Antibodies to 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 biologics, and in particular to multi-targeted monoclonal antibodies against Porphyromonas gingivalis infections. [Background technology]

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

[0003] Porphyromonas gingivalis is considered in the research field to be the causative agent of "adult" periodontal disease. Published literature has shown that periodontal disease caused by Porphyromonas gingivalis infection in children is less severe than in adults. The survival rate of dental implants is closely related to the health of the periodontal tissues in the oral cavity. Porphyromonas gingivalis is the primary pathogen causing peri-implantitis. In reality, most patients with periodontal disease experience a chronic, progressive, and recurrent course of the disease, which means that the bacterial infection is never eradicated.

[0004] Numerous clinical and statistical studies have shown that P. gingivalis not only causes oral and periodontal lesions, but is also associated with cardiovascular disease, Alzheimer's disease, diabetes, and tumors of the digestive and respiratory systems. P. gingivalis infection is also closely associated with an increased incidence of low birth weight and premature birth, and is closely associated with many chronic systemic diseases. To date, the pathogenetic mechanisms underlying the relationship between local infection and systemic diseases remain unclear, and experimental results sometimes contradict the intended research direction.

[0005] There are many different subtypes of P. gingivalis, and these subtypes have different bacterial virulence. When examining the pathogenic mechanisms of bacteria, bacterial virulence and abnormal immune responses have been the focus of controversy in the scientific community.

[0006] Currently, the main clinical method for treating periodontal disease is to remove local lesions using broad-spectrum antibiotics and physical / mechanical methods, with surgical treatment performed if necessary. In clinical practice, comprehensive solutions are needed to address issues such as how to prevent, treat, and control P. gingivalis infections, alleviate local and systemic damage caused by bacterial infection, how to select biological products with immune defense effects, and how to evaluate the function of new drugs using animal experimental models. Summary of the Invention [Problem to be solved by the invention]

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

[0008] The technical problem that the present invention aims to solve is to provide evidence that antibodies against P. gingivalis outer membrane proteins 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 P. gingivalis outer membrane proteins and the pathogenesis of local and systemic disorders caused by P. gingivalis infection.

[0010] A further technical problem that the present 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 P. gingivalis, specific monoclonal antibodies, and methods for producing the same. The present invention also discloses specific antigen targets corresponding to the 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 NOs: 1-10). The present invention also provides methods for producing recombinant proteins from plasmids containing artificial synthetic genes, expression cassettes, and recombinant vector or cell designs. In some embodiments, the specific antigen target sequences of the recombinant proteins are as set forth in SEQ ID NOs: 11-126.

[0012] The monoclonal antibodies described herein are directed to multiple antigen targets disclosed herein. In some embodiments, the nucleotide and amino acid sequences of the monoclonal antibodies corresponding to the antigen targets are as set forth in SEQ ID NOs: 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 as set forth in SEQ ID NOs: 168-174, and the present invention also provides methods for producing these bispecific antibodies.

[0013] The present invention also provides methods for preventing and treating periodontal disease / peri-implantitis and other diseases caused by chronic P. gingivalis infection using one or more monoclonal antibodies disclosed herein. In some embodiments, the combined antibody formulation comprises a RagB-4-1B11-4-7-7 monoclonal antibody and a RagB-4-1C3-7-8 monoclonal antibody in a mass ratio of 1:0.5-5. The present invention also provides expression cassettes, recombinant vectors, and eukaryotic cells for producing monoclonal and / or bispecific antibody pharmaceuticals for the prevention or treatment of periodontal disease / peri-implantitis and diseases caused by chronic P. gingivalis infection. The monoclonal and / or bispecific antibodies of the present invention are used in patients to prevent primary disease, recurrent disease, and chronic infectious diseases and diseases other than oral / dental diseases caused by P. gingivalis infection.

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

[0015] The present invention also provides novel animal models, which in some embodiments are infected with P. gingivalis and used to assess the effect of monoclonal antibodies and / or antibody-antigen reactions or cross-reactions of the present invention on the reproduction of the animals.

[0016] The present invention also provides a serological diagnostic kit for diagnosing and evaluating the therapeutic efficacy and prognosis of P. gingivalis infection and related diseases using a specific diagnostic reagent. In some embodiments, the amino acid sequence of the diagnostic reagent is as set forth in SEQ ID NOs. 2, 4, 6, 8, and 10. The diagnostic kit also provides instructions for using the reagent to test for specific antibodies in biological samples, including, but not limited to, body fluids such as blood, gingival crevicular fluid, urine, saliva, cerebrospinal fluid, pleural effusion / ascites, and amniotic fluid. [Brief explanation of the drawings]

[0017] The various aspects of the present disclosure can be better understood by reference to the drawings. The contents of the drawings are not necessarily drawn to scale, and it is important to clearly explain the principles of the present invention. Furthermore, parts corresponding to reference numerals in the drawings have multiple meanings. The drawings (hereinafter abbreviated as "figures") apply the principles of the invention, and when combined with the detailed description of the embodiments, the features and advantages of the present disclosure can be better understood. [Figure 1A] 1A shows SEQ ID NO: 1, which has a total length of 1461 bases and an average C+G content of 55.10%. [Figure 1B] Figure 1B shows the nucleic acid and amino acid sequences of Porphyromonas gingivalis RagB-1. Figure 1B shows SEQ ID NO: 2, which is 482 amino acids in length. For details, see Example 1. [Figure 2A] Figure 2 shows the nucleic acid and amino acid sequences of RagB-2 from Porphyromonas gingivalis. Figure 2A shows SEQ ID NO: 3, which has a total length of 1449 bases and an average C+G content of 51.62%. For details, see Example 1. [Figure 2B] Figure 2 shows the nucleic acid and amino acid sequences of RagB-2 from Porphyromonas gingivalis. Figure 2B shows SEQ ID NO: 4, which is 480 amino acids in length. For details, see Example 1. [Figure 3A]3A shows the nucleic acid and amino acid sequences of RagB-3 from Porphyromonas gingivalis. Figure 3A shows SEQ ID NO: 5, which has a total length of 1470 bases and an average C+G content of 51.29%. For details, see Example 1. [Figure 3B] Figure 3B shows the nucleic acid and amino acid sequences of RagB-3 from Porphyromonas gingivalis. Figure 3B shows SEQ ID NO: 6, which has a full length of 485 amino acids. For details, see Example 1. [Figure 4A] Figure 4 shows the nucleic acid and amino acid sequences of RagB-4 from Porphyromonas gingivalis. Figure 4A shows SEQ ID NO: 7, which has a total length of 1455 bases and an average C+G content of 51.00%. For details, see Example 1. [Figure 4B] Figure 4B shows the nucleic acid and amino acid sequences of RagB-4 from Porphyromonas gingivalis. Figure 4B shows SEQ ID NO: 8, which is 480 amino acids in length. For details, see Example 1. [Figure 5A] Figure 5 shows the nucleic acid and amino acid sequences of Porphyromonas gingivalis Cra4S1. Figure 5A shows SEQ ID NO: 9, which has a total length of 411 bases and an average C+G content of 52.55%. For details, see Example 1. [Figure 5B] Figure 5B shows the nucleic acid and amino acid sequences of Porphyromonas gingivalis Cra4S1. Figure 5B shows SEQ ID NO: 10, which is 132 amino acids in length. For details, see Example 1. [Figure 6] Figure 6 shows the polypeptide sequence alignment among the four major outer membrane proteins, including RagB-1, RagB-2, RagB-3, and RagB-4, by multiple sequence alignment using CLUSTALW (1.81). Figure 6 discloses the sequences of SEQ ID NO: 2 (new name: RagB-1, formerly named W50 RagB), SEQ ID NO: 4 (new name: RagB-2, formerly named ThaiRagB), SEQ ID NO: 6 (new name: RagB-3, formerly named QMULRagB), and SEQ ID NO: 8 (new name: RagB-4, formerly named 381RagB). For details, see Example 1. [Figure 7A] Expression, testing, purification, and identification of RagB-1 recombinant protein are shown. Figure 7A shows the expression of recombinant protein after induction. Lane M is a protein molecular weight marker (15-120 kDa), lanes 1-3 are BSA standards (0.5 μg, 1 μg, and 2 μg), lanes 4-6 are total bacterial protein (total bacterial lysate) after culturing seed cells for 7.4 and 24 hours, lanes 7-9 are the supernatant of total bacterial lysate after culturing seed cells for 7.4 and 24 hours, and lanes 10-12 are the pellet of total bacterial lysate after culturing seed cells for 7.4 and 24 hours. For details, see Example 1. [Figure 7B] Expression, testing, purification, and identification of recombinant RagB-1 protein are shown. Figure 7B shows the purified recombinant protein examined by SDS-PAGE. Lane M is a protein molecular weight marker (15-120 kDa), lane 1 is the non-reduced recombinant RagB-1 protein, and lane 2 is the reduced recombinant RagB-1 protein. For details, see Example 1. [Figure 8A] Expression, testing, purification, and identification of RagB-2 recombinant protein are shown. Figure 8A shows the expression of recombinant protein after induction. Lane M is a protein molecular weight marker (15-120 kDa), lanes 1-3 are BSA standards (0.5 μg, 1 μg, and 2 μg), lanes 4-6 are total bacterial proteins (total bacterial lysate) from seed cells cultured uninduced and for 22 hours, and lanes 7-9 are total bacterial lysate supernatants from seed cells cultured uninduced and for 22 hours. For details, see Example 1. [Figure 8B] Expression, testing, purification, and identification of recombinant RagB-2 protein are shown. Figure 8B shows the purified recombinant protein examined by SDS-PAGE. Lane M is a protein molecular weight marker (15-120 kDa), lane 1 is the non-reduced recombinant RagB-2 protein, and lane 2 is the reduced recombinant RagB-2 protein. For details, see Example 1. [Figure 9A]Expression, testing, purification, and identification of RagB-3 recombinant protein are shown. Figure 9A shows the expression of recombinant protein after induction. Lane M is a protein molecular weight marker (15-120 kDa), lanes 1-3 are BSA standards (0.5 μg, 1 μg, and 2 μg), lanes 4-6 are total bacterial proteins (total bacterial lysate) from uninduced and 22-hour cultures of seed cells, and lanes 7-9 are the supernatants of total bacterial lysate from uninduced and 22-hour cultures of seed cells. For details, see Example 1. [Figure 9B] Expression, testing, purification, and identification of recombinant RagB-3 protein are shown. Figure 9B shows the purified recombinant protein examined by SDS-PAGE. Lane M is a protein molecular weight marker (15-120 kDa), lane 1 is the non-reduced recombinant RagB-3 protein, and lane 2 is the reduced recombinant RagB-3 protein. For details, see Example 1. [Figure 10A] Expression, testing, purification, and identification of RagB-4 recombinant protein are shown. Figure 10A shows the expression of recombinant protein after induction. Lane M is a protein molecular weight marker (15-120 kDa), lanes 1-3 are 0.5 μg, 1 μg, and 2 μg of BSA standard, lanes 4-6 are total bacterial protein (total bacterial lysate) after culturing seed cells for 6 and 15.5 hours, lanes 7-9 are the supernatant of total bacterial lysate after culturing seed cells for 6 and 15.5 hours, and lanes 10-12 are the pellet of total bacterial lysate after culturing seed cells for 6 and 15.5 hours. For details, see Example 1. [Figure 10B] Expression, testing, purification, and identification of recombinant RagB-4 protein are shown. Figure 10B shows the purified recombinant protein examined by SDS-PAGE. Lane M is a protein molecular weight marker (15-120 kDa), lane 1 is the non-reduced recombinant RagB-4 protein, and lane 2 is the reduced recombinant RagB-4 protein. For details, see Example 1. [Figure 11A]Expression, testing, purification, and identification of the Cra4S1 recombinant protein are shown. Figure 11A shows the expression of the recombinant protein after induction. Lane M is a protein molecular weight marker (15-120 kDa), lanes 1-3 are BSA standards (0.5 μg, 1 μg, and 2 μg), lanes 4-6 are total bacterial proteins (total bacterial lysate) after culturing seed cells for 14 and 23 hours, lanes 7-9 are the supernatants of the total bacterial lysate after culturing seed cells for 14 and 23 hours, and lanes 10-12 are the pellets of the total bacterial lysate after culturing seed cells for 14 and 23 hours. For details, see Example 1. [Figure 11B] Figure 11B shows the expression, testing, purification, and identification of the Cra4S1 recombinant protein. Figure 11B shows the purified recombinant protein examined by SDS-PAGE. Lane M is a protein molecular weight marker (15-120 kDa), lane 1 is the non-reduced recombinant Cra4S1 protein, and lane 2 is the reduced recombinant Cra4S1 protein. For details, see Example 1. [Figure 12A] Figure 12 shows the distribution of antibodies against different outer membrane proteins of P. gingivalis in 51 serum samples from healthy individuals. Figure 12A shows that the mean OD value of the lowest quintile (10 / 51) of samples is set as the negative value. The positive rate is set as the OD value greater than 2.5 times the negative value. The positive rate for RagB-1 antibodies was 9.8% (5 / 51), the positive rate for high-titer antibodies (OD values ​​greater than 4 times the negative value) was 2% (1 / 51), the positive rate for RagB-2 antibodies was 0% (0 / 51), the positive rate for RagB-3 antibodies was 0% (0 / 51), but the positive rate for high-titer antibodies was 2% (1 / 51), the positive rate for RagB-4 antibodies was 17.6% (9 / 51), and the positive rate for high-titer antibodies was 13.7% (7 / 51), the positive rate for Cra4S1 antibodies was 9.8% (5 / 51), and the positive rate for high-titer antibodies was 0% (0 / 51), and the positive rate for antibodies to the unrelated protein GST was 0% (0 / 51). For details, see Example 5. [Figure 12B]Figure 12 shows the distribution of antibodies against different outer membrane proteins of P. gingivalis in 51 serum samples from healthy individuals. Figure 12B shows the percentage distribution of antibodies in the analyzed samples. For details, see Example 5. [Figure 13] This figure shows the distribution of the P. gingivalis ragB gene in gingival crevicular fluid samples from 107 patients with periodontal disease. Results: 28 patients were negative for P. gingivalis 16s RNA by PCR (28 / 107, 26.2%). Of the 16s RNA-positive samples, PCR ragB amplification tests showed that 7 patients were positive for the RagB-1 subtype (7 / 107, 6.6%), 30 patients were positive for the RagB-2 subtype (30 / 107, 28.0%), 32 patients were positive for the RagB-3 subtype (32 / 107, 29.9%), 6 patients were positive for the RagB-4 subtype (6 / 107, 5.6%), and 4 patients were of an unknown ragB subtype (4 / 107, 3.7%). For details, see Example 5. [Figure 14A] The distribution of antibodies against different outer membrane proteins of P. gingivalis in 62 serum samples from elderly patients is shown in Figure 14A. The mean OD value of the samples in the lowest quintile (12 / 62) was set as the negative value. The positive rate was set as the OD value greater than 2.5 times the negative value. The positive rate of RagB-1 antibody was 25.8% (16 / 62), and the high-titer antibody (OD value greater than 4 times the negative value) was 22.6% (14 / 62). The positive rate of RagB-2 antibody was 24.2% (15 / 62), and the high-titer antibody was 24.2% (15 / 62). The positive rate of RagB-3 antibody was 22.6% (14 / 62), and the high-titer antibody was 24.2% (15 / 62). The positive rate for RagB-4 antibodies was 16.1% (10 / 62), with high-titer antibodies in 22.6% (14 / 62). The positive rate for Cra4S1 antibodies was 22.6% (14 / 62), with high-titer antibodies in 21% (13 / 62). The positive rate for antibodies to the unrelated protein GST was 19.4% (12 / 62), with high-titer antibodies in 21% (13 / 62). For details, see Example 5. [Figure 14B]Figure 14 shows the distribution of antibodies against different outer membrane proteins of P. gingivalis in 62 serum samples from elderly patients. Figure 14B shows the percentage distribution of antibodies in the analyzed samples. For details, see Example 5. [Figure 15A] The distribution of antibodies against different outer membrane proteins of P. gingivalis in 49 serum samples from cardiovascular patients is shown in Figure 15A. The mean OD value of the samples in the lowest quintile (10 / 49) was set as the negative value. The positive rate was set as the OD value greater than 2.5 times the negative value. The positive rate of RagB-1 antibodies was 36.7% (18 / 49), with high-titer antibodies (OD value greater than 4 times the negative value) being 8.2% (4 / 49). The positive rate of RagB-2 antibodies was 32.7% (16 / 49), with high-titer antibodies being 8.2% (4 / 49). The positive rate of RagB-3 antibodies was 38.8% (19 / 49), with high-titer antibodies being 8.2% (4 / 49). 9), the positive rate for RagB-4 antibodies was 46.9% (23 / 49), with 10.2% (5 / 49) showing high titer antibodies, the positive rate for Cra4S1 antibodies was 36.7% (18 / 49), with 4.1% (2 / 49) showing high titer antibodies, and the positive rate for antibodies to the unrelated protein GST was 49% (24 / 49), with 4.1% (2 / 49) showing high titer antibodies. For details, see Example 5. [Figure 15B] Figure 15 shows the distribution of antibodies against different outer membrane proteins of P. gingivalis in 49 serum samples from cardiovascular patients. Figure 15B shows the percentage distribution of antibodies in the analyzed samples. For details, see Example 5. [Figure 16] Figure 1 shows a protein sequence alignment between P. gingivalis RagB-4 and the outer membrane protein of P. gulae strain COT-052 OH2179. When the outer membrane protein sequence (PubMed ID: 25858832, GenBank: JRAJ01000005.1) was aligned with RagB-4 (SEQ ID NO: 8), the two proteins shared 93% similarity. For details, see Example 5. [Figure 17]This figure shows the passive immune function of a combination of two target-specific antibodies. Photographs were taken 12 days after P. gingivalis challenge. Figure 17A shows mice in group G1 were administered monoclonal antibody (MAb) 1D2-2-1-3. Figure 17B shows mice in group G2 were administered MAb 1D2-2-1-3 and a mouse polyclonal antibody against Cra4S1. Figure 17C shows mice in group G3 were administered a mouse polyclonal antibody against Cra4S1. Figure 17D shows mice in group G4 were administered normal mouse serum. Figure 17E shows mice in group G5 were administered PBS as a control group for bacterial challenge. For details, see Example 6. [Figure 18A] The passive immune protective function of the conjugated antibody is shown (see Example 6 for details). The test animal group is shown on the X axis, and the skin and soft tissue lesion area (mm2) is shown on the Y axis. Figure 18A shows the results 5 days after challenge (see Example 6 for details). Statistical analysis was performed using one-way ANOVA, and *P<0.05 indicates a significant difference. [Figure 18B] The passive immune protective function of the conjugated antibody is shown (see Example 6 for details). The test animal group is plotted on the X axis, and the skin and soft tissue lesion area (mm) is plotted on the Y axis. Figure 18B shows the results 12 days after challenge (see Example 6 for details). Statistical analysis was performed using one-way ANOVA, and *P<0.05 indicates a significant difference. [Figure 19] Figure 19 shows the sequence of 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 the 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 the VL. For details, see Example 7. [Figure 20]The sequence of monoclonal antibody RagB-3-1D2-2-1-3 and examination of the recombinant antibody expressed in eukaryotic HEK293 cells and purified are shown. 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 the 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 the VL. Figure 20E shows the peak at 280 nm obtained by HPLC purification of the recombinant antibody and collecting the eluate. Figure 20F shows the purified recombinant antibody confirmed by SDS-PAGE, where lane M is a protein molecular weight marker (15-120 kDa) and lane 1 is purified RagB-3-1D2-2-1-3. For details, see Example 7. [Figure 21] Figure 21 shows the sequence of monoclonal antibody RagB-4-1B11-4-4 and the analysis of RagB-4-1B11-4-4 recombinant antibody expressed in eukaryotic HEK293 cells and purified. 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 the 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 the VL. Figure 21E shows the peak at 280 nm obtained by HPLC purification of the recombinant antibody and collecting the eluate. Figure 21F shows the purified recombinant antibody confirmed by SDS-PAGE, where lane M is a protein molecular weight marker (15-120 kDa) and lane 1 is purified RagB-4-1B11-4-4. For details, see Example 7. [Figure 22]Figure 22 shows the sequence of monoclonal antibody RagB-4-1C3-7-8 and the analysis of RagB-4-1C3-7-8 recombinant antibody expressed in eukaryotic HEK293 cells and purified. 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 the 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 the VL. Figure 22E shows the peak at 280 nm obtained by HPLC purification of the recombinant antibody and collecting the eluate. Figure 22F shows the purified recombinant antibody confirmed by SDS-PAGE, where lane M is a protein molecular weight marker (15-120 kDa) and lane 1 is purified RagB-4-1C3-7-8. For details, see Example 7. [Figure 23A] Figure 23 shows the designed sequence of bispecific antibody 1B11-1C3 and the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23A shows the DNA sequence of the recombinant heavy chain variable region (VH) (SEQ ID NO: 168), which was confirmed by sequencing. For details, see Example 8. [Figure 23B] Figure 23B shows the designed sequence of bispecific antibody 1B11-1C3 and the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23B shows the amino acid sequence of VH (SEQ ID NO: 169). For details, see Example 8. [Figure 23C] Figure 23C shows the designed sequence of bispecific antibody 1B11-1C3 and the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23C shows the sequence diagram of the heavy chain of the bispecific antibody, with the bold and underlined parts indicating the original specific MAb. For details, see Example 8. [Figure 23D]Figure 23D shows the designed sequence of bispecific antibody 1B11-1C3 and the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23D shows the DNA sequence (SEQ ID NO: 173) of the light chain (mouse Kappa) variable region (VL), confirmed by sequencing. For details, see Example 8. [Figure 23E] Figure 23E shows the designed sequence of bispecific antibody 1B11-1C3 and the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23F shows the amino acid sequence of the VL (SEQ ID NO: 174). For details, see Example 8. [Figure 23F] Figure 23F shows the designed sequence of bispecific antibody 1B11-1C3 and the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23F shows the sequence diagram of the light chain of the bispecific antibody, with the bold and underlined parts indicating the original specific MAb. For details, see Example 8. [Figure 23G] Figure 23G shows the designed sequence of bispecific antibody 1B11-1C3 and the recombinant plasmid expressing it in eukaryotic HEK293 cells. The recombinant bispecific antibody was purified by HPLC, and the peak at 280 nm obtained by collecting the eluate was shown. For details, see Example 8. [Figure 23H] Figure 23H shows the designed sequence of bispecific antibody 1B11-1C3 and the recombinant plasmid expressing it in eukaryotic HEK293 cells. Figure 23H shows the purified recombinant antibody as confirmed by SDS-PAGE. Lane M is a protein molecular weight marker (15-120 kDa), lane R is reduced 1B11-1C3 (76 kDa (heavy chain) / 24 kDa (light chain)), and lane NR is non-reduced 1B11-1C3 (200 kDa). For details, see Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0018] Although the present invention presents and describes various methods for implementing the invention, for those skilled in the art, these embodiments are provided as examples only. Those skilled in the art can make various changes, modifications and substitutions within the scope of the present invention. Some improvements and modifications may be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention. Definition of Terms

[0019] Numerous modifications and other embodiments will occur to those skilled in the art upon review of the foregoing description and associated drawings. Accordingly, it is to be understood that the present disclosure is not limited to the particular embodiments, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Those skilled in the art will recognize many variations and adaptations. These variations and adaptations are intended to be encompassed by the teachings of the present disclosure and are protected by the scope of the claims.

[0020] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not in a limiting sense.

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

[0022] Any recited method may be carried out in the order of events recited or in any other order which is logically possible. That is, unless expressly stated otherwise, no method or aspect of this invention requires that its steps be performed in a particular order. Thus, unless a method claim specifically recites that the steps are to be limited to a particular order, no order should be inferred. This rule applies to possible implicit bases of interpretation, including logical issues regarding the arrangement or operational flow of steps, ordinary meaning arising from grammatical construction or punctuation, or the number or type of aspects described in the specification.

[0023] All published publications and patents cited herein are incorporated by reference to disclose and describe the methods and / or materials in connection with such publications. Each publication or patent is specifically and individually identified as being incorporated by reference. Such citations are limited to the methods and / or materials described in the cited publications and patents, and do not extend to defining any terms in the cited publications and patents. Any term definitions provided in cited publications and patents are not considered definitions unless expressly repeated herein, and should not be construed as defining any term in the appended claims. The citation of any publication for its disclosure prior to the filing date should not be construed as an admission that the present disclosure is not antedated by prior disclosure. Further, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

[0024] Relevant publications and patents cited in the present specification for methods and / or materials are incorporated herein by reference. Each individual publication or patent is specifically and individually indicated by the form of reference. Such citation is expressly limited to the methods and / or materials described in the cited publications and patents, and does not extend to any dictionary definitions in the cited publications or patents. Dictionary definitions in cited publications and patents, unless expressly repeated herein, are not considered dictionary definitions and should not be construed to define any terms appearing in the appended claims. The citation of any publication prior to the filing date should not be construed as an admission that the present disclosure is not entitled to priority therefrom. Furthermore, the publication dates provided herein may differ from the actual publication dates, which may need to be independently confirmed.

[0025] Although the present disclosure may be described and claimed in certain legal categories, such as systems legal categories, this is for convenience only, and those skilled in the art will understand that aspects of the present disclosure may be described and claimed in any legal category.

[0026] It should also be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Furthermore, like terms defined in common dictionaries, they should be interpreted as having a consistent meaning in the context of this specification and the related technical field, and should not be interpreted as an idealized or overly formal meaning unless clearly defined herein.

[0027] Each aspect of the present disclosure employs techniques of molecular biology, microbiology, organic chemistry, biochemistry, physiology, cell biology, vascular biology, and the like, which, unless otherwise indicated, are mature in the art and are described in detail herein.

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

[0029] As used herein, "comprising" should be interpreted as specifying the presence of the stated features, integers, steps, or components, and does not negate the presence or addition of one or more features, integers, components, or groups thereof. Furthermore, the terms "from," "including," "includes," "comprises," "comprises," "including," "comprising," "relating to," "concerning," and "for example" are used in an open, non-limiting sense and can be used interchangeably. Furthermore, the term "comprising" is intended to include examples and embodiments covered by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of" is intended to include examples covered by the term "consisting of."

[0030] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Before a list of elements, a phrase such as "at least one" modifies the entire list of elements, not just each individual element within the list.

[0031] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context specifically dictates otherwise. Thus, for example, reference to a "therapeutic agent" includes one or more therapeutic agents and / or a combination of one or more therapeutic agents, and so forth.

[0032] References to "one / a" compound, therapeutic agent, and pharmaceutical composition refer to one or more molecules of the compound, therapeutic agent, and pharmaceutical composition, respectively, and are not limited to a single compound, therapeutic agent, and pharmaceutical composition. The one or more molecules may be the same or different, so long as they belong to the class of compound, therapeutic agent, and pharmaceutical composition. For example, "a" therapeutic agent is understood to include one or more therapeutic agent molecules that may be the same or different (e.g., having different isotopic abundances and / or different degrees of hydration, or equilibria with different conjugate base or conjugate acid forms).

[0033] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is further understood that the endpoints of each range are significant relative to, and independent of, the other endpoints. It is also understood that a number of values ​​are disclosed herein, and that in addition to the value itself, each value is herein disclosed as "with respect to" that particular value. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. Similarly, when values ​​are expressed as approximations, the use of the antecedent "about" will understand that the particular value forms another aspect. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0034] When a range is expressed, it specifically refers to one particular value and / or to another particular value. When a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value within that range is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those limits are also included in the invention. For example, the phrase "from x to y" includes not only a range from "x" to "y," but also a range from greater than "x" but less than "y." If the stated range includes one or both of the limits, ranges excluding either or both of those limits are also included in the invention. The range may also be expressed as an upper limit, for example, "about x, y, z or less" should be interpreted to include the ranges "less than x," "less than y," and "less than z," as well as the specific ranges of "about x," "about y," and "about z." Similarly, the phrase "about x, y, z or more" should be interpreted to include the ranges "greater than x," "greater than y," and "greater than z," as well as the specific ranges of "about x," "about y," and "about z." Furthermore, the phrase "from about 'x' to 'y'," where 'x' and 'y' are numerical values, includes "from about 'x' to about 'y'."

[0035] It should be understood that such range formats are used for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly recited. For convenience of description, a numerical range such as "about 0.1% to 5%" should be interpreted to include not only the explicitly recited values ​​from about 0.1% to about 5%, but also individual values ​​(e.g., about 1%, about 2%, about 3%, and about 4%) and subranges (e.g., about 0.5% to about 1.1%, about 0.5% to about 2.4%, about 0.5% to about 3.2%, and about 0.5% to about 4.4%, as well as other possible subranges) within the indicated range.

[0036] As used herein, the terms "about," "approximately," "substantially," and the like, when associated with a numerical variable, generally refer to the value of that variable, as well as all values ​​within experimental error (e.g., within a 95% confidence interval) or within + / - 10% of the indicated value. As used herein, "about," "approximately," "or approximately," and "substantially" mean that the associated quantity or value may be an exact value or a value that will produce an equivalent result or effect. That is, it is understood that quantities, dimensions, formulations, parameters, and other quantities and characteristics need not be completely precise and may reflect approximations and / or variations, including tolerances, conversion factors, rounding, measurement error, and other factors, to produce an equivalent result or effect. In some cases, a value that will produce an equivalent result or effect may not be reasonably determinable. Generally, whether explicitly stated or not, a quantity, size, formulation, parameter, or other quantity or characteristic preceded by "about," "approximately," or "or approximately" is "about," "approximately," or "or approximately," and includes the value of the particular quantity itself, unless otherwise specified.

[0037] As used herein, when used in connection with a numerical variable, "about," "approximate," "substantially," and the like may generally refer to the value of the variable and the greater of all variables within experimental error (e.g., within a 95% confidence interval of the mean) or within + / - 10% of the indicated value. As used herein, the terms "about," "approximate," "equal to or about," and "substantially" may mean that the amount or value under consideration is likely to be the exact value or a value that will produce an equivalent result or effect as claimed or set forth herein. That is, it will be understood that amounts, volumes, formulations, parameters, and other quantities and characteristics need not be exact, but can be approximated and / or larger or smaller, as necessary, to achieve equivalent results or effects, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, as well as other factors known to those skilled in the art. In some cases, a value that will produce an equivalent result or effect cannot be reasonably determined, and generally, the amount, volume, formulation, parameter, or other quantity or characteristic cannot be expressly described as "about," "approximate," or "equal to or about." When "about," "approximately," or "equal to or about" is used in front of a quantitative value, unless otherwise specified, it is understood that the parameter also includes the particular quantitative value itself.

[0038] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both cases where the event or circumstance occurs and cases where it does not occur.

[0039] As used herein, "administration" can refer to routes of administration such as oral, topical, intravenous, subcutaneous, transdermal, transcutaneous, intramuscular, intraarticular, parenteral, intraarteriolar, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernosal, intradural, intravitreal, intracerebral and lateral ventricles, intratympanic, intracochlear, rectal, intravaginal, by inhalation, by catheter, stent, or implanted reservoir or other device, active or passive (e.g., by diffusion) into the perivascular space and adventitia. For example, medical devices such as stents can contain compositions or formulations disposed on their surface, which can dissolve or otherwise distribute to surrounding tissues and cells. The term "parenteral" can include subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Administration can be continuous or intermittent. In various embodiments, the formulations can be administered therapeutically, i.e., used to treat an existing disease or condition. Additionally, the formulations can 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 that may produce a pharmacological, immunogenic, biological, and / or physiological effect in a subject by local and / or systemic action. A therapeutic agent may be a primary active agent, i.e., the component that produces all or part of the effect of a composition. A therapeutic agent may also be a secondary therapeutic agent, i.e., the component that produces additional portions of a composition and / or other effects. Thus, the term includes compounds or chemicals traditionally considered to be drugs, vaccines, and biologics, including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, and the like.

[0041] Examples of therapeutic agents are found in well-known references such as, for example, The Merck Index (14th ed.), The Physician's Desk Reference (64th ed.), and Fundamentals of Therapeutic Medicine (12th ed.), and include, but are not limited to, drugs, vitamins, mineral supplements, substances used in the treatment, prevention, diagnosis, cure, or mitigation of a disease or medical condition, 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 agent" includes adjuvants, anti-infective agents such as antibiotics and antivirals, analgesics and analgesic combinations, appetite suppressants, anti-inflammatory agents, antiepileptics, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergics and cholinergic mimetics, antimuscarinics and muscarinic receptor agonists, antiadrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, antiarthritics, antiasthmatics, anticonvulsants, antihistamines, analgesics, antineoplastics, antipruritics, antipyretics, antispasmodics, cardiovascular agents (including calcium channel blockers, beta blockers, beta agonists and antiarrhythmics), antihypertensives, diuretics, vasodilators, This includes all major compounds or compositions used in the therapeutic field, including, but not limited to, central nervous system stimulants, cough and cold remedies, decongestants, diagnostic reagents, hormones, bone growth promoters and bone resorption inhibitors, immunosuppressants, muscle relaxants, psychostimulants, sedatives, tranquilizers, proteins, peptides and fragments thereof (naturally occurring, chemically synthesized, or recombinantly produced), and nucleic acid molecules (ribonucleotides (RNA) or deoxyribonucleotides (DNA) including polymeric forms of two or more nucleotides, double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin), and other bioactive macromolecules such as proteins and enzymes. Therapeutic agents may be bioactive agents used in medicine, including veterinary applications and agriculture (e.g., plants), 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 in the treatment, prevention, diagnosis, cure or amelioration of a disease or medical condition, substances that affect the structure or function of the body, or prodrugs that become biologically active or more active when in a particular physiological environment.

[0042] As used herein, a "kit" refers to a package containing at least two components. These components together constitute a functional unit for a specific purpose. The individual components may be physically packaged together or separately. For example, instructions included in a kit may or may not be physically packaged with the other individual components. The instructions may be provided separately from the other components in paper or electronic form, stored in a computer storage device, downloaded from an internet website, or recorded as a presentation.

[0043] The "instructions" referred to above refer to documents 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 (e.g., purchasing information), brief or detailed protocols for using the kit, troubleshooting, references, technical support, and other related documentation. The instructions may be provided with the kit or as a separate component, and may be provided in paper or electronic format, on a computer-readable storage device, downloaded from an internet website, or as a recorded presentation. The instructions may include one or more documents and may include future updates.

[0044] The terms "subject," "individual," or "patient," used interchangeably herein, may refer to a vertebrate, such as a mammal (e.g., a human). "Subject" refers to a cell, a cell population, a tissue, an organ, or an organism, and preferably refers to humans and their components.

[0045] As used herein, the terms "treatment" and "therapeutic" may generally refer to achieving a desired pharmacological and / or physiological effect. This effect may (but is not necessarily) be prophylactic treatment to prevent or partially prevent a disease, symptom, or associated condition. This effect may also be curative, i.e., partially or completely curing a disease, symptom, or associated condition. As used herein, the term "treatment" includes any treatment of inflammation associated with any disease in a subject, particularly a human, and may include one or more of: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., preventing its progression; and (c) palliating the disease, i.e., reducing or ameliorating the disease and / or its symptoms or conditions. As used herein, the term "treatment" may refer to treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need of treatment) include those already suffering from the disease and / or those in need of prevention of the disease. As used herein, the term "treatment" includes inhibiting a disease, discomfort, or condition. For example, alleviating a disease, discomfort, or condition, such as preventing its progression and causing regression of the disease, discomfort, and / or condition. Treating a disease, discomfort, or condition can also include ameliorating at least one symptom of a particular disease, discomfort, or condition, even if the underlying pathophysiology is not affected; for example, administering a painkiller may treat a subject's pain even if the drug does not treat the cause of the pain.

[0046] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or affect undesired symptoms, but usually not sufficient to cause adverse side effects. The specific therapeutically effective dosage level will vary from patient to patient depending on a variety of factors, such as the disease being treated and its severity, the specific composition used, the patient's age, weight, overall health, sex, and diet, the time of administration, the route of administration, the excretion rate of the specific compound used, the duration of treatment, drugs used in combination with or concomitantly with the specific compound used, and similar factors within the knowledge and expertise of a general practitioner, 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 inhibition of the progression of the disease or condition. This may only temporarily slow the progression of the disease. However, in other cases, it may be necessary to permanently prevent the progression of the disease. This can be monitored by conventional diagnostic methods for any particular disease known to those skilled in the art. The expected response to treatment of a disease or condition may be delaying or even preventing the onset of the disease or condition.

[0047] For example, it is within the skill of a skilled artisan to schedule the dosage of a drug required to achieve a therapeutic effect, starting at a low level and gradually increasing the dosage until the desired effect is achieved. If necessary, a therapeutically effective daily dose can be administered in multiple doses. Thus, a single-dose composition can comprise these amounts or multiples thereof to constitute a daily dose. If there are contraindications, a specialist physician can adjust the dosage. Generally, 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 highest safe dose based on reasonable medical judgment. However, one skilled in the art will understand that a patient may require a lower or tolerated dose for medical reasons, psychological reasons, or virtually any other reason.

[0048] For example, the response to a therapeutically effective amount of the disclosed compounds and / or pharmaceutical compositions can be measured by determining the physiological effect of the treatment or agent, such as the reduction or elimination of disease symptoms after administration of the treatment or pharmacological agent. Measuring the level of response is within the skill of one of ordinary skill in the art. The amount of treatment can be varied, for example, by increasing or decreasing the amount of the disclosed compounds and / or pharmaceutical compositions, changing the disclosed administered compounds and / or pharmaceutical compositions, changing the route of administration, or changing the administration schedule. Dosages can vary and can be administered one or more times daily, for one or several days. Guidelines regarding appropriate dosages for particular types of pharmaceuticals can be found in the literature.

[0049] As used herein, the term "prophylactically effective amount" refers to an amount that effectively prevents the development or onset of a disease or condition.

[0050] As used herein, the terms "prevent" or "prevention" refer to taking proactive action to eliminate, avoid, eliminate, prevent, hinder or inhibit something from occurring. Unless otherwise indicated, when mitigating, inhibiting or preventing is used 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" describes a substance that does not cause adverse biological or other side effects, i.e., does not cause unacceptable levels of adverse biological effects or interact in a deleterious way.

[0052] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from an active agent and an acid or base that can be tolerated by the body, or a salt prepared from an active agent and an acid or base that can be tolerated by the subject or body when used in an effective therapeutic dose range. When compounds described in this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting these compounds in their neutral form or with a sufficient amount of base, either in pure form or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, ammonium salts, organic amine salts, magnesium salts, lithium salts, strontium salts, or similar salts. When compounds described in this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of these compounds with a sufficient amount of the desired acid, either in pure form or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphoric acid, and the like, and 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, methanesulfonic acid, etc. Also included are salts of amino acids such as arginate, and salts of organic acids such as glucuronate or galacturonate.

[0053] As used herein, the term "pharmaceutically acceptable ester" refers to an ester of a compound disclosed herein, including those that are hydrolyzed in vivo and readily decompose in the human body to leave 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, with C1-C4 alkyl esters being preferred. Esters of compounds disclosed herein can be prepared according to conventional methods. Pharmaceutically acceptable esters can be attached to a hydroxyl group by reacting the compound with acid and an alkyl carboxylic acid, such as acetic acid, or acid and an aryl carboxylic acid, such as benzoic acid. For compounds containing a carboxylic acid group, pharmaceutically acceptable esters can be prepared from the compound by reacting the compound with a base, such as triethylamine, and an alkyl halide, such as methyl iodide, benzyl iodide, or cyclopentyl iodide, or an alkyl trifluoromethanesulfonate. They can also be prepared by reacting the compound with an acid, such as hydrochloric acid, and an alcohol, such as ethanol or methanol.

[0054] The term "pharmaceutically acceptable amide" refers to the non-toxic amides disclosed in the present invention derived from ammonia, primary C1-C6 alkylamines, and secondary C1-C6 dialkylamines. In the case of secondary amines, the amines may be in the form of a five- or six-membered heterocycle 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 the present invention can be prepared according to conventional methods. Pharmaceutically acceptable amides can be prepared from compounds containing primary or secondary amine groups by reacting the amino group-containing compound with an alkyl anhydride, aryl anhydride, acyl halide, or aroyl halide. In the case of compounds containing carboxylic acid groups, pharmaceutically acceptable amides can be prepared from compounds containing carboxylic acid groups by reacting the compound with a base such as triethylamine, a dehydrating agent such as dicyclohexylcarbodiimide or carbonyldiimidazole, and an alkyl or dialkylamine such as methylamine, diethylamine, or piperidine. They can also be prepared under dehydrating conditions, such as the addition of molecular sieves, by reacting a compound with an acid such as sulfuric acid and an alkylcarboxylic acid such as acetic acid, or an arylcarboxylic acid such as benzoic acid, etc. The composition can include a pharmaceutically acceptable prodrug form of the compound disclosed in the present invention.

[0055] The term "pharmaceutically acceptable prodrug" or "prodrug" refers to a prodrug of a compound disclosed herein, which is, within the scope of reasonable medical judgment, suitable for use in contact with the tissues of humans and lower animals, does not produce excessive toxicity, irritation, or allergic reactions, and is effective for its intended use, consistent with a reasonable benefit-risk ratio. The prodrugs disclosed herein may be rapidly converted in vivo, e.g., by hydrolysis in blood, to the parent compound having the disclosed structure. This is discussed in detail in T. Higuchi and V. Stella's ACS Symposium Series, Vol. 14, "Prodrugs as Novel Delivery Systems," and in "Bioreversible Carriers in Drug Design," edited by Edward B. Roche, 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 in the present invention), the structure of which is sufficiently similar to the structure disclosed herein, such that it is expected that, based on this similarity, it would be within the skill of a person skilled in the art to prepare a compound that has the same or similar activity and use as the compound in question, or that induces the same or similar activity and use as a precursor. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound, which should also be included in the scope of protection of this patent.

[0057] As used herein, the nomenclature of compounds, including organic compounds, can be designated using the common name IUPAC, IUBMB, or CAS naming recommendations. When one or more stereochemical features are present, the Cahn-Ingold-Prelog stereochemical rules can be employed to designate stereochemical priority, E / Z conventions, etc. When defining the name of a compound, one skilled in the art can use the naming rules to systematically reduce the structure of the compound or use CHEMDRAW TMThe structure of a compound can be readily determined by commercially available software such as (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 methods for treating and / or preventing various diseases and / or related conditions caused by Porphyromonas gingivalis infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of one or more monoclonal and / or bispecific antibodies disclosed herein. In some embodiments, the present invention provides methods for reducing the level of infection caused by Porphyromonas gingivalis. Methods for measuring the level of infection are well known in the art. In one embodiment, the level of infection is reduced by about 5% to about 100%. In another embodiment, the level of infection in a subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. Monoclonal and bispecific antibodies

[0060] As disclosed herein, 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 comprises a heavy chain (mouse IgG1) variable region (VH) having the nucleotide sequence (357 bases) set forth in SEQ ID NO: 128 and the encoded amino acid sequence (119 amino acids) set forth in SEQ ID NO: 129, and a light chain (mouse kappa) variable region (VL) having the nucleotide sequence (282 bases) set forth in SEQ ID NO: 133 and the encoded amino acid sequence (94 amino acids) set forth in SEQ ID NO: 134. The nucleotide sequence of the heavy chain of the RagB-2-1A4-3-7-7 monoclonal antibody is set forth in SEQ ID NO: 175, the encoded amino acid sequence is set forth in SEQ ID NO: 176, the nucleotide sequence of the light chain is set forth in SEQ ID NO: 177, and the encoded amino acid sequence is set forth in SEQ ID NO: 178.

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

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

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

[0065] 5. The 1B11-1C3 bispecific antibody comprises a heavy chain (mouse IgG1) having the nucleotide sequence (2,232 bases) set forth in SEQ ID NO: 168 and the encoded amino acid sequence (728 amino acids) set forth in SEQ ID NO: 172, and a light chain (mouse kappa type) having the nucleotide sequence (744 bases) set forth in SEQ ID NO: 173 and the encoded amino acid sequence (232 amino acids) set forth in SEQ ID NO: 174. Administration and Dosage

[0066] While the active ingredient can be administered alone, it may be more desirable to administer it as a pharmaceutical formulation or composition. As described below, the disclosed formulations, whether for veterinary or human use, contain at least one active ingredient, as well as one or more acceptable carriers and optional other therapeutic ingredients. The carrier must be "acceptable," i.e., compatible with the other ingredients in the formulation and not physiologically harmful to the recipient.

[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, if delivered by a method other than oral administration, are typically isotonic. All formulations are used in accordance with the "Pharmaceutical Excipients Handbook" (1986). Formulations may include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkyl cellulose, hydroxyalkylmethyl cellulose, and stearic acid. The pH range of the formulation is about 3 to about 11, but is typically about 7 to 10. A skilled clinician can easily determine a therapeutically effective amount of the active ingredient using conventional dose-escalation studies. Typically, the dosage of the active ingredient is 0.01 mg to 2 g. In one embodiment, the dosage is about 10 mg to 450 mg. In another embodiment, the dosage is about 25 mg to about 250 mg. In another embodiment, the dosage will be about 50 or 100 mg. For example, in one embodiment, the dose is about 100 mg, and it is expected that the active ingredient can be administered once, twice, or three times daily. Additionally, 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 pharmaceutical ingredient includes a suitable route of administration. The formulations can be conveniently presented in unit dosage form and prepared by any method known in the art of pharmacy. For techniques and formulations, see "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, Pa.). These methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more accessory ingredients. Typically, the formulations are finally shaped by uniformly and intimately admixing the active ingredient with liquid carriers, or finely divided solid carriers, or both.

[0069] Formulations suitable for oral administration are provided as individual packages such as capsules, coated tablets, or tablets containing a predetermined amount of the active pharmaceutical ingredient, which may be a powder or granules, an aqueous or non-aqueous liquid or suspension, or a water-in-oil liquid or oil-in-water liquid emulsion. The active ingredient may also be administered as a pill, ointment, or paste. In some embodiments, the active ingredient is administered by subcutaneous injection.

[0070] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be made by compressing in a suitable machine the active ingredient in a free-flowing form (such as a powder or granules), optionally mixed with a binder, lubricant, inert diluent, preservative, or surfactant. Molded tablets can be made by molding in a suitable machine a mixture of an inert liquid-containing diluent and a powdered active pharmaceutical ingredient. Tablets can optionally be coated or scored, and can be formulated as sustained- or controlled-release tablets so that the active ingredient is slowly released.

[0071] The active ingredient can be administered by a route appropriate for the condition to be treated. Suitable routes include oral, anal, 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 condition of the recipient. In some embodiments, the active ingredient can be administered orally due to its high oral bioavailability. In an example, the patient is a human. Active Pharmaceutical Ingredients

[0072] The active pharmaceutical ingredients disclosed herein include an effective amount of one or more 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 prepared. Oral compositions can be prepared according to any method known in the pharmaceutical art, and such compositions may contain one or more excipients, including sweeteners, flavorings, colorings, and preservatives, to provide a palatable formulation. Tablets containing the active ingredient mixed with non-toxic, pharmaceutically acceptable excipients are acceptable. These excipients may include, for example, inert diluents such as calcium or sodium carbonate, lactose, lactose monohydrate, crospovidone sodium, polyvinylpyrrolidone, calcium 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. Tablets may be uncoated or may be coated by known techniques, such as microencapsulation, to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be used.

[0074] Formulations for oral administration 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 oil medium (such as peanut oil, liquid paraffin, or olive oil).

[0075] Aqueous suspensions disclosed by the present invention contain the active substance mixed with excipients suitable for the preparation of aqueous suspensions. These excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum arabic, and gum tragacanth, as well as dispersing 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 hexitols (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or propyl p-hydroxybenzoate) and one or more coloring agents, flavoring agents, 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 a mineral oil (e.g., liquid paraffin). Oily suspensions may contain thickening agents such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners and flavorings may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant, such as ascorbic acid.

[0077] The dispersible powders and granules disclosed herein are suitable for preparation of an aqueous suspension by the addition of water, providing a composition of the active ingredient mixed with a dispersing agent, wetting agent, suspending agent, and one or more preservatives. Suitable dispersing and suspending agents are exemplified above. Further excipients such as sweeteners, flavorings, and coloring agents may also be present.

[0078] The active pharmaceutical ingredient disclosed in the present 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 soybean lecithin), esters or partial esters of fatty acids and hexitols (such as sorbitan monooleate), and condensation products of these partial esters with ethylene oxide (such as polyoxyethylene sorbitan monooleate). Emulsions may contain sweeteners and flavor enhancers. Syrups and elixirs may be prepared using sweeteners such as glycerol, sorbitol, or sucrose. In addition, these formulations may contain mucosal protectants, preservatives, flavorings, or coloring agents.

[0079] The active pharmaceutical ingredients disclosed in the present invention may be prepared as sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. Such suspensions can be prepared according to known techniques using the above-mentioned suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be prepared as sterile injectable solutions or suspensions using non-toxic, acceptable diluents or solvents (such as 1,3-butanediol solution), or as lyophilized powders. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid can also be used in injectable preparations.

[0080] The amounts of active ingredient and carrier material to be combined into a single dosage form will vary depending on the host being treated and the particular method of administration (e.g., oral administration or subcutaneous injection). For example, a sustained-release formulation for oral administration to humans may contain approximately 1 to 1,000 mg of active ingredient mixed with a suitable and convenient carrier material, which may represent approximately 5% to 95% (weight:weight) of the total composition. Pharmaceutical compositions can be prepared to provide easily measurable dosages. For example, an aqueous solution suitable for intravenous infusion may contain approximately 3 to 500 μg of active ingredient per mL of solution, allowing an appropriate amount to be infused at a rate of approximately 30 mL / hour. Formulations for subcutaneous administration 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 which may contain antioxidants, buffers, bactericides and solutes which render the formulation isotonic with the blood of the recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents.

[0082] The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials, and may be stored in a lyophilized condition requiring only the addition of a sterile liquid carrier, such as water for injection, prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets as described above. Preferred unit dosage formulations are those containing a daily dose or daily sub-dose (or an appropriate fraction 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 administration method. In some embodiments, the provided method comprises administering a pharmaceutical composition comprising one or more antibodies disclosed herein and a pharmaceutically acceptable carrier or excipient. The combination formulations and / or treatment regimens disclosed herein comprise an antibody disclosed herein together with one or more pharmaceutically acceptable carriers or excipients, and optionally other therapeutic agents, whether now known or developed in the future, for the treatment and / or prevention of infectious diseases. The combination formulation containing the active ingredients can be in any form suitable for the intended administration method.

[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 include administering a pharmaceutical composition comprising one or more vaccines disclosed herein and a pharmaceutically acceptable carrier or excipient. The combination formulations and / or treatment regimens disclosed herein include the vaccines disclosed herein together with one or more pharmaceutically acceptable carriers or excipients, and optionally other therapeutic agents, whether currently known or developed in the future, for the treatment and / or prevention of infectious diseases. The combination formulation containing the active ingredients can be 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 proteins of P. gingivalis consists of two co-transcribed and independent genes, ragA and ragB. Data demonstrated the existence of four major distinct subtypes of the outer membrane proteins encoded by the ragB gene. These four subtypes accounted for more than 95% of the clinical samples from 197 periodontal disease patients obtained from a global collaborative study. The four subtypes of P. gingivalis were named W50ragB protein, ThairagB protein, QMLragB protein, and 381ragB protein, respectively, based on their RagB outer membrane proteins.

[0086] These four allotypic proteins differ significantly in sequence, with pairwise similarities ranging from only 42% to 59% (Figure 6). RagA, like RagB, was found to have allotypic proteins. However, Cra4S1 is a polypeptide fragment near the N-terminus of the RagA protein encoded by the ragA gene, and is a conserved domain, meaning that the Cra4S1 polypeptide is present in all allotypic strains of P. gingivalis.

[0087] In some embodiments, the present invention discloses a genetically modified sequence of Porphyromonas gingivalis outer membrane protein RagB, and pilot-scale production of untagged RagB and Cra4S1 recombinant proteins has been successfully completed (Example 1, Figures 1-5 and Figures 7-11).

[0088] Although different RagB outer membrane protein subtypes each have their own antigenic properties, studies have shown that polyclonal antibody sera obtained by immunizing animals with a single recombinant RagB protein exhibit significant cross-reactivity with other RagB protein isoforms under certain dilution conditions (Example 2).

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

[0090] Despite the sequence similarity of RagB subtypes being only 42-59%, cross-reactivity between different RagB proteins suggests the possibility of the existence of common antigens or compatible epitopes. Are these cross-reactivities caused by linear protein sequences or by two-dimensional planar and / or multidimensional structures? What is the molecular basis for these cross-reactivities? Where are the target antigens? Do monoclonal antibodies provide cross-immunoprotective effects?

[0091] In the present invention, a method for constructing synthetic peptide libraries is provided. These peptide libraries contain peptide fragments from five different subtypes of the P. gingivalis outer membrane protein RagB and Cra4S1, and are used to screen for target antigens of monoclonal antibodies. The immune reaction between the monoclonal antibodies and peptides at different locations in the corresponding proteins is utilized to identify antigen targets. In the present example, several monoclonal antibodies with cross-reactive properties all found their corresponding antigen targets in the peptide library. This important discovery has profound implications for manufacturing processes and costs in future product development. While previous approaches have used specific monoclonal antibodies to target homologous bacteria due to differences in P. gingivalis strains, a new approach uses antibody products that can simultaneously target four P. gingivalis subtypes (Example 4).

[0092] More specifically, the present invention elucidates the pathogenesis of P. gingivalis based on the research methods and results of the pathogenesis of P. gingivalis. In the examples, it is believed that the heterophilic antigenic characteristics of P. gingivalis and individual heterophilic antibodies are the causes of chronic persistence of the disease and multiple organ damage.

[0093] In the Examples, serological test results of clinical specimens show that outer membrane proteins of P. gingivalis (including RagB and Cra4S1) have heterophilic antigenic properties, and the test results indicate that the local lesions and systemic disorders caused by P. 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 P. gingivalis infection, which is used to evaluate systemic damage caused by pathogen infection and methods for treating P. gingivalis infection.

[0095] Experimental results of the conjugated antibody in a mouse reproductive model have shown broad significance, facilitating the development of products for the prevention and treatment of Porphyromonas gingivalis and related chronic systemic diseases. Of particular importance, two monoclonal antibodies with adjacent antigen targets were discovered, each targeting a different subtype of bacteria, and the location and sequence of the antigen target in each outer membrane protein were identified. As research in this field continues to expand, further therapeutic applications will be established, and the antibodies and methods of use disclosed in this invention open up broad market prospects for products for the prevention and / or treatment of various diseases related to periodontal disease. Therefore, the present invention provides stable and efficient biological preparations, solving the problem of treating different subtypes of pathogenic bacteria, simplifying manufacturing processes, reducing production costs, and ultimately benefiting public health overall (Example 6).

[0096] In the Examples, the present invention discloses the nucleotide and protein sequences of mouse monoclonal antibody genes generated by immunization with the Porphyromonas gingivalis outer membrane protein RagB. Recombinant antibody plasmids constructed using these antibody sequences were successfully expressed in eukaryotic cells. These retain the antigen-binding ability of the original monoclonal antibodies, providing a molecular basis for therapeutic drug development and industrial production of the products.

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

[0098] In the examples, the present invention provides two specific monoclonal antibodies whose antigen targets are closely related and belong to the same subtype of Porphyromonas gingivalis. Importantly, these two antibodies exhibit cross-reactivity with outer membrane proteins of different isoforms, and these cross-reactive antigens differ in polypeptide sequence and location on the outer membrane protein. This discovery significantly simplifies the manufacturing process and effectively improves product production efficiency.

[0099] In the Examples, the present invention discloses the design and engineering of bispecific antibodies targeting different subtypes of P. gingivalis, and recombinant antibody plasmids were successfully constructed using the designed antibody sequences and expressed in eukaryotic cells. Data presented in the present invention demonstrate that the recombinant bispecific antibodies not only retain the antigen-binding ability of the original monoclonal antibodies, but also compensate for the shortcomings of the original monoclonal antibodies. These data demonstrate the feasibility and potential of bispecific antibody product development (Example 8).

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

[0101] The present invention discloses a diagnostic kit reagent for the diagnosis, treatment, and prognosis of diseases associated with Porphyromonas gingivalis infection. In some embodiments, the amino acid sequences of the diagnostic reagents are set forth in SEQ ID NOs. 2, 4, 6, 8, and 10. The diagnostic kit also provides instructions for using the reagents to test for specific antibodies in biological samples, including, but not limited to, blood, gingival crevicular fluid, urine, saliva, cerebrospinal fluid, pleural effusion, peritoneal fluid, and amniotic fluid. For details on how to prepare, use, and analyze the results of the diagnostic kit, see Example 9 of the present invention.

[0102] The present invention also discloses polypeptide vaccines that can mobilize an individual's active immune function. In the examples, the sequences of the polypeptide vaccines are determined by the target of neutralizing antibodies, and the antigenic properties of the polypeptide vaccines are preserved while irrelevant and harmful components are removed. The polypeptide vaccines disclosed in the present invention reduce the side effects of commonly used vaccines, can be administered repeatedly, and provide efficient and long-lasting protection against P. gingivalis infection.

[0103] The present invention further provides polypeptide vaccines and conjugate antibodies for active and passive immunization of infected animals. For details of the preparation, use and results of these polypeptide vaccines and / or conjugate antibodies, see Example 10 of the present invention.

[0104] The above generally describes various aspects of the present invention, and the following examples describe some additional and / or more detailed aspects of the present invention. The various aspects of the present invention are expressed in conjunction with the following examples and corresponding text descriptions and illustrations, but the present invention is not limited to these descriptions. Rather, the meaning of the aspects of the present invention is to encompass all alternatives, modifications, and equivalents that fall within the spirit and scope of the present invention.

[0105] The following examples are intended to provide those of skill in the art with complete schematics and descriptions of how to make 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. Efforts have been made to ensure accuracy of numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise noted, ingredients are expressed by weight, temperatures are in degrees Celsius (°C) or are at ambient temperature, and pressures are at or near atmospheric. [Example]

[0106] Example 1 Expression of RagB and Cra4S1 in E. coli The P. gingivalis outer membrane protein RagB exists primarily in four isoforms in clinical periodontal disease samples, and these four isoforms have been named W50RagB, ThaiRagB, QMLRagB, and 381RagB.

[0107] In this study, the names of the four RagB subtypes were simplified: W50RagB was designated RagB-1, ThaiRagB was designated RagB-2, QMLRagB was designated RagB-3, and 381RagB was designated RagB-4. As shown in Figure 6 and Table 1, the protein sequence similarity between RagB isoforms ranges from only 42% to 57%, with the sequence similarity between RagB-1 and RagB-2 being 47%, between RagB-1 and RagB-3 being 57%, between RagB-1 and RagB-4 being 49%, between RagB-2 and RagB-3 being 45%, between RagB-2 and RagB-4 being 42%, and between RagB-3 and RagB-4 being 49%.

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

[0109] Table 1 RagB multiple sequence alignment (percentage of identical sequences) JPEG2025531306000001.jpg27156

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

[0111] Specifically, the nucleic acid and amino acid sequences of artificially synthesized codon-optimized RagB-1, RagB-2, RagB-3, RagB-4, and Cra4S1 are shown in SEQ ID NOs: 1-10 and Figures 1A-1B (particularly referring to RagB-1, SEQ ID NOs: 1-2), 2A-2B (particularly referring to RagB-2, SEQ ID NOs: 3-4), 3A-3B (particularly referring to RagB-3, SEQ ID NOs: 5-6), 4A-4B (particularly referring to RagB-4, SEQ ID NOs: 7-8), and 5A-5B (particularly referring to Cra4S1, SEQ ID NOs: 9-10), respectively.

[0112] Table 2. Expression of recombinant outer membrane proteins in E. coli JPEG2025531306000002.jpg26161

[0113] All five P. gingivalis outer membrane proteins, including RagB-1 (W50 RagB), RagB-2 (ThaiRagB), RagB-3 (QMLRagB), RagB-4 (381RagB), and Cra4S1, were successfully expressed in the host Escherichia coli. Specifically, after successful plasmid construction, the cloned genes (including optimized nucleotide sequences) were confirmed by sequencing to ensure the accuracy of the protein sequences. After transforming the plasmids into E. coli, recombinant protein expression was promoted by induction. Bacteria expressing the recombinant proteins were centrifuged to collect the precipitate, and the resulting pellet was mechanically disrupted. The supernatant was then purified by ion-exchange chromatography to obtain the target proteins. All recombinant proteins were tag-free and soluble (see Table 2).

[0114] Pilot-scale production of the five untagged recombinant proteins was completed by a third-party contract research organization (CRO) (GenScript, USA, Contract No. C9458FG140). Specifically, the recombinant plasmids and / or seed bacteria were sent to the CRO for confirmation and identification before fermentation. During high-density fermentation, the medium was free of animal-derived proteins and antibiotics. After fermentation was completed, the proteins were purified using 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 for the five untagged target proteins.

[0115] Table 3. Small-scale prototyping of recombinant proteins JPEG2025531306000003.jpg16167

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

[0117] Example 2 Cross-reactivity of polyclonal antibodies to Porphyromonas gingivalis outer membrane proteins Although the protein sequences of different P. gingivalis RagB subtypes are different, animal serological tests have shown that antibodies exhibit cross-reactivity with different RagB subtypes. Therefore, a step to test for antibody cross-reactivity with different RagB subtypes was added to the detection experiment design. In this study, we primarily used enzyme-linked immunosorbent assay (ELISA) to detect different antibody affinities for antigens.

[0118] Specifically, the preparation of mouse and rabbit antibody serum is briefly described as follows: Mice and rabbits are immunized three to four times with an antigen protein (RagB / Cra4S1), and the specific antibody titer in the serum is measured using an indirect ELISA method. After the antibody level reaches a predetermined standard, whole blood samples are collected from the animals, incubated, and centrifuged to obtain serum, which is then frozen and stored.

[0119] ELISA is an enzyme-linked immunosorbent assay. The basic principle is to immobilize a certain concentration of antigen on the surface of a polystyrene microplate by physical adsorption. Diluted serum (host antibody) is added to the reaction plate. After allowing sufficient time for the antigen and antibody to bind, unbound free antibody molecules are washed away. An enzyme-labeled secondary antibody (anti-host antibody) is then added, and the free molecules are washed away again. Finally, an enzyme substrate solution is added, and the enzyme reaction is allowed to proceed for a certain period of time. The color reaction of the substrate is measured, which indirectly reflects the qualitative and semi-quantitative characteristics of the substrate. The specific steps are as follows:

[0120] 1. Antigen coating: Prepare a protein (antigen) solution in PBS to a concentration of 4 μg / mL. 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 per well and tapping off any remaining buffer in 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 per well and tapping off any remaining buffer in the wells with absorbent paper. The coated ELISA plate can be stored in a -20°C freezer for 4 to 6 weeks.

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

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

[0123] 4. Color development: Prepare the substrate solution. Dissolve 4-nitrophenyl phosphate disodium hexahydrate in 1M Tris-MgCl2 at 1 mg / mL. Add 100 μL of the substrate solution to each well. Incubate at room temperature in the dark for 50 to 180 minutes. Set a dual wavelength (405 nm and 630 nm) program on the microplate reader. Read the data on the preheated microplate reader. Save the data for analysis.

[0124] In traditional ELISAs, there are many different detection systems available. In this example, alkaline phosphatase-conjugated secondary antibodies were selected for enzyme labeling due to their long stability and low false positive rate. Specifically, for ELISA plate number 17003 shown in Table 4, the antigen layout design described here is 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 a negative, unrelated protein control. All sera in the test and control groups were diluted 1:2000, allowing each serum sample to detect six antigens, including RagB-1, RagB-2, RagB-3, RagB-4, Cra4S1, and GST. The optical density (OD) values ​​of the ELISA plate number 17003 are shown in Table 4, with PBS and normal serum as negative controls.

[0125] Table 4. Detection of polyclonal antibodies in animals infected with P. gingivalis JPEG2025531306000004.jpg77161

[0126] The ELISA results show that antibodies against the corresponding outer membrane proteins are detectable in the sera of animals infected with homologous P. gingivalis bacteria. 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 P. gingivalis RagB-2 subtype. Their sera 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, the higher the corresponding antibody levels. For example, four mice in group G8 (G8-1, G8-2, G8-3, and G8-4) were infected with P. gingivalis only once, while the other four mice (G6-1, G6-2, G7-1, and G7-2) were infected with P. gingivalis three times. The OD values ​​of antibody detection levels in mice infected multiple times were higher than those in mice infected only once. Furthermore, high titers of specific antibodies were produced after inoculation of RagB-2 protein in animals previously infected with P. gingivalis (Tables 4 and 5, G6-3, G6-4, G7-3, and G7-4). Compared with animals in the vaccination-only protocol, the antibody OD values ​​after vaccination in animals previously infected with bacteria were significantly elevated. 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 JPEG2025531306000005.jpg68161

[0128] The results showed that after inoculation of RagB-2 protein into animals pre-infected with P. gingivalis RagB-2 (G6-3, G6-4, G7-3, and G7-4), the serum antibodies not only showed a strong immune response to RagB-2, but also cross-reacted with different subtypes of RagB proteins (including RagB-1, RagB-3, and RagB-4), and further showed weak but visible cross-reaction with Cra4S1 protein, but did not react with the negative control GST protein.

[0129] Table 6. Detection of polyclonal antibodies in animals infected with P. gingivalis JPEG2025531306000006.jpg77161

[0130] Table 7. Information on experimental animals JPEG2025531306000007.jpg65153

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

[0132] This example demonstrated that recombinant P. gingivalis outer membrane proteins, including RagB and Cra4S1, have good antigenicity and can stimulate animals to produce high-titer antibodies. Furthermore, it was observed that the polyclonal serum antibodies cross-react with RagB proteins of other subtypes. Although the gene and protein sequences of the P. gingivalis outer membrane protein RagB differ significantly between different subtypes, they are homologous alleles. These results suggest that there may be common antigens or matching antigenic determinants among P. gingivalis subtypes. In particular, the antibodies produced by immunization with RagB protein cross-reacted with Cra4S1 protein, a surprising and unexpected result.

[0133] Example 3 Cross-reactivity of RagB monoclonal antibodies Monoclonal antibodies against P. gingivalis outer membrane proteins, including RagB and Cra4S1 proteins, were prepared by a third-party contract research organization (CRO). Specifically, the general process for preparing mouse hybridoma cells and obtaining monoclonal antibodies is briefly described as follows: First, animals are immunized five times with antigen protein, with 1-2 weeks between immunizations. Mouse spleen cells are fused with well-grown SP2 / 0 myeloma cells, and the fused cells are grown in culture for 7 days. Subsequently, positive clones are screened using the immunizing antigen and then subcloned three times to confirm that the positive cell lines produce antibodies continuously and stably. The final hybridoma seed cell lines are stored in liquid nitrogen.

[0134] Table 8. RagB hybridoma cell lines JPEG2025531306000008.jpg165138

[0135] Table 9. Cra4S1 hybridoma cell line JPEG2025531306000009.jpg20161

[0136] Each antigen item provides 5 to 10 monoclonal antibody-positive hybridoma cell lines (Tables 8 and 9). Monoclonal antibodies are primarily extracted and purified from cell culture supernatants and ascites fluid produced after intraperitoneal inoculation of mouse hybridoma cells. Immunoglobulin subclass detection was performed on the purified monoclonal antibodies, and positive results are indicated in the tables with a "P."

[0137] Table 10. Detection of monoclonal antibody cross-reactive antigens JPEG2025531306000010.jpg78161

[0138] Table 11. Detection of monoclonal antibody cross-reactive antigens JPEG2025531306000011.jpg76161

[0139] Tables 10 and 11 show the original results of cross-reactivity between monoclonal antibodies and RagB recombinant proteins. The ELISA procedure was as described in Example 2. The antigen adsorption plate was designed as follows: columns 1, 5, and 9 were adsorbed with RagB-1 recombinant protein; columns 2, 6, and 10 with RagB-2; columns 3, 7, and 11 with RagB-3; and columns 4, 8, and 12 with RagB-4. The antibodies were derived from hybridoma culture supernatants or purified antibodies, with dilutions ranging from 1:1000 to 1:4000. Each antibody sample was allowed to react with the antigen proteins RagB-1, RagB-2, RagB-3, and RagB-4 in four wells.

[0140] A positive result was determined when the OD measurement value was more than twice that of the normal serum negative control group. The data presented in this example showed that all tested monoclonal antibodies demonstrated a positive immune response to the antigen, except for one hybridoma (wells B5-8 listed in Table 11, cell name RagB-4-1C11-1-5, showed a negative reaction). Furthermore, several hybridoma antibodies were identified that not only reacted with the corresponding RagB protein but also cross-reacted 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 monoclonal antibody specific for RagB-2 protein but also cross-reacts with RagB-1 protein. RagB-3-1D2-2-1-3 in plate wells E5-E8 is a monoclonal antibody specific for RagB-3 protein but also cross-reacts with RagB-1 and RagB-2 proteins. In Table 11, 1B11-4-4 antibody and 1C3-7-8 antibody in wells H1-H4 and A5-A8 are monoclonal antibodies specific for RagB-4 protein but cross-react with RagB-1, RagB-2, and RagB-3 proteins, respectively.

[0142] Because there are significant differences in nucleic acid and protein sequences between P. gingivalis RagB subtypes and no clear contiguous consensus sequences in pairwise alignments, previous bacterial immunotherapy focused on precision, first identifying the subtype and then selecting the corresponding specific monoclonal antibody. However, this example discloses several monoclonal antibodies that can cross-react with RagB proteins of different subtypes, indicating that different P. gingivalis subtypes 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 peptide libraries To identify the antigenicity of P. gingivalis RagB outer membrane proteins 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. Synthetic peptides were produced by a third-party CRO (GenScript, USA). Specifically, based on the amino acid sequence of each protein, peptide chains consisting of 18–25 amino acids were artificially synthesized starting from the N-terminus. The next peptide chain contained five amino acids from the C-terminus of the previous peptide chain, forming a sequence overlap, and so on. Except for the peptides at both ends of each protein (each peptide chain has only a single overlapping sequence), all other peptide chains overlap by five amino acids with the peptide chains on both sides, upstream and downstream, to avoid missing 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 by adding a number to the RA, e.g., RA-1, RA-2, etc. RagB-2 peptides are named by adding a serial number to the RB, RagB-3 peptides are named by adding a serial number to the RC, and RagB-4 peptides are named by adding a serial number to the RD. Cra4S1 peptides are named by adding a serial number to the Cra and NewCra. Most synthetic peptide chain fragments are soluble 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 P. gingivalis outer membrane proteins, all monoclonal antibodies were reacted with fragments in the peptide library to identify targets and important sequence fragments for antigen-antibody binding. Specifically, a 1 mg / ml soluble solution was prepared based on the peptide's solubility. The polypeptide solution was then further diluted to 20 μg / ml and adsorbed onto an ELISA plate. Subsequent antibody testing steps were as described in Example 2. Each monoclonal antibody reacted with its corresponding outer membrane protein polypeptide fragment of the RagB subtype. To avoid missing antigens and determine the binding site, each synthetic peptide chain had overlapping portions with the upstream and downstream polypeptides, as described above.

[0145] The construction of the four RagB subtype peptide libraries was aimed at capturing antigen epitopes and antigen targets that cross-react with monoclonal antibodies. As described in Example 3, some monoclonal antibodies targeting specific RagB subtypes showed cross-reactivity to other RagB subtypes. These antibodies were screened with the peptide libraries to identify antibody-antigen targets.

[0146] Table 12 RagB-1 (SEQ ID NO: 2) peptide library (GenScript, USA, C4965GB230) JPEG2025531306000012.jpg90153

[0147] Table 13. RagB-2 (SEQ ID NO: 4) peptide library (GenScript, USA, C4965GB230) JPEG2025531306000013.jpg82161

[0148] Table 14. RagB-3 (SEQ ID NO: 6) peptide library (GenScript, USA, C4965GB230) JPEG2025531306000014.jpg81155

[0149] Table 15. RagB-4 (SEQ ID NO: 8) Peptide Library (GenScript, USA, C4965GB230) JPEG2025531306000015.jpg94161

[0150] Table 16. RagB-4 (SEQ ID NO: 10) peptide library JPEG2025531306000016.jpg16696

[0151] The antigen-antibody binding reaction was determined by the OD value of the indirect ELISA experiment. An OD value higher than twice that of the negative serum control was considered a positive result.

[0152] Table 17 shows the capture of antigen targets by a peptide library of RagB-1 (SEQ ID NO: 2) and monoclonal antibodies, including monoclonal antibodies against RagB-1 and those recognized to react with other RagB subtypes. ELISA results showed that three RagB-1 hybridoma antibody strains 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 generated by inoculation with RagB-1 protein, designated 2019-G85-1 serum (P), reacted with the RA-1 peptide chain. Interestingly, monoclonal antibodies of other subtypes cross-reacted with RagB-1 protein, revealing that target antigens were also found in the RagB-1 peptide library. For example, 1A4-3-7-7 (specific for RagB-2 protein) bound to the RA-19 peptide chain, 1D2-2-1-3 (specific for RagB-3 protein) bound to the RA-17 peptide chain, 2C1-4-6-2 (specific for RagB-3 protein) bound to RA-9 and RA-17, 2B7-4-6-7 (specific for RagB-3 protein) bound to RA-9, while 1B11-4-4 (specific for RagB-4 protein) bound to the RA-21 peptide chain.

[0153] Table 17. Screening of target antigens with RagB-1 (SEQ ID NO: 2) peptide library JPEG2025531306000017.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 against RagB-2 and recognized monoclonal antibodies reactive with other RagB subtypes. ELISA results showed that three RagB-2 hybridoma antibody strains detected the peptide antigen target. 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). A mouse serum of a polyclonal antibody generated by inoculation with RagB-2 protein, designated 2019-G65-1, reacted with the RB-17 peptide chain.

[0155] Monoclonal antibodies cross-reactive with RagB-2 protein also found peptide targets in the RagB-2 peptide library: for example, 1D2-2-1-3 (specific for RagB-3 protein) binds to the RB-17 peptide chain, whereas 1C3-7-8 (specific for RagB-4 protein) binds to the RB-21 peptide chain.

[0156] Table 18. Screening of target antigens with RagB-2 (SEQ ID NO: 4) peptide library JPEG2025531306000018.jpg163111

[0157] Table 19. Screening of target antigens with RagB-3 (SEQ ID NO: 6) peptide library JPEG2025531306000019.jpg164117

[0158] Table 20. Screening of target antigens with RagB-3 (SEQ ID NO: 6) peptide library JPEG2025531306000020.jpg156120

[0159] Tables 19 and 20 show the capture of antigen targets by monoclonal antibodies, including a peptide library of RagB-3 (SEQ ID NO: 6) and monoclonal antibodies against RagB-3 and those identified to react with other RagB subtypes. ELISA results showed that nearly all of the RagB-3 hybridoma antibodies tested detected the peptide antigen target, and 15 of the 16 hybridoma antibodies reacted with peptide chains from 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 raised 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) failed to detect distinct polypeptide targets in the peptide library. The 1D2-2-1-3 recombinant plasmid antibody reacted highly specifically with the RC-17 peptide chain but failed to react with other polypeptide fragments.

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

[0162] Table 21. Screening of target antigens using RagB-4 (SEQ ID NO: 8) peptide library JPEG2025531306000021.jpg161119

[0163] Table 21 shows the capture of antigen targets by monoclonal antibodies, including a peptide library of RagB-4 (SEQ ID NO: 8) and monoclonal antibodies against RagB-4 and those identified to react with other RagB subtypes. ELISA results showed that the RagB-4 hybridoma antibodies detected the polypeptide antigen target, and six of the seven hybridoma antibodies 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 RagB-3 protein, but no cross-reactivity was observed between these antibodies and RagB-4 polypeptide fragments. Mouse serum containing polyclonal antibodies generated by inoculation with RagB-4 protein was designated G90 serum (P) and reacted with the RD-1 peptide chain.

[0164] In an antigen target capture study of the Cra4S1 monoclonal antibody, the culture supernatants of over 40 positive hybridoma cell lines were tested to screen for antigen targets. Although these antibodies reacted with high titers to the recombinant protein, they did not react with the artificially synthesized peptide antigens created in the Cra4S1 peptide library. Only a few clones showed weak antigen-antibody reactions. Table 16 provides two peptide libraries of Cra4S1. Even after adjusting the length and position of the peptide chain, no antigen-antibody binding reaction was still detected.

[0165] As mentioned above, several monoclonal antibody lines have shown cross-reactivity to other RagB proteins and their fragments in peptide libraries. Important 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] Monoclonal antibody 1A4-3-7-7 specifically reacts with the RagB-2 protein and with the RA-19 and RB-18 peptide chains. The underlined amino acids are completely identical in both sequences, indicating that the specific antigen-binding site of the RagB-2-1A4-3-7-7 monoclonal antibody is located in 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 site of the RagB-3-1D2-2-1-3 monoclonal antibody is located in RA-17&18 (SEQ ID NO: 123) and / or RB-17 (SEQ ID NO: 51), and / or RC-17&18 (SEQ ID NO: 124). The underlined amino acids are completely identical in both sequences, indicating that the monoclonal antibody RagB-3-1D2-2-1-3 has cross-reactivity with the other two RagB subtypes, but the epitope and length of the antibody-antigen binding site 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 site of the RagB-4-1B11-4-4 monoclonal antibody is located in RA-21 (SEQ ID NO: 31), RB-21 (SEQ ID NO: 55), RC-21 (SEQ ID NO: 79), and RD-21 (SEQ ID NO: 103). The underlined amino acids indicate the exact sequence identity of the two sequences, indicating that the monoclonal antibody RagB-4-1B11-4-4 exhibits cross-reactivity with the other three RagB subtypes, although the epitope and length of the antibody-antigen binding site are not identical.

[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 monoclonal antibody RagB-4-1C3-7-8 to the 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 the 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 site of the RagB-4-1C3-7-8 monoclonal antibody is located in RA-21&22 (SEQ ID NO: 125), RB-21 (SEQ ID NO: 55), RC-22 (SEQ ID NO: 80), and RD-21&22 (SEQ ID NO: 126). The underlined amino acids indicate the exact sequence identity of the two sequences, indicating that the monoclonal antibody RagB-4-1C3-7-8 exhibits cross-reactivity with the other three RagB subtypes, but the epitope and length of the antibody-antigen binding site are not identical.

[0197] The discovery of polyclonal antibodies cross-reactive with different subtypes of RagB proteins, as well as monoclonal antibodies with multiple antigenic targets, was surprising and unexpected. For two monoclonal antibody lines with multiple cross-reactivity derived from RagB-4 immunization, 1B11-4-4 (SEQ ID NOs: 148-149 and 153-154) and 1C3-7-8 (SEQ ID NOs: 158-159 and 163-164), data confirmed that their antigenic targets are located in the RD-21 (SEQ ID NO: 103) and RD-21&22 (SEQ ID NO: 126) regions. Although the locations of these antigenic targets are close to each other, each antigenic target has a unique location and sequence. Importantly, they share antigenic determinants located at different locations in the RagB-1, RagB-2, and RagB-3 proteins.

[0198] Thus, antigen-antibody reactions between monoclonal antibodies and peptide chains in the RagB peptide library confirmed the antigenicity of RagB polypeptide fragments, providing a molecular basis for the development of antibodies and vaccines that react with these antigenic fragments and aiding in the development of therapeutics for controlling diseases caused by P. gingivalis infection. Monoclonal antibodies 1B11-4-4 (SEQ ID NOs: 148-149 and 153-154) and 1C3-7-8 (SEQ ID NOs: 158-159 and 163-164) have their respective antigen targets within 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. Adjacent antigen targets provide ideal conditions for the preparation of mixed monoclonal antibodies and bispecific antibodies.

[0199] Example 5 Heterophile antigens and the pathogenesis of Porphyromonas gingivalis In recent years, clinical and statistical studies have shown that periodontal disease is associated with various systemic diseases, especially chronic diseases and tumors. Despite many detailed studies, the direct relationship between P. gingivalis and chronic diseases and the underlying mechanism remain unclear.

[0200] In Example 4, when monoclonal antibodies against the Cra4S1 protein were screened, the antibodies were shown to react strongly with the full-length recombinant Cra4S1 protein, but were unable to identify specific antigenic target fragments of this protein. The Cra4S1 polyclonal antibody serum also failed to reveal major polypeptide targets 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 diffuse and is only represented by its three-dimensional spatial structure. Furthermore, in Example 4, monoclonal antibodies against the RagB-3 protein identified multiple target antigenic polypeptide sequence fragments in the RagB-3 peptide library. However, testing with the polyclonal antibody serum failed to reveal major peptide fragments and / or epitopes 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 antigenic polypeptide fragments / targets RA-1 and RD-1 at the N-terminus of each peptide, respectively, whereas RagB-2 polyclonal antibodies were able to react with peptide RB-17. Overall, these results indicate that the antigenic properties of different outer membrane protein subtypes of P. gingivalis are not identical.

[0201] Looking back at the study of the Cra4S1 recombinant protein, Example 2 shows that animal sera from P. gingivalis infection reacted with RagB but not with Cra4S1, suggesting that the antigenicity of the Cra4S1 protein may not be dominant. The data in Example 2 also show that when RagB protein was used to immunize animals with a history of bacterial infection, serum antibodies immunoreacted with Cra4S1 but did not react with an unrelated protein (GST negative control), suggesting that RagB antibodies cross-react with the Cra4S1 protein. In several examples, the RagB-4-1B11-4-4 monoclonal antibody was found to indeed cross-react with Cra4S1.

[0202] Surprisingly, when human serum samples were used to detect antibodies against P. gingivalis, antibodies against RagB and Cra4S1 were detected in significant proportions in the samples. Specifically, the human serum samples collected in this example included healthy individuals, patients with cardiovascular disease, and geriatric patients. Tables 22, 26, and 29 show the ELISA results for these human serum samples, detecting antibodies against P. gingivalis outer membrane proteins. The design and implementation of the antigen adsorption on the ELISA plate were the same as those described in Example 2. The antigen layout design is briefly described as follows: columns 1 and 7 were adsorbed 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, and columns 6 and 12 with GST recombinant protein as an unrelated protein negative control.

[0203] The dilution of these serum samples from Group 1 (healthy subjects) was 1:300. Each serum sample was tested in a six-well antigen protein test containing RagB-1, RagB-2, RagB-3, RagB-4, Cra4S1, and GST. The secondary antibody was alkaline phosphatase-conjugated mouse anti-human IgG for human samples and goat anti-mouse IgG for controls. Each ELISA plate included a PBS negative control, and a positive control (labeled M-PC) was obtained from the serum of immune mice immunized with a pentavalent protein vaccine containing RagB-1, RagB-2, RagB-3, RagB-4, and Cra4S1.

[0204] Because this was the first time that P. gingivalis outer membrane protein antibodies were detected using human serum, the mean OD value of the lowest one-fifth (lowest 20%) of all samples was preliminarily defined as negative, and the cutoff value was preliminarily set at 2.5 times that value. OD values ​​greater than 2.5 times the negative value were considered positive. Table 22 shows the ELISA results for serum samples from 51 healthy subjects. Table 23 shows the lowest OD value for each protein group detected in 10 subjects (20% of 51 samples). Table 23 also shows the number of samples with OD values ​​greater than 2.5 times the negative OD value (considered a positive result), as well as data for false-positive subjects with OD values ​​between 1.8 and 2.5 times the negative value. OD values ​​greater than four times the negative value (4x) were considered strong positive results, quantitatively reflecting higher protein titers.

[0205] The human serum samples in Group 1 consisted of 51 healthy individuals (blood tests from young adults, numbers H1–H51). The positive rate for RagB-1 antibody tests was 11.8% (6 / 51), of which 2% (1 / 51) were strongly positive, with OD values ​​exceeding four times the negative value. The positive rate for RagB-2 antibody tests was 0% (0 / 51), the positive rate for RagB-3 antibody tests was 2% (1 / 51), and the positive rate for RagB-4 antibody tests was 31.3% (16 / 51), of which 13.7% (7 / 51) were strongly positive. 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 accounted for a relatively large proportion 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), whereas the positive rate for antibodies to the unrelated protein GST was 0% (0 / 51).

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

[0207] An etiological study was conducted on periodontal disease patients who visited the dental outpatient clinic of the Royal London Hospital, UK, from 2002 to 2004. A total of 107 samples were analyzed using PCR to detect P. gingivalis and its subtypes in gingival crevicular fluid samples from periodontal disease patients. PCR nucleic acid testing showed that P. gingivalis ragB-2 and ragB-3 alleles were the most common group in the periodontal disease patient population. Detailed methods are described in patent application PCT / GB2005 / 001976, which is incorporated herein by reference. Briefly, total DNA was extracted from gingival crevicular fluid samples, and P. gingivalis 16S RNA 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 P. gingivalis. The results showed that the RagB-2 and RagB-3 subtypes accounted for approximately 58% of the samples combined (Figure 13, Table 25). The results of these clinical specimens indicate that the RagB-2 and RagB-3 P. gingivalis subtypes are relatively common in the local East London periodontal disease population.

[0208] Virulence tests of different bacterial strains using animal models of soft tissue injury have not revealed any outer membrane protein subtypes that are significantly more or less virulent than others. The mechanisms by which bacterial virulence 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 previous examples. For plates 20-05-P5, 20-05-P6, and 20-05-P7, the dilution of the primary antibody (patient serum) was 1:1000, and for plate 20-05-P8, the dilution of the primary antibody (patient serum) was 1:500. To avoid false positive results, the sample dilution was increased from 1:300 to 1:500 and 1:1000.

[0210] Table 22. Survey of antibodies to Porphyromonas gingivalis in serum of healthy subjects JPEG2025531306000022.jpg163106

[0211] Table 23. Data analysis of serum surveys of healthy individuals JPEG2025531306000023.jpg149155

[0212] Table 24. Analysis of antibody distribution of outer membrane proteins JPEG2025531306000024.jpg68161

[0213] Table 25. Distribution analysis of RagB subtypes in patients with periodontal disease JPEG2025531306000025.jpg17145

[0214] Table 26. Serum antibody survey of Porphyromonas gingivalis in elderly patients JPEG2025531306000026.jpg95163

[0215] Table 27 shows the data analysis of serum samples from elderly patients (samples 201-255 in Table 26 and samples 256-262 in Table 29), a total of 62 samples. Based on the pre-established criteria, Table 28 shows the data analysis of serum samples 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 antibodies was 43.5% (27 / 62), of which 21% (13 / 62) were strongly positive, while the positive rate for antibodies to the unrelated protein GST 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 JPEG2025531306000027.jpg138160

[0217] Table 28. Distribution analysis of outer membrane protein antibodies JPEG2025531306000028.jpg67140

[0218] Table 29. Serum antibody survey of Porphyromonas gingivalis in cardiovascular patients JPEG2025531306000029.jpg96161

[0219] Table 30. Data analysis of survey results in cardiovascular patients JPEG2025531306000030.jpg132160

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

[0221] Table 30 shows the data analysis of serum samples from cardiovascular patients (numbers 101-149), a total of 49 samples. Based on the predefined criteria, the positive rate for RagB-1 antibody detection was 44.9% (22 / 49), of which 8.2% (4 / 49) were strongly positive. The positive rate for RagB-2 was 40.8% (20 / 49), of which 8.2% (4 / 49) were strongly positive. The positive rate for RagB-3 antibody was 46.9% (23 / 49), of which 8.2% (4 / 49) were strongly positive. The positive rate for RagB-4 antibody was 57.1% (28 / 49), of which 10.2% (5 / 49) were strongly positive. The positive rate for Cra4S1 antibody was 40.8% (20 / 49), of which 4.1% (2 / 49) were strongly positive. On the other hand, the positive rate for antibodies to the unrelated protein GST 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 with healthy controls, serological surveys of patients with cardiovascular disease and geriatric diseases have three notable features. First, the detection rate of RagB-2 and RagB-3 antibodies is significantly increased. Second, the detection rate of GST protein antibodies (i.e., antibodies against unrelated antigens) is significantly increased. Third, the proportion of patients with high-titer antibodies in samples from cardiovascular disease and geriatric patients is much higher than that of healthy controls.

[0223] Table 31. Distribution analysis of outer membrane protein antibodies JPEG2025531306000031.jpg73153

[0224] Antibodies to the unrelated antigen GST are not detected in healthy individuals, but the detection rate of GST antibodies is significantly increased in patients with cardiovascular disease and geriatric diseases. Furthermore, the detection of GST antibodies is directly correlated with the levels of RagB and Cra4S1 antibodies and is only seen 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 their GST antibodies. No independent cases of GST antibody positivity were found.

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

[0226] Evidence from multiple sources indicates that P. gingivalis outer membrane proteins, including RagB and Cra4S1, share characteristics of heterophile antigens. Heterophile antigens are common antigens that are structurally similar to but completely unrelated to antigens present in host tissues. Numerous scientific literature has reported diseases caused by heterophile antigens. For example, the cell membrane of Streptococcus pyogenes shares antigens with glomerular basement membrane and myocardial tissue, potentially leading to tubulitis or myocarditis after streptococcal infection. Similarly, Escherichia coli (E. coli) lipopolysaccharide shares antigens with human colonic mucosa, and infection with the former has been associated with the development of ulcerative colitis.

[0227] Heterophilic antibodies are polyspecific immunoglobulins with a certain potency that are induced by known or unknown antigenic substances (heterophilic antigens) and can bind to a variety of proteins, although the affinity may not be strong. These antibodies are endogenous and naturally occurring, and can react with molecules unrelated to the original antigen. These immunoglobulins, which have different chemical structures but have multiple binding capacities for the test substance, are called heterophilic antibodies.

[0228] Heterophilic antibodies can be understood as polyspecific or nonspecific immune enhancement and may have immune protective functions. However, multiple immune responses against heterophilic antigens and antigenic components shared by different tissues may cause immunopathology.

[0229] Serological surveys have revealed that RagB, Cra4S1, and GST antibody positive cases account for the majority of all samples in patients with cardiovascular disease and geriatric diseases.Based on epidemiological survey data, P. gingivalis outer membrane proteins, including RagB and Cra4S1, exhibit heterophile antigenic characteristics.

[0230] Since the RagB-4 (381RagB) DNA / protein sequence (SEQ ID NO: 8) was first disclosed in 2004, scientists have discovered high similarity of protein sequences similar to RagB-4 in other species. For example, the outer membrane protein (WP_039431148) of Porphyromonas gulae (a major periodontal pathogen in dogs and cats) has 93% similarity with RagB-4 (see Figure 16), further supporting the heterophile antigen properties of P. gingivalis and its wide application value. Furthermore, although RagB-4 is unrelated to the Cra4S1 molecular sequence, as described in Example 4 and in Example 7 below, the RagB-4-1B11-4-4 monoclonal antibody cross-reacts with the Cra4S1 protein, and this cross-reactivity with the full-length Cra4S1 protein and not with a specific polypeptide suggests the possibility of diffusive or weak binding, a characteristic commonly observed with 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 has a history of Porphyromonas gingivalis infection.Periodontal disease is usually chronic and progressive, and although the body's immune response to Porphyromonas gingivalis varies in degree, it usually cannot completely eliminate the infection.

[0232] This example shows that the detection rate of P. gingivalis outer membrane protein antibodies is relatively high in the serum of patients with cardiovascular disease and geriatric disease. Furthermore, most of these patients also show reactions between serum immunoglobulins and unrelated proteins. This suggests that a significant portion of these patients are in an immune-sensitized state, and these antibodies are likely to be heterophile antibodies, which are strongly correlated with chronic diseases and pathological damage, and may even play a pathogenic role in the pathogenesis of certain diseases.

[0233] As shown in our experimental results, P. gingivalis appears to be less toxic to young individuals. It is speculated that heterophile antibodies do not accumulate to harmful levels in young individuals. Human serum samples also showed that P. gingivalis outer membrane protein antibodies were not detected in some individuals across all three groups (including healthy individuals, patients with cardiovascular disease, and elderly patients) (Tables 22, 26, and 29). These individuals either have natural / nonspecific resistance to P. gingivalis or have never been infected with P. gingivalis. This finding highlights the possibility that P. gingivalis may be one of the multifactorial contributors to complex systemic disease, and that differences in individual immune responses play an important 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 feasible method for predicting the onset of P. gingivalis infection and related diseases, which can prompt timely attention to the onset of disease, provide significant benefits for early treatment of patients, and potentially reduce morbidity and mortality.

[0235] Further research will enhance our understanding of the relationship between P. gingivalis infection and chronic disease. The data provided in this example suggest that the heterophile antigenic properties of P. gingivalis, combined with individual characteristics, may explain the heterophile antibody and / or hypersensitivity reactions that are important factors in chronic P. gingivalis infection and systemic disease. Therefore, clearing the pathogenic infection, reducing the release of heterophile antigens, and / or lowering circulating heterophile antibody levels may mitigate tissue damage caused by harmful heterophile antibodies, proactively treat associated diseases, and control and prevent further deterioration of multiple tissues.

[0236] Example 6 Application of conjugated antibodies in animal breeding models Animal experimental data have shown that a RagB protein and Cra4S1 protein combination vaccine provides superior local tissue protection compared with single-protein vaccines. However, the combination vaccine did not provide cross-protection against other P. gingivalis subtypes. For example, vaccination with a combination vaccine containing RagB-1, RagB-3, and Cra4S1 protected animals against P. gingivalis infection with RagB-1 and RagB-3 subtypes, but not against RagB-2 subtype infection. Passive immunization has confirmed that antibody combinations, such as a mixture of anti-RagB and anti-Cra4S1, provide superior protection compared with either single antibody and control groups. Further details of the 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 to provide precise treatment based on the diagnosis of P. gingivalis outer membrane protein RagB subtype. As described in Example 3, monoclonal antibodies against four different RagB proteins and the Cra4S1 protein have been identified and described.

[0238] An antibody combination experiment was designed. In this example, the RagB-3-specific 1D2-2-1-3 monoclonal antibody was combined with the mouse serum-derived Cra4S1 polyclonal antibody. Specifically, 5-6-week-old male Balb / c mice (weighing 15-18 g) were grouped into six groups. Four days and one day before bacterial infection, mice were intraperitoneally injected with 100 μl of antibody solution (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. The animals were then challenged with bacteria.

[0239] The preparation of bacterial suspensions is briefly described below. Porphyromonas gingivalis containing various subtypes (RagB-1, RagB-2, RagB-3, and RagB-4) was grown on strictly anaerobic agar (FAA) containing 5% defibrinated horse blood and placed in an anaerobic incubator at 37°C, with the culture gas set to 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 h until the OD600nm reached 1–1.2. The cultures were then centrifuged, washed twice, and cultured at different concentrations (2–8 x 10) using BHI medium. 10 Bacterial suspensions of 1000 CFU / ml were prepared and used in animal experiments.

[0240] Table 32. Information on animals participating in passive immunization experiments JPEG2025531306000032.jpg27161

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

[0242] The data show that animals receiving the combination of RagB-3 monoclonal antibody 1D2-2-1-3 and Cra4S1 polyclonal antibody achieved better local immune protection at the site of bacterial infection, with less soft tissue damage and faster healing compared to other groups of animals that did not receive the antibody combination. The data also demonstrate that the antibody combination achieved local efficacy even within the body's fluid circulation. In this example, intraperitoneal injection (systemic administration technique) provided local subcutaneous protective effects in subsequent P. gingivalis bacterial infection.

[0243] Table 33. Monoclonal Antibody Immune Protection Experimental Data Record JPEG2025531306000033.jpg124161

[0244] Table 34 Data collection JPEG2025531306000034.jpg33161

[0245] Periodontal disease caused by P. gingivalis may appear as local damage visible to the naked eye. Clinicians can determine the severity of periodontal disease by examining the depth of periodontal pockets and the susceptibility to gingival bleeding. However, the association between P. gingivalis infection and a series of systemic diseases suggests that periodontal disease may be only the tip of the iceberg of the harm caused by P. gingivalis infection.

[0246] Currently, conventional animal models of P. gingivalis infection primarily focus on soft tissue destruction and alveolar bone resorption. The virulence of different P. gingivalis strains is inconsistent, and local tissue damage caused by P. gingivalis infection is observed in almost all animals, with the extent of damage depending on the infection dose. Typically, the higher the bacterial concentration that an animal is infected with, the more severe the local tissue damage. In most cases, infected animals self-heal, although some abscesses may recur after initial wound healing. Bacterial infection alone does not generally result in high mortality rates. The pathogenic mechanism of P. gingivalis has never been conclusively proven, and the scientific community has so far been unsuccessful in establishing an animal model that correlates P. gingivalis infection with systemic disease.

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

[0248] In this first established animal model, three groups were established: Group 1, male and female mice received both the antibody combination and P. gingivalis infection; Group 2, male mice served as the control group, and female mice received the antibody combination and P. gingivalis infection; and Group 3, male mice received the antibody combination and P. gingivalis infection, and female mice served as the control group. The antibodies were derived from rabbit serum, and the details of the antibody combinations are shown in Table 35. Specifically, two intraperitoneal antibody injections were administered four days apart, after which the mice were infected with P. 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 contained one male and one female per cage.

[0249] In the initial experiments, due to the limited number of control female mice (i.e., neither antibody nor P. gingivalis infection), younger female mice (11-12 weeks old instead of 18-20 weeks old) were used; see Table 35 for details. As a control, control female mice of similar age were mated with control male mice, and it was shown that 11-12 week old female mice had fewer pups.

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

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

[0252] In Table 38, male mice administered the antibody combination and then infected with P. gingivalis were mated with normal control female mice. The results showed that the number of liveborn pups was improved compared to the previous case in which both males and females received the antibody combination and P. gingivalis infection. The control group consisted of male mice vaccinated with PBS and infected with P. gingivalis mated with normal control female mice. The average number of liveborn pups from infected male mice administered the antibody combination was similar to that of control mice vaccinated with PBS. Interestingly, the liveborn pups and survival rate in Table 38 were lower than those in Table 37, and the stillbirth rate was higher. These data suggest that the infection status of males after antibody administration and infection has a greater impact on the liveborn pup rate than the infection status of female mice.

[0253] Table 35. Information on animals participating in the experiment JPEG2025531306000035.jpg107161

[0254] Table 36. Reproduction experiment (1) - Antibody administration and infection of both male and female animals (a control group without antibody administration was added) JPEG2025531306000036.jpg32161

[0255] Table 37. Reproduction experiment (2) - Mating of female mice administered with antibody and infected (with a control not administered with antibody) with male control mice not administered with antibody and not infected JPEG2025531306000037.jpg33161

[0256] Table 38. Reproduction experiment (3) - Mating of male mice administered with antibody and infected (with a control not administered with antibody) with female control mice not administered with antibody and not infected JPEG2025531306000038.jpg32161

[0257] Table 39. Investigation of immune defense function of combined monoclonal antibodies JPEG2025531306000039.jpg45161

[0258] Based on the above results, the next step was to evaluate the effects of administering RagB monoclonal antibodies to these mouse models. As described in Example 4, three monoclonal antibodies showed potential as vaccines against P. gingivalis due to their cross-reactivity with multiple RagB subtypes: 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). Because the passive immunity provided by a single antibody is limited, as described in PCT / CN2019 / 124433, this example aimed to evaluate the effects of passive immunity using a combination of two (or more) monoclonal antibodies disclosed in the present invention.

[0259] The animal experiments involving antibody combinations and P. gingivalis infection were performed as described above. Specifically, mice were given two intraperitoneal injections of the antibodies at four-day intervals, and then the animals were infected with P. gingivalis. Survival rates and local lesions were observed and recorded.

[0260] Table 39 shows the results of passive immunization of animals administered antibody combinations followed by infection with P. gingivalis. Male and female mice were inoculated with the two monoclonal antibody combinations listed in Table 39, while control groups received normal mouse IgG or PBS and were then infected with P. gingivalis RagB-3 subtype. One mouse (1 / 8) administered normal mouse IgG died after bacterial infection, and two mice (2 / 8) died in each of the G942 and G944 groups. 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 received the immune challenge, and the other female mouse was not challenged or infected. The weight of the female mice, as well as the number of live and stillborn pups, were recorded. If a female mouse suddenly lost weight and no pups were found, abortion may have occurred. Because there were fewer female mice available than male mice, there was a shortage of female mice in each group. As a result, some female mice from other studies were used. These mice had records of antibody injection and P. gingivalis infection; specific details are shown in Table 40.

[0262] Table 40. Immune profile of female mice added to the reproductive model JPEG2025531306000040.jpg73161

[0263] Table 41. Reproductive Effects of Combined Monoclonal Antibodies (1) - Male and female mice were challenged with mouse IgG, and the experimental group included control female mice without antibody and without challenge. JPEG2025531306000041.jpg70161

[0264] G941 / G951 (Table 41) and G945 / G955 (Table 45) were control groups and received normal mouse IgG and PBS, respectively. These animals were not administered any protective antibodies prior to infection with P. gingivalis and therefore represent the baseline expected reproductive success of animals that recovered from infection. The number of liveborn pups observed in both groups was similar; however, in the G941 / G951 group, a high proportion of female mice across the four cages failed to conceive (4 / 7), whereas the G945 / G955 group experienced a high proportion of stillbirths.

[0265] Table 42. Reproductive Effects of Combined Monoclonal Antibodies (2) - Male and female mice received a combination of monoclonal antibodies 1B11 and 1D2 and were infected, and the experimental group included female mice controls without antibodies and without infection. JPEG2025531306000042.jpg61161

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

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

[0268] In the G944 / G954 group (Table 44), animals received a combination of 1D2 and 1C3 monoclonal antibodies prior to infection with P. gingivalis. As previously noted, male mice had a 25% mortality rate (2 / 8) following bacterial infection. Subsequent reproductive success was similar to that of the G945 / G955 control group, which received PBS prior to infection. Of the remaining six male mice, one was unable to impregnate any female mice (including the control female mice). Live pups observed: A total of 31 pups were born from 15 female mice. There were a high number of stillbirths (10 pups), and three female mice were suspected to have aborted.

[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 control mice without antibody and without infection. JPEG2025531306000043.jpg78161

[0270] Table 44. Reproductive Effects of Combined Monoclonal Antibodies (4) - Male and female mice received a combination of monoclonal antibodies 1C3 and 1D2 and were infected. The experimental group included female control mice without antibodies and without infection. JPEG2025531306000044.jpg61161

[0271] Table 45. Reproductive Effects of Combined Monoclonal Antibodies (5) - Male and female mice received monoclonal antibody PBS and infection, and the experimental group included female control mice without antibody and infection. JPEG2025531306000045.jpg78161

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

[0273] The results of the animal experimental model disclosed in this example demonstrate that Porphyromonas gingivalis infection causes reproductive disorders. Literature has reported that pregnant women with periodontal disease are three to seven times more likely to give birth prematurely and with low birth weight than those without periodontal disease. For many years, researchers have studied genetics, endocrinological characteristics, and tissue and organ infection, focusing on the effects of infection on the maternal reproductive system and fetal characteristics.

[0274] This example demonstrates that P. gingivalis infection causes reproductive disorders. The worst outcomes occur when both males and females are infected, with high rates of fertilization failure, stillbirth, and abortion. The data disclosed in this example also show that when infected females mate with uninfected males, their reproduction remains stable; conversely, when infected males mate with uninfected females, the end result is worse. These results clearly demonstrate that P. gingivalis infection and immune status in males adversely affect reproductive outcomes, as infected males consistently have poor reproductive outcomes in these animal models. A possible mechanism of action is via heterophile antigens on the surface of P. gingivalis, which resemble host tissues, such as sperm or tissues of the male reproductive system.

[0275] Furthermore, although P. gingivalis -specific antibodies can provide immune protection, they can also cause immune-mediated pathological damage. For example, the 1D2 monoclonal antibody exhibited strong antigen-antibody responses to multiple RagB subtypes, including the RA-17, RB-17, and RC-17 peptides (Example 4), demonstrating local immune protection. However, this monoclonal antibody was also noted to cause high mortality and reduced reproductive success in this example.

[0276] The data disclosed in this example also demonstrate that two other monoclonal antibodies, i.e., 1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3), are specific for RagB-4, can cross-react with multiple RagB subtypes, are safe, and do not harm the animal's reproductive system. These combined antibodies effectively improve the physical condition of male and female animals and directly or indirectly restore / cure fertility. The data suggest that these combined antibodies may be used for other diseases associated with P. gingivalis infection and / or systemic diseases, including, but not limited to, periodontal disease, cardiovascular disease, rheumatoid arthritis, oral and gastrointestinal cancer, ulcerative colitis, nervous system diseases, autoimmune encephalomyelitis, lung cancer, abnormal pregnancy, pancreatic cancer, diabetes, chronic kidney disease, bacterial pneumonia, and chronic obstructive pulmonary disease. One treatment strategy is to remove harmful antigens and / or antibodies associated with the development and occurrence of the disease and / or to increase the titer or concentration of beneficial antibodies in the body.

[0277] Example 7 Mouse Monoclonal Antibodies and Humanized Mutations Four monoclonal antibody hybridoma cell lines: RagB-2-1A4-3-7-7, RagB-3-1D2-2-1-3, RagB-4-1B11-4-4, and RagB-4-1C3-7-8, all showed cross-reactivity with outer membrane proteins of different subtypes of P. gingivalis. The nucleic acid sequences of these antibodies have been obtained. Briefly, total RNA from each cell line was extracted and reverse-transcribed using antisense primers according to conventional RNA extraction and transcription methods. The light and heavy chain variable region sequences were then amplified, and the PCR fragments were inserted into the pUC19T vector. PCR was then used to screen clones, and positive clones were selected for sequencing.

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

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

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

[0281] RagB-4-1B11-4-4 (Baiying, China, B583901, also referred to as "1B11") comprises 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 of which is shown in SEQ ID NO: 149, and 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 of which is shown in SEQ ID NO: 154 (see also Figures 21A-21D). The present invention also provides a bispecific version of this monoclonal antibody. Expression of the recombinant antibody is shown in Figures 21E and 21F.

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

[0283] Table 46. Physicochemical properties of recombinant antibodies JPEG2025531306000046.jpg14160

[0284] The recombinant plasmids of three RagB monoclonal antibodies, including 1D2, 1B11, and 1C3, were expressed in eukaryotic cells HEK293 by a CRO company (Baining, 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 listed in Table 46. The antigen-binding function of the recombinant antibodies was detected using ELISA (see Table 47).

[0285] The detection results of the recombinant antibody plasmids are shown in Table 47. 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 were diluted to 100 μg / ml and each antibody was added to one row of an ELISA plate, with 12 wells per row, to perform antigen testing, 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 JPEG2025531306000047.jpg35161

[0287] The data show that the binding patterns of the recombinant antibodies to their corresponding antigens are identical to those of the monoclonal antibodies generated 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 the combination of two monoclonal antibodies, 1B11-4-4 (1B11) and 1C3-7-8 (1C3), provides cross-immune protection against four different P. gingivalis subtypes. The 1B11-4-4 (1B11) antibody also cross-reacts with the Cra4S1 protein (Table 47), a conserved epitope in all P. gingivalis subtypes. Because Cra4S1 is thought to be a heterophile antigen, monoclonal antibody 1B11 may exert a synergistic effect by reducing the heterophile antigen of Cra4S1 in addition to providing cross-immune protection against other P. gingivalis outer membrane protein subtypes.

[0289] Therefore, humanized sequence mutations of 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. The present 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 antibody production.

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

[0291] Example 8 RagB Bispecific Monoclonal Antibodies 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 engineered into a variety of different structural patterns. Through different mechanisms of action, BsMAbs can be designed to recruit and activate immune cells, disrupt receptor signaling, inactivate signaling ligands, and bind to protein complexes.

[0292] The above data indicate 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&22 region of the Porphyromonas gingivalis outer membrane protein RagB-4, and that the combination of the two monoclonal antibodies provides stable systemic and local immune protective effects. Therefore, a bispecific monoclonal antibody 1B11-1C3 (Baiying, China, B745901) combining RagB-4-1B11-4-4 (1B11) and RagB-4-1C3-7-8 (1C3) was prepared, which comprises a heavy chain whose nucleotide sequence is shown in SEQ ID NO: 168 and whose encoded amino acid sequence is shown in SEQ ID NO: 172, and a light chain whose nucleotide sequence is shown in SEQ ID NO: 173 and whose encoded amino acid sequence is shown in SEQ ID NO: 174.

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

[0294] The heavy chain of the bispecific 1B11-1C3 antibody comprises the nucleotide sequence and amino acid sequence, in which the nucleotide sequence of the heavy chain variable region VH comprises 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 comprises 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 1C3 full-length light chain variable region shown in SEQ ID NO: 164 (see Figures 23A-23F).

[0295] Table 48. Physicochemical properties of recombinant bispecific antibodies JPEG2025531306000048.jpg16161

[0296] The recombinant plasmid was transfected into eukaryotic cells HEK293, and the recombinant antibody was induced, expressed, and purified by protein A affinity chromatography (see Figures 23G and 23H). The physicochemical properties of the recombinant bispecific antibody protein are shown in Table 48. Antigen-antibody reactions were detected using ELISA, and bispecific antibody data are also provided in Table 47 in Example 7. The ELISA experimental 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 reactivity with each of its corresponding target peptide fragments and recombinant proteins of different subtypes, and each monoclonal antibody also exhibits the same reactivity with 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 immunoglobulins and enhances the immune response to the antigen target. The bispecific monoclonal antibody disclosed in this invention is intended for the treatment of chronic diseases caused by Porphyromonas gingivalis infection, including, but not limited to, periodontal disease, cardiovascular disease, rheumatoid arthritis, oral and gastrointestinal cancer, ulcerative colitis, nervous system diseases, autoimmune encephalomyelitis, lung cancer, abnormal pregnancy, pancreatic cancer, diabetes, 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 offer a more efficient and simple solution for developing new therapies.

[0299] Example 9 Serum antibody diagnostic kit The data disclosed in Example 5 indicate that approximately 40-50% of patients with cardiovascular disease and geriatric disease have high titers of antibodies against the P. gingivalis outer membrane proteins RagB and Cra4S1, as well as unrelated proteins (such as GST recombinant proteins). Related data provide that qualitative and / or quantitative testing and / or measurement of these antibodies can be used to diagnose P. gingivalis infection early, assess the risk of developing chronic diseases associated with P. gingivalis infection, and evaluate the prognosis and treatment effects of the disease.

[0300] The present invention provides a method for diagnosing or evaluating the prognosis and / or therapeutic efficacy of P. gingivalis infection or chronic diseases caused by or associated with P. gingivalis. Specifically, patients can be screened for antibodies against RagB proteins (including Rag-1, RagB-2, RagB-3, and RagB-4), Cra4S1 proteins, and unrelated proteins (including GST and / or other tissue proteins, such as muscle or mucosal proteins). ELISA technology is used to detect the presence or absence of antibodies in individual patient serum. High levels of antibodies, especially high titers, indicate that the patient is in an immune-susceptible state, which increases the risk of developing a particular chronic disease. This screening allows for patient stratification and identification of high-risk individuals, facilitating prompt initiation of treatment.

[0301] Example 10 PgingiVacRD1B11-1C3, a polypeptide vaccine Data from animal experimental models show that most animals are able to self-repair the local damage caused by initial P. gingivalis infection, but repeated infections can gradually worsen the condition and make the local damage more difficult to heal. These phenomena indicate that the host's immune defenses and bacterial invasion enter into a continuous imbalance process of repair and deterioration, and that chronic P. gingivalis infection is usually not completely eliminated.

[0302] For certain diseases, patients need to take antibody drugs for a long period of time and cannot discontinue the treatment. However, a single vaccination could be a more effective, simple, and cost-effective treatment.

[0303] For the treatment of P. gingivalis infection, after a period of passive immunotherapy with specific antibodies, patients can be administered an efficient polypeptide vaccine, which stimulates the production of effective neutralizing antibodies. These antibodies can persist in the patient's serum for a long period of time, thereby preventing the proliferation and spread of the pathogen. The polypeptide vaccine of the present invention is cost-effective and can be safely administered by conventional routes. Furthermore, the vaccine product does not contain full-length proteins, which avoids harmful, allergenic, and irrelevant antigen fragments, thereby reducing the risk of heterophilic antigens producing heterophilic antibodies.

[0304] The present invention discloses a polypeptide vaccine, PgingiVacRD1B11-1C3, which is an RD21 to RD22 polypeptide fragment based on RagB-4-1B11-4-4 (1B11) and / or RagB-4-1C3-7-8 (1C3) antibody responses. Both ends of the polypeptide can be optimized appropriately. The polypeptide of the PgingiVacRD1B11-1C3 vaccine can be synthesized, or recombinant nucleotide sequences of the polypeptide can be used to construct an expression plasmid and express it under host cell-inducing conditions. Proteins expressed in host cells can be purified using conventional methods. The PgingiVacRD1B11-1C3 polypeptide consists of 75 amino acids, and its amino acid sequence is set forth in SEQ ID NO: 127.

[0305] While the present invention presents and describes various ways of carrying out the invention, those skilled in the art will recognize that these embodiments are provided by way of example only. The present invention is not limited to the specific examples provided in the specification. While the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Those skilled in the art will appreciate that certain changes, modifications, and substitutions may be made without departing from the principles of the present invention. Therefore, all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, and it should be understood that these descriptions, configurations, or relative proportions depend upon various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. Therefore, the present invention is intended to encompass all such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended to cover methods and structures within the scope of these claims and their equivalents.

Claims

1. Monoclonal antibodies that target one or more outer membrane proteins of P. gingivalis include: (i) a light chain variable region (VL) having the amino acid sequence set forth in the Sequence Listing, SEQ ID NO: 154, comprising (a) the amino acid sequences of SEQ ID NO: 155 as CDR-L1, (b) SEQ ID NO: 156 as CDR-L2, and (c) SEQ ID NO: 157 as CDR-L3; and a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 149, comprising (a) the amino acid sequences of SEQ ID NO: 150 as CDR-H1, (b) SEQ ID NO: 151 as CDR-H2, and (c) SEQ ID NO: 152 as CDR-H3; (ii) a light chain variable region (VL) having the amino acid sequence set forth in Sequence Listing SEQ ID NO: 164, comprising the amino acid sequences of (a) SEQ ID NO: 165 as CDR-L1, (b) SEQ ID NO: 166 as CDR-L2, and (c) SEQ ID NO: 167 as CDR-L3; and a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 159, comprising the amino acid sequences of (a) SEQ ID NO: 160 as CDR-H1, (b) SEQ ID NO: 161 as CDR-H2, and (c) SEQ ID NO: 162 as CDR-H3; (iii) a light chain variable region (VL) having the amino acid sequence set forth in Sequence Listing SEQ ID NO: 144, comprising the amino acid sequences of (a) SEQ ID NO: 145 as CDR-L1, (b) SEQ ID NO: 146 as CDR-L2, and (c) SEQ ID NO: 147 as CDR-L3; and a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 139, comprising the amino acid sequences of (a) SEQ ID NO: 140 as CDR-H1, (b) SEQ ID NO: 141 as CDR-H2, and (c) SEQ ID NO: 142 as CDR-H3; (iv) A light chain variable region (VL) having the amino acid sequence set forth in Sequence Listing SEQ ID NO: 134, which comprises the amino acid sequences of (a) SEQ ID NO: 135 as CDR-L1, (b) SEQ ID NO: 136 as CDR-L2, and (c) SEQ ID NO: 137 as CDR-L3, and a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 129, which comprises the amino acid sequences of (a) SEQ ID NO: 130 as CDR-H1, (b) SEQ ID NO: 131 as CDR-H2, and (c) SEQ ID NO: 132 as CDR-H3.

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

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

4. 4. The monoclonal antibody of claim 3, wherein the bispecific antibody comprises: A light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO:174, wherein: (a) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:165; (b) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:166; and (c) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:

167. A mixed chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO:172, wherein: (a) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:160; (b) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:161; and (c) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:162; (d) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:150; (e) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:151; (f) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:152; (g) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:155; (h) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:156; and (i) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:

157.

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

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

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

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

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

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

11. A method for treating a patient with a Porphyromonas gingivalis infection or a chronic disease caused by a Porphyromonas gingivalis infection, the method comprising administering to the subject an effective amount of the cells or cell line described in claim 7.

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

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

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

15. A method for treating a patient with a Porphyromonas gingivalis infection or a chronic disease caused by a Porphyromonas gingivalis infection, the method comprising administering to the subject an effective amount of the composition of any one of claims 12 to 14.

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

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

18. An antigen of a Porphyromonas gingivalis outer membrane protein, which reacts or cross-reacts with one or more monoclonal antibodies according to any one of claims 1 to 4.

19. The antigen of claim 18, wherein the antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 11-127.

20. 20. A vaccine comprising one or more antigens according to claim 18 or 19.

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

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

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

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

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

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

27. An animal model for evaluating Porphyromonas gingivalis infection, therapeutic efficacy, and prognosis, wherein an animal is infected with Porphyromonas gingivalis, and the monoclonal antibody, antibody-antigen reaction, or cross-reaction according to any one of claims 1 to 3 is used to evaluate the effects on the reproduction of the animal.

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

29. (a) an antigenic reagent according to claim 18 or 19, and (b) A kit for the diagnosis and prognosis of infection by P. gingivalis, including instructions for using the reagent to test for specific antibodies in a biological sample.

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

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