-21 peptide, Column 6: RD-22 peptide, Column 7: RagB-1 recombinant protein, Column 8: RagB-2 recombinant protein, Column 9: RagB-3 recombinant protein, Column 10: RagB-4 recombinant protein, Column 11: Cra4S1 recombinant protein, Column 12: RC-17 peptide. The antibodies are diluted to 100 μg / ml, and each antibody is added to one row of an ELISA plate. Antigen testing is then performed 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).

[0286] Table 47 Detection of target polypeptides of monoclonal antibodies JPEG0007917864000047.jpg35161

[0287] The data show that the binding reaction pattern of the recombinant antibodies to their corresponding antigens is the same as that of the monoclonal antibodies produced from hybridoma cells. Under identical antibody dilution conditions, the absorbance values ​​and patterns of the ELISA reaction plates are very similar, indicating that the recombinant antibodies retain their antigen-binding function and have the same stability as the original monoclonal antibodies. These recombinant antibodies are candidates for further development as therapeutic agents.

[0288] Example 6 discloses that a 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 a conserved epitope in all Porphyromonas gingivalis subtypes. Since Cra4S1 is considered a heterophilic antigen, the monoclonal antibody 1B11 can exert a synergistic effect by reducing the heterophilic properties of Cra4S1, in addition to providing cross-immunoprotection against other Porphyromonas gingivalis outer membrane protein subtypes.

[0289] Therefore, the humanized sequence mutations for the RagB-4-1B11-4-4(1B11) and RagB-4-1C3-7-8(1C3) monoclonal antibodies were designed by a third-party CRO company (Genscript, USA) according to conventional methods. This invention provides analysis of mouse chimeric antibodies, humanized gene synthesis, antibody expression and affinity screening, antibody production, and establishment of a seed cell bank, enabling industrial production of antibodies.

[0290] The monoclonal antibodies disclosed in this invention are used to treat chronic diseases caused by Porphyromonas gingivalis infection, including but not limited to periodontal disease, cardiovascular disease, rheumatoid arthritis, oral gastrointestinal cancer, ulcerative colitis, neurological disorders, autoimmune encephalomyelitis, lung cancer, abnormal pregnancy, pancreatic cancer, diabetes mellitus, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease.

[0291] Example 8: RagB bispecific monoclonal antibody Bispecific monoclonal antibodies (BsMAbs) are artificially synthesized proteins that can simultaneously bind to two different antigens or two or more different epitopes. Natural monoclonal antibodies typically target only one antigen. Through development, BsMAbs can be designed with a variety of different structural patterns. Through different mechanisms of action, BsMAbs can be designed to recruit and activate immune cells, interfere with receptor signaling, inactivate signaling ligands, and bind to protein complexes.

[0292] The data above shows that the antigenic targets of the two monoclonal antibodies RagB-4-1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3) are two adjacent epitopes within the peptide RD-21 & RD-22 region of the Porphyromonas gingivalis outer membrane protein RagB-4, and that the combination of the two monoclonal antibodies exhibits stable systemic and local immunoprotective effects. Therefore, a bispecific monoclonal antibody 1B11-1C3 (Baiying, China, B745901) was prepared by combining RagB-4-1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3). This antibody contains a heavy chain whose nucleotide sequence is shown at SEQ ID NO: 168 and whose encoded amino acid sequence is shown at SEQ ID NO: 172, and a light chain whose nucleotide sequence is shown at SEQ ID NO: 173 and whose encoded amino acid sequence is shown at SEQ ID NO: 174.

[0293] Specifically, the bispecific antibody 1B11-1C3 uses an scFv pattern, where the single-chain variable fragment (scFv) is a fusion protein of the heavy chain (VH) and light chain (VL) variable regions of an immunoglobulin linked by a commercially available short-chain peptide. This fusion protein includes a heavy chain containing the variable region of a specific monoclonal antibody linked by a short-chain peptide and the mouse IgG1 heavy chain constant region (CH), and a light chain containing the variable region of a specific monoclonal antibody and the mouse immunoKappa constant region.

[0294] The heavy chain of the bispecific 1B11-1C3 antibody contains 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 sequence includes 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 1C3 light chain variable region shown at SEQ ID NO: 164 (see Figures 23A-23F).

[0295] Table 48 Physicochemical properties of recombinant bispecific antibodies JPEG0007917864000048.jpg16161

[0296] Recombinant plasmids were transfected into eukaryotic cells HEK293, and recombinant antibodies were induced and purified by protein A affinity chromatography (see Figures 23G and 23H). The physicochemical properties of the recombinant bispecific antibody proteins are shown in Table 48. Antigen-antibody reactions were detected using ELISA, and data for bispecific antibodies are also provided in Table 47 of Example 7. The ELISA procedure was as described in Example 2, and the antigen adsorption design was as described in Example 7. The antibodies were diluted to 100 μg / ml, and 12 wells of antigen were tested with each antibody.

[0297] The data provided in this invention demonstrate that the bispecific 1B11-1C3 antibody exhibits antigen-antibody binding reactions to their respective corresponding target peptide fragments and different subtypes of recombinant proteins, and that each monoclonal antibody also exhibits the same reaction to its corresponding peptide target. The bispecific antibody exhibits the same effect as the combination of monoclonal antibodies 1B11-4-4 and 1C3-7-8. Compared to the use of combined antibodies and single antibodies, the fusion protein retains the specificity of the original immunoglobulin and enhances the immune response to the antigen target. The bispecific monoclonal antibodies disclosed in this invention are intended for the treatment of chronic diseases caused by Porphyromonas gingivalis infection, including but not limited to periodontal disease, cardiovascular disease, rheumatoid arthritis, oral gastrointestinal cancer, ulcerative colitis, neurological disorders, autoimmune encephalomyelitis, lung cancer, abnormal pregnancy, pancreatic cancer, diabetes mellitus, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease.

[0298] If therapeutic efficacy can be ensured and stable production can be achieved, bispecific antibodies will be a more efficient and simpler solution for developing new therapies.

[0299] Example 9: Serum antibody diagnostic kit The data disclosed in Example 5 show that approximately 40–50% of patients with cardiovascular disease and geriatric disease have high titer antibodies against the Porphyromonas gingivalis outer membrane proteins RagB and Cra4S1, as well as unrelated proteins (such as GST recombinant proteins). The relevant data suggest that qualitative and / or quantitative testing and / or measurement of these antibodies may be useful for the early diagnosis of Porphyromonas gingivalis infection, for assessing the risk of developing chronic diseases associated with Porphyromonas gingivalis infection, and for evaluating disease prognosis and treatment efficacy.

[0300] This invention provides a method for diagnosing or evaluating the prognosis and / or therapeutic effect of porphyromonas gingivalis infection or chronic diseases caused by or associated with porphyromonas gingivalis. Specifically, it allows for screening of antibodies against patients' RagB proteins (including Rag-1, RagB-2, RagB-3, and RagB-4), Cra4S1 protein, and unrelated proteins (including GST and / or other tissue proteins, e.g., muscle proteins or mucosal proteins). ELISA technology is used to detect the presence or absence of antibodies in the serum of individual patients. High antibody levels, especially high titers, indicate that the patient is immunosusceptible, thereby increasing the risk of developing certain chronic diseases. This screening allows for patient stratification and identification of high-risk individuals, facilitating the prompt initiation of treatment.

[0301] Example 10: PgingiVacRD1B11-1C3, polypeptide vaccine Data from animal models indicate that while most animals can self-repair local damage from a first-time Porphyromonas gingivalis infection, repeated infections can gradually worsen the condition and make local damage more difficult to heal. These phenomena suggest that the host's immune defenses and bacterial invasion enter a continuous imbalance process of repair and deterioration, and that chronic Porphyromonas gingivalis infections are not normally completely eliminated.

[0302] For certain diseases, patients need to use antibody medications for extended periods and cannot discontinue treatment. However, a single dose of vaccine may be a more effective, simpler, and cost-effective treatment option.

[0303] For the treatment of Porphyromonas gingivalis infection, after a period of passive immunotherapy with specific antibodies, patients can receive an efficient polypeptide vaccine, which stimulates the production of their own effective neutralizing antibodies. These antibodies can remain in the patient's serum for a long period, thereby preventing the proliferation and transmission of the pathogen. The polypeptide vaccine of the present invention is cost-effective and can be safely administered via conventional routes. Furthermore, the vaccine product does not contain full-length proteins and avoids particularly harmful, allergic, and irrelevant antigenic fragments, thus reducing the risk of heterophilic antigens producing heterophilic antibodies.

[0304] This invention discloses a polypeptide vaccine, namely PgingiVacRD1B11-1C3, which is an RD21 to RD22 polypeptide fragment based on a RagB-4-1B11-4-4(1B11) and / or RagB-4-1C3-7-8(1C3) antibody reaction. Both 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 recombinant nucleotides of the polypeptide and expressed under host cell induction conditions. The protein expressed in host cells 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.

[0305] This invention presents and describes various methods for carrying out the invention, but to those skilled in the art, these embodiments are provided only as examples. The invention is not limited to the specific examples provided in the specification. Although the invention has been described with reference to the above specification, the descriptions and illustrations of embodiments herein are not intended to be constrained. Those skilled in the art can also make several changes, modifications, and substitutions without departing from the principles of the invention. Accordingly, it should be understood that not all aspects of the invention are limited to the specific descriptions, structures, or relative proportions described herein, and that these descriptions, structures, or relative proportions depend on various conditions and variables. When carrying out the invention, it should be understood that various alternatives to the embodiments of the invention described herein can be adopted. Accordingly, the invention should also encompass such alternatives, modifications, changes, or equivalents. The following claims define the scope of the invention and are intended to encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. Monoclonal antibodies targeting one or more outer membrane proteins of P. gingivalis, selected from the group consisting of (i) to (iii) below: (i) Light chain variable regions (VL) whose amino acid sequence is listed in sequence listing SEQ ID NO: 154, and which include the amino acid sequences of (a) CDR-L1 as SEQ ID NO: 155, (b) CDR-L2 as SEQ ID NO: 156, and (c) CDR-L3 as SEQ ID NO: 157; and heavy chain variable regions (VH) whose amino acid sequence is listed in SEQ ID NO: 149, and which include the amino acid sequences of (a) CDR-H1 as SEQ ID NO: 150, (b) CDR-H2 as SEQ ID NO: 151, and (c) CDR-H3 as SEQ ID NO: 152; (ii) Light chain variable regions (VL) whose amino acid sequence is listed in sequence listing SEQ ID NO: 164, and which include the amino acid sequences of (a) CDR-L1 as SEQ ID NO: 165, (b) CDR-L2 as SEQ ID NO: 166, and (c) CDR-L3 as SEQ ID NO: 167; and heavy chain variable regions (VH) whose amino acid sequence is listed in SEQ ID NO: 159, and which include the amino acid sequences of (a) CDR-H1 as SEQ ID NO: 160, (b) CDR-H2 as SEQ ID NO: 161, and (c) CDR-H3 as SEQ ID NO: 162; (iii) Light chain variable region (VL) whose amino acid sequence is listed in sequence listing SEQ ID NO: 144, and which includes the amino acid sequences of (a) CDR-L1 as SEQ ID NO: 145, (b) CDR-L2 as SEQ ID NO: 146, and (c) CDR-L3 as SEQ ID NO: 147; and heavy chain variable region (VH) whose amino acid sequence is listed in SEQ ID NO: 139, and which includes the amino acid sequences of (a) CDR-H1 as SEQ ID NO: 140, (b) CDR-H2 as SEQ ID NO: 141, and (c) CDR-H3 as SEQ ID NO:

142.

2. A monoclonal antibody according to claim 1, which is Fab, Fab', F(ab)'2, single-chain Fv(scFv), Fv fragment, or IgG class.

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

4. An isolated nucleic acid molecule encoding the monoclonal antibody described in claim 1.

5. An expression vector comprising the isolated nucleic acid molecule described in claim 4.

6. A cell or cell line comprising an isolated nucleic acid molecule according to claim 4 or an expression vector according to claim 5.

7. A composition comprising the monoclonal antibody described in claim 1 and a pharmaceutically acceptable carrier.

8. A composition comprising an isolated nucleic acid molecule according to claim 4 and a pharmaceutically acceptable carrier.

9. A composition comprising the cells or cell line described in claim 6 and a pharmaceutically acceptable carrier.

10. The composition according to claim 7, for use in the treatment of porphyromonas gingivalis infection or chronic diseases caused by porphyromonas gingivalis infection.

11. The composition according to claim 10, wherein the chronic disease is periodontal disease, cardiovascular disease, rheumatoid arthritis, oral gastrointestinal cancer, ulcerative colitis, neurological disease, autoimmune encephalomyelitis, cancer, abnormal pregnancy, malignant pancreatic cancer, diabetes mellitus, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease.

12. A method for producing a monoclonal antibody according to claim 1, comprising culturing cells or a cell line containing the isolated nucleic acid molecule described in claim 4, and recovering a monoclonal antibody from the cultured cells.

13. An antigen target peptide to which the monoclonal antibody according to claim 1 binds, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 55, 79, and 103, the group consisting of SEQ ID NOs: 125, 55, 80, and 126, and the group consisting of SEQ ID NOs: 123, 51, and 124, derived from isolated Porphyromonas gingivalis outer membrane protein.

14. A method for evaluating the therapeutic effect of Porphyromonas gingivalis infection, comprising administering the monoclonal antibody described in claim 1 to a non-human animal infected with Porphyromonas gingivalis, and evaluating the effect on the reproduction of the non-human animal.

15. A method for detecting antibodies in a biological sample, (a) Performing an antigen-antibody reaction or cross-reaction with the antigen peptide described in claim 13 to detect a specific antibody of Porphyromonas gingivalis, and (b) A detection method comprising detecting heterophilic antibodies against heterophilic antigens unrelated to Porphyromonas gingivalis.

16. (a) The antigen peptide reagent according to claim 13, (b) Reagents for heterophilic antigens unrelated to Porphyromonas gingivalis, and (c) A kit for monitoring the prognosis of Porphyromonas gingivalis infection, including instructions for testing specific antibodies in a biological sample using reagents.

17. The kit according to claim 16, wherein the biological sample is selected from the group consisting of blood, gingival crevicular exudate, urine, saliva, cerebrospinal fluid, pleural fluid / ascites fluid, and amniotic fluid.

18. The composition according to claim 8, for use in the treatment of an infection caused by Porphyromonas gingivalis or a chronic disease caused by Porphyromonas gingivalis infection.

19. The composition according to claim 9, for use in the treatment of an infection caused by Porphyromonas gingivalis or a chronic disease caused by Porphyromonas gingivalis infection.

Images