CN121513160A - Application of Fusobacterium-specific antimicrobial peptides in the preparation of drugs that enhance the efficacy of antitumor immunotherapy - Google Patents

Abstract

The invention discloses application of a fusobacterium specific antibacterial peptide in preparation of a medicament for improving anti-tumor immunotherapy effect of a PD-1 antibody, wherein the fusobacterium specific antibacterial peptide and the PD-1 antibody are found to have a remarkable effect of inhibiting tumor growth. The invention expands the application range of the specific antibacterial peptide of the fusobacterium, and has important significance for developing novel antitumor drug compositions and establishing novel high-efficiency immunotherapy methods.

Description

Application of fusobacterium specific antibacterial peptide in preparation of medicine for promoting anti-tumor immunotherapy efficacy

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of fusobacterium specific antibacterial peptide in preparation of a medicine for promoting anti-tumor immunotherapy efficacy.

Background

The fusobacterium specific antibacterial peptide is a class of antibacterial peptide which specifically kills bacteria of the phylum fusobacterium and has weak inhibition on other bacteria. CN114989254B discloses a polypeptide P8 having an inhibitory effect on fusobacterium nucleatum (Fusobacterium nucleatum, abbreviated as f.nucleatum), and oral administration of the polypeptide P8 to mice shows a preventive effect on colorectal cancer. However, the therapeutic effect of fusobacterium-specific antibacterial peptides is unknown for colorectal cancer that has developed.

In recent years, immunotherapy has shown remarkable therapeutic effects in various tumors, and immune checkpoint inhibitors represented by PD-1 (programmed death 1)/PD-L1 (programmed DEATH LIGAND, programmed death molecule ligand 1) antibodies are classified into 20 tumor subtypes, and are widely used in clinical treatment. Monoclonal antibodies to the PD-1 receptor (PD-1 antibodies) on the market include KEYTRUDA ^TM (pembrolizumab (Pembrolizumab)), OPDIVO ^TM (Nivolumab), and the like.

Because of the resistance and resistance of immunotherapy, only a small fraction of tumor patients can currently benefit from immunotherapy. The effective rate of immune checkpoint inhibitors is low, only about 20%. Meanwhile, a part of patients with effective treatment can generate drug resistance in the treatment process and lose the treatment effect. Resistance and drug resistance of immunotherapy severely hamper the use of immunotherapy. Therefore, the use of immune checkpoint inhibitors, such as PD-1 antibodies, in combination with other effective drugs to increase the effective rate of immunotherapy is a common approach in the field of anti-tumor immunotherapy. Therefore, searching for potential effective drugs capable of being combined with PD-1 antibodies to further improve the effective rate of immunotherapy is a current urgent problem to be solved, and is also a consensus in the field of anti-tumor immunotherapy.

Disclosure of Invention

The present invention aims to solve at least one of the above technical problems in the prior art. Therefore, the invention provides a technical scheme capable of effectively improving the anti-tumor immunotherapy efficacy of the PD-1 antibody.

In order to achieve the above object, according to a first aspect of the present invention, there is provided use of a fusobacterium specific antibacterial peptide in the preparation of a medicament for improving the antitumor immunotherapeutic efficacy of a PD-1 antibody.

In some embodiments of the invention, the fusobacterium-specific antibacterial peptide includes a peptide having good specific antibacterial activity against fusobacterium nucleatum (f.nucleic), i.e., the peptide has no or only low antibacterial activity against test bacteria other than f.nucleic.

In some embodiments of the invention, the fusobacterium-specific antibacterial peptide comprises polypeptide P8 disclosed in chinese patent document CN 114989254B. The polypeptide P8 has good specific antibacterial activity against Fusobacterium nucleatum (F.nucleic), namely has no or low antibacterial activity against test bacteria except F.nucleic. The amino acid sequence of the polypeptide P8 is shown as SEQ ID NO. 1.

PD-1 is an inhibitory receptor expressed on activated B cells, T cells and bone marrow cells. In the present invention, a "PD-1 antibody" refers to an antibody or an antigen-binding fragment thereof having a specific binding to a PD-1 receptor, and the PD-1 antibody has a specific inhibitory or antagonistic effect on the PD-1 receptor, which is a PD-1 inhibitor or antagonist. Still further, the PD-1 antibody is a PD-1 antagonist that blocks human PD-L1 from binding to human PD-1 or blocks both human PD-L1 and PD-L2 from binding to human PD-1.

An "antigen binding fragment" refers to an antigen binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen to which a full length antibody binds, e.g., a fragment that retains one or more CDR regions, e.g., three heavy chain CDRs and three light chain CDRs. Examples of antibody binding fragments include, but are not limited to, fab ', F (ab') ₂, and Fv fragments.

In some embodiments of the invention, the PD-1 antibody is a monoclonal antibody.

In some embodiments of the invention, the PD-1 antibody is an antibody that specifically binds to human PD-1.

In some embodiments of the invention, the PD-1 antibody is a commercially available PD-1 antibody, including, but not limited to, nivolumab, pembrolizumab (Pembrolizumab), terlipressin Li Shan antibody (Toripalimab), melittizumab (Sintilimab), carlizumab (Camrelizumab), tirelimumab (Tislelizumab), pe An Puli mab (Penpulimab), saprolimumab (Zimberelimab), s Lu Lishan antibody (Serplulimab), and putirimumab (Pucotenlimab).

Pembrolizumab (also known as palbocavib, pembrolizumab) is a PD-1 inhibitor developed by the company moxadong and comprises the Light Chain (LC) Complementarity Determining Regions (CDRs) LC-CDR1, LC-CDR2 and LC-CDR3 of the amino acid sequences set forth in SEQ ID Nos 2, 3 and 4, and the Heavy Chain (HC) CDRs HC-CDR1, HC-CDR2 and HC-CDR3 of the amino acid sequences set forth in SEQ ID Nos 5, 6 and 7, the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of pembrolizumab are set forth in SEQ ID Nos 8 and 9, respectively, and the amino acid sequences of the heavy chain and the light chain of pembrolizumab are set forth in SEQ ID Nos 10 and 11, respectively. See chinese patent publication CN117279952a for sequence information of pembrolizumab.

Generally, the variable domains of both the heavy and light chains comprise three hypervariable regions, also known as Complementarity Determining Regions (CDRs), which are located within relatively conserved Framework Regions (FR). CDRs are typically aligned by framework regions to enable binding to specific epitopes. In general, from N-terminal to C-terminal, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Amino acid allocation of each domain is generally defined according to Sequences of Proteins of Immunological Interest, kabat et al, national Institutes of Health, bethesda, md., 5 th edition, NIH publication No.91-3242 (1991), kabat (1978) adv. Prot. Chem.32:1-75, kabat et al, (1977) J.biol. Chem.252:6609-6616, chothia et al, (1987) J mol. Biol.196:901-917, or Chothia et al, (1989) Nature 342:878-883.

In some embodiments of the invention, the PD-1 antibodies comprise Light Chain (LC) Complementarity Determining Regions (CDRs) LC-CDR1, LC-CDR2 and LC-CDR3 of the amino acid sequences as set forth in SEQ ID Nos. 2,3 and 4, and Heavy Chain (HC) CDRs HC-CDR1, HC-CDR2 and HC-CDR3 of the amino acid sequences as set forth in SEQ ID Nos. 5,6 and 7.

In some embodiments of the invention, the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the PD-1 antibodies are shown in SEQ ID Nos. 8 and 9, respectively.

In some embodiments of the invention, the amino acid sequences of the heavy and light chains of the PD-1 antibodies are shown in SEQ ID Nos. 10 and 11, respectively.

In some embodiments of the invention, the PD-1 antibody comprises pembrolizumab (Pembrolizumab).

In some embodiments of the invention, the tumor comprises a colorectal tumor.

In a second aspect of the invention, there is provided a pharmaceutical composition for improving the anti-tumor immunotherapeutic efficacy of a PD-1 antibody, comprising a PD-1 antibody and a fusobacterium-specific antibacterial peptide.

In some embodiments of the invention, the fusobacterium-specific antibacterial peptide includes a peptide having good specific antibacterial activity against fusobacterium nucleatum (f.nucleic).

In some embodiments of the invention, the fusobacterium specific antibacterial peptide comprises a polypeptide P8, and the amino acid sequence of the polypeptide P8 is shown in SEQ ID NO. 1.

The definition of "PD-1 antibody" is as described above.

In some embodiments of the invention, the PD-1 antibodies comprise Light Chain (LC) Complementarity Determining Regions (CDRs) LC-CDR1, LC-CDR2 and LC-CDR3 of the amino acid sequences as set forth in SEQ ID Nos. 2,3 and 4, and Heavy Chain (HC) CDRs HC-CDR1, HC-CDR2 and HC-CDR3 of the amino acid sequences as set forth in SEQ ID Nos. 5,6 and 7.

In some embodiments of the invention, the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the PD-1 antibodies are shown in SEQ ID Nos. 8 and 9, respectively.

In some embodiments of the invention, the amino acid sequences of the heavy and light chains of the PD-1 antibodies are shown in SEQ ID Nos. 10 and 11, respectively.

In some embodiments of the invention, the PD-1 antibody comprises pembrolizumab (Pembrolizumab).

In some embodiments of the invention, the tumor comprises a colorectal tumor.

In some embodiments of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Preferably, the auxiliary materials comprise pharmaceutically acceptable solvents, diluents, excipients, carriers, binders, lubricants, suspending agents, coating agents and solubilizers.

The drug may be in various forms, such as liquid, semi-solid, and solid dosage forms. According to another embodiment of the invention, the pharmaceutical forms include liquid solutions (e.g., injectable and infusible solutions), sprays, dispersions or suspensions. Preferably, the medicament is an injectable liquid solution.

In a third aspect of the invention, a kit for improving the efficacy of an anti-tumor immunotherapy is provided, comprising a PD-1 antibody and a fusobacterium-specific antibacterial peptide.

In some embodiments of the invention, the fusobacterium-specific antibacterial peptide includes a peptide having good specific antibacterial activity against fusobacterium nucleatum (f.nucleic).

In some embodiments of the invention, the fusobacterium specific antibacterial peptide comprises a polypeptide P8, and the amino acid sequence of the polypeptide P8 is shown in SEQ ID NO. 1.

The definition of "PD-1 antibody" is as described above.

In some embodiments of the invention, the PD-1 antibodies comprise Light Chain (LC) Complementarity Determining Regions (CDRs) LC-CDR1, LC-CDR2 and LC-CDR3 of the amino acid sequences as set forth in SEQ ID Nos. 2,3 and 4, and Heavy Chain (HC) CDRs HC-CDR1, HC-CDR2 and HC-CDR3 of the amino acid sequences as set forth in SEQ ID Nos. 5,6 and 7.

In some embodiments of the invention, the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the PD-1 antibodies are shown in SEQ ID Nos. 8 and 9, respectively.

In some embodiments of the invention, the amino acid sequences of the heavy and light chains of the PD-1 antibodies are shown in SEQ ID Nos. 10 and 11, respectively.

In some embodiments of the invention, the PD-1 antibody comprises pembrolizumab (Pembrolizumab).

In some embodiments of the invention, the tumor comprises a colorectal tumor.

In a fourth aspect of the present invention, there is provided a method for improving the anti-tumor immunotherapy efficacy of a PD-1 antibody, comprising the step of applying an effective amount of a fusobacterium-specific antibacterial peptide in the anti-tumor immunotherapy of a PD-1 antibody.

Preferably, the effective amount is from 5 to 20mg/kg body weight, and more preferably, the effective amount is about 10mg/kg body weight. The term "effective amount" as used herein refers to an amount of the fusobacterium-specific antibacterial peptide that is administered sufficient to provide the desired effect, as compared to the case where the fusobacterium-specific antibacterial peptide is not administered. The expected effects include a significant increase in anti-tumor immunotherapy efficacy and response rate of PD-1 antibodies, or a significant decrease in tumor volume.

In some embodiments of the invention, the fusobacterium specific antibacterial peptide comprises a polypeptide P8, and the amino acid sequence of the polypeptide P8 is shown in SEQ ID NO. 1.

In some embodiments of the invention, the PD-1 antibody comprises pembrolizumab (Pembrolizumab).

In some embodiments of the invention, the tumor comprises a colorectal tumor.

The beneficial effects of the invention are as follows:

(1) The invention discloses application of the specific antibacterial peptide of the clostridium in preparation of a medicament for improving the anti-tumor immunotherapy effect of the PD-1 antibody for the first time, and expands the application range of the specific antibacterial peptide of the clostridium.

(2) The invention discovers that the combination of the specific antibacterial peptide of the fusobacterium and the PD-1 antibody has obvious effect of inhibiting the growth of tumors, and is beneficial to developing novel antitumor pharmaceutical compositions and establishing a novel high-efficiency immunotherapy method.

Drawings

Fig. 1 shows the therapeutic effect of P <0.05, "n.s." on colorectal cancer in the no treatment group (i.e., mock treatment group), the polypeptide P8 treatment group, the PD-1 antibody treatment group, and the PD-1 antibody + polypeptide P8 treatment group of example 1.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The starting materials, reagents or apparatus used in the examples and comparative examples were either commercially available from conventional sources or may be obtained by prior art methods unless specifically indicated. Unless otherwise indicated, assays or testing methods are routine in the art.

Example 1 treatment of colorectal cancer by polypeptide P8 in combination with PD-1 antibodies

The present invention was administered to 40C 57BL/6 female mice of 3 weeks of age (days of operation indicated by "_√") according to the dosing regimen shown in Table 1 below. Specifically, 3 week old C57BL/6 female mice were given 2.5% dextran sodium sulfate drinking water for 3 days to aid in subsequent f.nucleic colonization. The final experiment was then started and designated Day 1.Day 1 began f.nucleic lavage treatment of mice to simulate the fact that f.nucleic was present in the human gut. The intragastric dose was 10 ⁹ CFU/ml f.nucleic, 200 μl per mouse intragastric, 3 times per week for 3.5 weeks to Day 24.

At week 2.5, day 11, MC38 tumor cells were injected to mimic carcinogenesis at a dose of 2 x 10 ⁵ cells per subcutaneous injection. Mice were then divided into 4 groups of 10 animals each, which were the no treatment group (i.e., mock treatment group), the polypeptide P8 treatment group, the PD-1 antibody treatment group, and the PD-1 antibody + polypeptide P8 treatment group, respectively.

At week 3.5, day 18, the tumor volume stabilized to about 50mm ³, and corresponding treatment was initiated on each of the 4 groups of mice for 2 weeks to Day 31. Wherein the therapeutic dose of the polypeptide P8 treatment group is 10mg/kg each time, the stomach is irrigated 3 times a week, the therapeutic dose of the PD-1 antibody (pembrolizumab Pembrolizumab; selleck China, catalog number A2005) is 10mg/kg, and the injection frequency is 2 times/week through intraperitoneal injection.

TABLE 1 dosing regimen for polypeptide P8 in combination with PD-1 antibody therapy

After the start of treatment (starting from Day 18), tumor volumes were measured every 2 days until the end of the experiment, day 39, and the treatment results are shown in fig. 1.

From the results shown in fig. 1, it can be seen that the tumor volumes of mice in the untreated group and the polypeptide P8 treated group were continuously increased, and the polypeptide P8 treated group did not produce significant therapeutic effects (P > 0.05) compared to the untreated group, indicating that the polypeptide P8 alone had no therapeutic effect on colorectal tumors. The PD-1 antibody treatment group controls the tumor volume to be about 250mm ³, and has remarkable effect of inhibiting tumor growth (p < 0.05) compared with the non-treatment group, but is insufficient for shrinking the tumor, and can only control the tumor within the volume range. Compared with the PD-1 antibody treatment group, the PD-1 antibody+polypeptide P8 treatment group can obviously enhance the treatment effect (P < 0.05) of tumors, reduce the tumor volume to about 50mm ³ at the experimental endpoint Day 39, and show the tendency of curing tumors. From the experimental results, the independent polypeptide P8 has no therapeutic effect on colorectal tumor, however, the polypeptide P8 has a synergistic effect on colorectal tumor immunotherapy of the PD-1 antibody, namely, the combined use of the polypeptide P8 and the PD-1 antibody can enhance the effective rate of the PD-1 antibody on colorectal cancer therapy.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. Application of Fusobacterium specific antibacterial peptide in preparing medicament for improving anti-tumor immunotherapy effect of PD-1 antibody.

2. The use according to claim 1, wherein the fusobacterium specific antibacterial peptide comprises a fusobacterium nucleatum (Fusobacterium nucleatum) specific antibacterial peptide.

3. The use according to claim 1, wherein the amino acid sequence of the fusobacterium specific antibacterial peptide is as shown in seq id No. 1.

4. The use according to claim 1, wherein the PD-1 antibody comprises the Light Chain (LC) Complementarity Determining Regions (CDRs) LC-CDR1, LC-CDR2 and LC-CDR3 of the amino acid sequences as set forth in SEQ ID Nos 2, 3 and 4, and the Heavy Chain (HC) CDRs HC-CDR1, HC-CDR2 and HC-CDR3 of the amino acid sequences as set forth in SEQ ID Nos 5, 6 and 7.

5. The use according to claim 1, wherein the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the PD-1 antibody are shown in SEQ ID nos 8 and 9, respectively.

6. The use of claim 1, wherein the PD-1 antibody comprises pembrolizumab (Pembrolizumab).

7. The use of claim 1, wherein the tumor comprises a colorectal tumor.

8. A pharmaceutical composition for improving the anti-tumor immunotherapeutic efficacy of a PD-1 antibody, the pharmaceutical composition comprising a PD-1 antibody and a fusobacterium-specific antibacterial peptide.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises a pharmaceutically acceptable adjuvant.

10. A kit for improving the efficacy of anti-tumor immunotherapy, comprising a PD-1 antibody and fusobacterium-specific antibacterial peptide.