Ofloxacin or its derivatives and pd-1 monoclonal antibody conjugates and uses thereof

Abstract

The application discloses an ofloxacin or derivative thereof and PD-1 monoclonal antibody conjugate and application thereof, and comprises a tumor targeting polypeptide, a non-cleavable linker, a cleavable linker, ofloxacin or a derivative thereof and a PD-1 monoclonal antibody; the C terminal of the tumor targeting polypeptide is connected with the ofloxacin or derivative thereof through the non-cleavable linker; and the N terminal of the tumor targeting polypeptide is connected with the PD-1 monoclonal antibody through the cleavable linker. The application can not only significantly improve the targeting property of ofloxacin, PD-1 monoclonal antibody and other tumor treatment drugs, reduce systemic toxic side effects, but also improve the tumor immune microenvironment from multiple aspects, improve the immunotherapy effect, and play a role of reducing the amount, increasing the efficiency and reducing the toxicity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an ofloxacin or its derivative conjugate with a PD-1 monoclonal antibody and its application. Background Technology

[0002] Peptide-drug conjugates (PDCs) are a novel type of drug conjugate that can serve as a platform for cancer therapy. They primarily consist of a peptide, a linker, and a payload, similar to antibody-drug conjugates (ADCs), selectively delivering drugs to tumors. By targeting the peptide to bind to tumor-specific receptors, cell selectivity is improved, and therapeutic efficacy is enhanced. Compared to the monoclonal antibodies used in ADCs, PDC peptides are easier to synthesize and purify, have lower costs, exhibit lower immunogenicity, and are rapidly cleared by the kidneys, reducing toxicity. However, the development of novel PDCs remains limited due to the lack of target receptors.

[0003] Tumor cells and their secreted exosomes can express PD-L1, which binds to PD-1 on the surface of T cells, inhibiting the recognition and killing of tumor cells by T cells. In recent years, tumor immunotherapy based on this mechanism has become a research hotspot and achieved significant breakthroughs. Studies on anti-PD-1 / PD-L1 therapy have been shown to have universal applicability to various tumors. However, the generalizability of responses to this therapy is not ideal, with only 10-30% of cancer patients benefiting. Furthermore, due to individual differences among cancer patients and the complexity of tumors themselves, many patients develop resistance after ICB treatment. Therefore, improving immunotherapy remains a hot topic and a challenge in this field.

[0004] Therefore, given the bottlenecks and shortcomings of existing technologies, it is necessary to develop a drug that can provide safe and effective immunotherapy for various malignant tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a conjugate of ofloxacin or its derivatives with a PD-1 monoclonal antibody and its application. This conjugate combines a targeting peptide with ofloxacin and the PD-1 monoclonal antibody. Through this design, the drug can actively recognize and precisely target tumor cells, significantly increasing the drug concentration within tumor tissue while effectively reducing non-specific distribution in normal tissues. This achieves precision and efficiency in targeted therapy, thereby improving the efficacy of immunotherapy and reducing systemic toxicity.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] In a first aspect of the invention, a conjugate of ofloxacin or a derivative thereof with a PD-1 monoclonal antibody is provided, comprising:

[0008] The tumor-targeting peptide, an insoluble linker, an insoluble linker, ofloxacin or a derivative thereof, and a PD-1 monoclonal antibody, wherein the C-terminus of the tumor-targeting peptide is linked to ofloxacin or a derivative thereof via the insoluble linker, and the N-terminus of the tumor-targeting peptide is linked to the PD-1 monoclonal antibody via the insoluble linker.

[0009] Furthermore, the ofloxacin or its derivatives include one of ofloxacin, a first derivative, and a second derivative;

[0010] The first derivative includes quinolone drugs such as norfloxacin, levofloxacin, pefloxacin, enoxacin, ciprofloxacin, lomefloxacin, guapafloxacin, moxifloxacin, trovafloxacin, clinfloxacin, sparfloxacin, and nemonoxacin malate, as well as levofloxacin hydrochloride, levofloxacin lactate, levofloxacin sulfate, levofloxacin maleate, levofloxacin propenone derivative, levofloxacin aldehyde thiourea derivative, ofloxacin α,β-unsaturated ketone derivative, levofloxacin aldehyde 4-arylaminothiourea derivative, bis-fluoroquinolone thiazolidinyl levofloxacin derivative, and ofloxacin amide derivative;

[0011] The second derivative includes ofloxacin or a derivative obtained by combining the fluoroquinolone skeleton of the first derivative with an α,β-unsaturated ketone or amide group.

[0012] Preferably, the ofloxacin or its derivatives are selected from compounds containing the typical structures of formula (I) or (II):

[0013]

[0014] Furthermore, the tumor-targeting peptide is selected from one of cell-penetrating peptides (CPPs) or cell-targeting peptides (CTPs). Cell-penetrating peptides are typically selected from Pep-1, Pentratin, PepFact14, and Transportan; cell-targeting peptides are typically selected from PEGA, somatostatin analogs, dinoflagellin analogs, RGD peptides, etc.

[0015] Furthermore, the non-cleavable linkers are not triggered to break by chemical or enzymatic stimulation. The mechanism of action of the non-cleavable linkers is primarily the metabolism of the polypeptide / protein, leaving behind the payload—the linker fragment—which can escape from the endosome / lysosome to kill tumor cells. The cleavage of the cleavable linkers can be enzymatically or chemically mediated, such as by changes in pH. These linkers are characterized by their stability in blood circulation to prevent premature and nonspecific drug release.

[0016] Preferably, the non-cleavable linker is selected from linkers containing oxime bonds or thioether bonds, and the cleavable linker is selected from pH-sensitive (containing hydrazine bonds or acetaldehyde bonds), redox-sensitive (containing glutathione), or enzyme-sensitive (specific peptide chain) linkers.

[0017] Furthermore, the PD-1 monoclonal antibody is selected from one of pembrolizumab, nivolumab, sintilimab, toripalimab, and camrelizumab; the Fc segment of the PD-1 monoclonal antibody is conjugated to the polypeptide of the cleavable linker.

[0018] In a second aspect of the invention, a pharmaceutical composition is provided comprising the ofloxacin or a derivative thereof conjugate with a PD-1 monoclonal antibody, and medically acceptable excipients.

[0019] In a third aspect of the invention, the use of the said ofloxacin or its derivatives conjugates with PD-1 monoclonal antibodies or the said pharmaceutical composition in the preparation of antitumor drugs is provided.

[0020] Furthermore, the tumor in the antitumor drug includes at least one of colon cancer, oral squamous cell carcinoma, and malignant melanoma.

[0021] Furthermore, the dosage form of the antitumor drug includes at least one of granules, tablets, pills, capsules, injections, and dispersants.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] 1. This invention provides a conjugate of ofloxacin or its derivatives with a PD-1 monoclonal antibody and its application. The conjugate combines a targeting peptide with ofloxacin and a PD-1 monoclonal antibody. Through this design, the drug can actively recognize and precisely target tumor cells, significantly increasing the drug concentration in tumor tissues while effectively reducing non-specific distribution in normal tissues. This achieves precision and efficiency in targeted therapy, thereby improving the efficacy of immunotherapy and reducing systemic toxicity.

[0024] 2. The ofloxacin derivative and PD-1 monoclonal antibody peptide conjugate has a linker that is sensitive to the tumor microenvironment and can cleave in the tumor microenvironment, increasing the concentration of PD-1 monoclonal antibody in the tumor. PD-1 monoclonal antibody can bind to PD-1 on the surface of T cells in the tumor and prevent it from binding to PD-L1 expressed on the tumor surface and tumor-derived exosomes, thus enabling it to recognize and kill tumors more efficiently.

[0025] 3. The ofloxacin derivative conjugate with PD1 monoclonal antibody peptide allows ofloxacin to be internalized by tumor cells, thereby exerting a more efficient anti-tumor effect. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a structural design diagram of the ofloxacin derivative and PD-1 monoclonal antibody peptide conjugate described in this invention;

[0028] Figure 2 This is a schematic diagram of the decomposition of the ofloxacin derivative and PD-1 monoclonal antibody peptide conjugate described in this invention;

[0029] Figure 3 The number of exosomes released by oral squamous cell carcinoma cells after treatment with the ofloxacin derivative and PD-1 monoclonal antibody polypeptide conjugate described in this invention.

[0030] Figure 4 The percentage of cell survival after oral squamous cell carcinoma cells were treated with the ofloxacin derivative and PD-1 monoclonal antibody peptide conjugate described in this invention.

[0031] Figure 5 This invention demonstrates the inhibitory effect of the ofloxacin derivative and PD-1 monoclonal antibody peptide conjugate on the growth of oral squamous cell carcinoma in mice.

[0032] Figure 6 The effect of the ofloxacin derivative and PD-1 monoclonal antibody peptide conjugate described in this invention on the survival of mice with oral squamous cell carcinoma. Detailed Implementation

[0033] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0034] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0035] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0036] The following will provide a detailed description of an ofloxacin or its derivative conjugate with a PD-1 monoclonal antibody and its application, in conjunction with embodiments and experimental data. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the technical methods or conditions described in literature, standards, or other technical materials in the field, or according to the product instructions. Unless otherwise specified, the methods described in this section are conventional methods known in the art, and the consumables and reagents used are commercially available unless otherwise specified.

[0037] The overall concept of this invention is as follows;

[0038] Ofloxacin is a fluoroquinolone antibiotic. Its fluoroquinolone compounds can achieve antitumor activity through poisoning by type II human DNA topoisomerase.

[0039] The inventors of this application have discovered that ofloxacin can inhibit the expression of tumor PD-L1 by inhibiting signal transduction linker molecule 2 (STAM2) and can reduce the secretion of tumor cell exosomes. However, the use of existing ofloxacin does not have cell selectivity, which often leads to an increase in systemic adverse reactions.

[0040] Therefore, ofloxacin-PD-1 monoclonal antibody-targeting peptide-drug conjugates hold an attractive prospect for achieving safe and effective immunotherapy against various malignant tumors.

[0041] According to a typical embodiment of the present invention, a conjugate of ofloxacin or a derivative thereof with a PD-1 monoclonal antibody is provided, comprising:

[0042] The tumor-targeting peptide, an insoluble linker, an insoluble linker, ofloxacin or a derivative thereof, and a PD-1 monoclonal antibody, wherein the C-terminus of the tumor-targeting peptide is linked to ofloxacin or a derivative thereof via the insoluble linker, and the N-terminus of the tumor-targeting peptide is linked to the PD-1 monoclonal antibody via the insoluble linker.

[0043] This application utilizes a linker peptide to conjugate ofloxacin to a PD-1 monoclonal antibody. The linker peptide comprises a tumor microenvironment-sensitive linker and a tumor-targeting peptide. The conjugate targets the tumor site, and the linker cleaves in response to the tumor microenvironment, releasing the two conjugated ofloxacin and PD-1 monoclonal antibody. These target the tumor cells and immune cells respectively. On one hand, ofloxacin inhibits PD-L1 membrane stability and exosome secretion on tumor cells; on the other hand, the PD-1 monoclonal antibody binds to the PD-1 protein on the surface of immune cells, further blocking immunosuppressive signals and reactivating the tumor-killing function of the immune system. Ofloxacin or its derivatives can disrupt intracellular PD-L1, and the PD-1 monoclonal antibody can target PD-L1 on the cell membrane, thus producing an unexpected synergistic effect.

[0044] The key points for preparing an ofloxacin or its derivative conjugate with a PD-1 monoclonal antibody according to this application are as follows:

[0045] (1) Ofloxacin derivatives: They are characterized by having effects comparable to ofloxacin, and their molecules contain the functional active structure of fluoroquinolone drugs, possessing both antibacterial and antitumor activities.

[0046] (2) Peptide: The targeting portion of ofloxacin derivatives and PD-1 monoclonal antibody conjugates can specifically recognize and bind to receptors or markers on the surface of tumor cells, thereby guiding the conjugates to the tumor microenvironment. It has advantages such as small molecular weight, low immunogenicity, and ease of synthesis and modification.

[0047] (3) Linker: In the ofloxacin derivative and PD-1 monoclonal antibody conjugate, the C-terminal peptide is linked to the ofloxacin derivative, and the ofloxacin derivative is precisely delivered into the tumor cells to exert its drug effect using an incletable linker; the N-terminal peptide is linked to the PD-1 monoclonal antibody, and the cleavable linker is used to break it in the tumor microenvironment (pH-sensitive, enzyme-sensitive, reduction-sensitive and temperature-sensitive), so as to effectively release the ofloxacin derivative and PD-1 monoclonal antibody at specific sites and ensure drug concentration.

[0048] (4) PD-1 monoclonal antibody: The drug portion that protects immune cells in the tumor microenvironment, which can be a selected chemotherapeutic drug, radionuclide, or other active molecule. Through a peptide conjugate delivery system, PD-1 monoclonal antibody can accumulate at high concentrations in the tumor microenvironment, protecting immune cells from the aggression of tumor cells, thereby enhancing the therapeutic effect.

[0049] Through the above methods, this invention can not only significantly improve the targeting of tumor treatment drugs such as ofloxacin and PD-1 monoclonal antibodies and reduce systemic toxic side effects, but also improve the tumor immune microenvironment from multiple aspects, enhance the efficacy of immunotherapy, and play a role in reducing dosage, increasing efficacy, and reducing toxicity.

[0050] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those well known to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may be used in the implementation of the related technologies involved in this invention.

[0051] Example 1:

[0052] This embodiment presents a structural design for an ofloxacin derivative and a PD-1 monoclonal antibody peptide conjugate, as follows: Figure 1As shown, it mainly includes four parts: (1) ofloxacin derivative with the functional active structure of fluoroquinolone drugs (with STAM2 functional domain recognition characteristics); (2) a polypeptide with tumor cell targeting (C-terminus-AEQNPIYWARYADWLFTTPLLLLDLALLADADEGT-N-terminus), SEQ ID NO.1; (3) a non-cleavable linker with an oxime bond (specific structural formula is C=NOH); (4) a cleavable linker with tumor microenvironment enzyme sensitivity (Gly-Phe-Leu-Gly), SEQ ID NO.2; (5) PD-1 monoclonal antibody.

[0053] In the above technical solution, the tumor-targeting polypeptide has a certain directionality when penetrating the cell membrane. It enters the cytoplasm through the bilayer via the C-terminus. The C-terminus is linked to an ofloxacin derivative via a non-cleavable linker, which transports it into the tumor cell. Through polypeptide degradation, the ofloxacin derivative is efficiently released into the tumor cell. After the targeting polypeptide penetrates the membrane, the N-terminus remains on the extracellular side, i.e., in the tumor microenvironment. Under the catalysis of enzymes, the cleavable linker Gly-Phe-Leu-Gly linker breaks, effectively releasing the PD-1 antibody targeted by immune cells.

[0054] The specific preparation method is as follows: The target peptide is mainly synthesized using solid-phase peptide synthesis (SPPS), by gradually adding amino acid residues to a solid support to form a target peptide chain. During the synthesis process, reaction conditions such as temperature, pH, and solvent need to be controlled to ensure the correct folding and functionality of the peptide chain. Linkers are connected to specific positions of the target peptide to form a target peptide-linker complex. Ofloxacin or its derivatives are coupled to the non-cleavable linker end, and PD-1 monoclonal antibody is coupled to the cleavable linker end to form a complete ofloxacin derivative and PD-1 monoclonal antibody peptide conjugate.

[0055] Ofloxacin derivative-PD-1 monoclonal antibody peptide conjugates primarily target and deliver ofloxacin derivatives and PD-1 monoclonal antibodies into tumor cells via targeted peptide conjugation. The targeted peptide and non-cleavable linker precisely deliver the ofloxacin derivative into tumor cells. Within the tumor microenvironment, the cleavable linker breaks, releasing the PD-1 monoclonal antibody targeted by immune cells. Figure 2 This method increases the drug concentration within the tumor and reduces the concentration in other normal sites, thereby improving the efficacy of immunotherapy and reducing systemic toxicity.

[0056] Example 2

[0057] The effects of ofloxacin derivatives obtained in Example 1 on PD-1 monoclonal antibody peptide conjugate on exosome release and tumor cell killing in oral squamous cell carcinoma cells were analyzed.

[0058] 1. The inhibitory effect of the conjugate on the release of exosomes from oral squamous cell carcinoma cells.

[0059] (1) The concentration of the conjugate was investigated using cell viability as an auxiliary detection indicator. On the one hand, the conjugate can inhibit oral squamous cell carcinoma CAL27 cells; on the other hand, the conjugate does not have a toxic effect on normal cells.

[0060] (2) The optimal concentration of the conjugate (preferred concentration range is 1 nM-100 nM, the specific concentration in this case is 20 nM) was added to CAL27 cells cultured in vitro and treated for 12 hours. The number of exosomes derived from CAL27 cells before and after the conjugate treatment was determined by transmission electron microscopy (TEM) and nanoparticle tracking NTA method. Figure 3 The results showed that the conjugate could significantly inhibit the release of exosomes from oral squamous cell carcinoma CAL27 cells.

[0061] 2. Killing effect of the first linker of the conjugate on oral squamous cell carcinoma cells.

[0062] The optimal concentration of the conjugate was added to normal oral mucosal cells and oral squamous cell carcinoma CAL27 cells cultured in vitro, and the cells were treated for 12 hours. Flow cytometry analysis was performed on the cells before and after conjugate treatment using the reactive dye Zombie. Figure 4 The results showed that the conjugate had a certain selective killing effect on oral squamous cell carcinoma CAL27 cells.

[0063] 3. The effect of ofloxacin derivative obtained in Example 1 on the growth of oral squamous cell carcinoma in mice was analyzed.

[0064] (1) A spontaneous oral tumor model was established in C57 mice using the chemical inducer 4NQO. After 16 weeks of continuous feeding with 4NQO, obvious tumor formation was observed in the oral mucosa of the mice. The mice that were successfully induced were randomly divided into groups and then treated with drugs.

[0065] (2) Mice with oral squamous cell carcinoma were treated with ofloxacin alone, PD-1 monoclonal antibody alone, and the conjugate of Example 1 of this invention, respectively. Mice were administered ofloxacin or the conjugate (1 mg per mouse) by gavage daily for a total of 22 times; anti-PD-1 monoclonal antibody (200 μg per mouse) was injected intraperitoneally every three days for a total of 4 injections. (3) The changes and size of the white lesions on the tongue of the mice were photographed and recorded.

[0066] The results are as follows Figure 5 As shown, PD-1 monoclonal antibody therapy alone cannot inhibit tumor growth, while conjugate therapy significantly reduces tumor growth and tumor burden. Figure 5 Compared to PD-1 monoclonal antibodies and ofloxacin alone, the conjugates in this application have a synergistic effect.

[0067] 4. Analysis of the effect of the ofloxacin derivative obtained in Example 1 on the survival of mice with oral squamous cell carcinoma. After treating mice with oral squamous cell carcinoma according to steps 1) and 2) of Example 3, the survival time of the mice was recorded.

[0068] Statistical results are as follows Figure 6 As shown, compared with PD-1 monoclonal antibody alone and ofloxacin alone, the ofloxacin derivative of this application combined with PD-1 monoclonal antibody peptide conjugate significantly improved the overall survival rate of mice with oral squamous cell carcinoma.

[0069] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0070] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0071] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An ofloxacin-PD-1 monoclonal antibody conjugate, characterized in that, include: The invention comprises a tumor-targeting polypeptide, a non-cleavable linker, a cleavable linker, ofloxacin, and a PD-1 monoclonal antibody. The C-terminus of the tumor-targeting polypeptide is linked to ofloxacin via the non-cleavable linker, and the N-terminus of the tumor-targeting polypeptide is linked to the PD-1 monoclonal antibody via the cleavable linker. The non-cleavable linker is an oxime bond (C=NOH), and the cleavable linker is an enzyme-sensitive peptide chain. The amino acid sequence is shown in SEQ ID NO.

2. The amino acid sequence of the tumor-targeting polypeptide is shown in SEQ ID NO.1; The PD-1 monoclonal antibody is selected from one of pembrolizumab, nivolumab, sintilimab, toripalimab, and camrelizumab; the Fc segment of the PD-1 monoclonal antibody is conjugated to the polypeptide of the cleavable linker.

2. A pharmaceutical composition, characterized in that, It includes the ofloxacin and PD-1 monoclonal antibody conjugate as described in claim 1, and medically acceptable excipients.

3. The use of the ofloxacin-PD-1 monoclonal antibody conjugate of claim 1 or the pharmaceutical composition of claim 2 in the preparation of an antitumor drug, wherein the tumor in the antitumor drug is oral squamous cell carcinoma.

4. The application according to claim 3, characterized in that, The dosage forms of the antitumor drugs include at least one of granules, tablets, pills, capsules, injections, and dispersants.

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