Patent Application
20230210967
The present application contains a Sequence Listing, which is being submitted via EFS-Web on even date herewith. The Sequence Listing is submitted in a file entitled “SEQLISTING-UIPS001.001APC.txt” which was created on Oct. 21, 2022, and is approximately 45,056 bytes in size, and further updated by a file entitled “2023-02-08 Sequence Listing - UIPS001.001APC.txt” which was created on Feb. 8, 2023, and is approximately 41,205 bytes in size. This Sequence Listing is hereby incorporated by reference.
The present invention relates to a new type of cancer peptide vaccine for the treatment or prevention of cancer. More specifically, the present invention relates to a cancer peptide vaccine that can be specifically delivered to immune cells with which the cancer peptide vaccine is intended to come into contact.
A cancer peptide vaccine has been studied as a new treatment method for cancer. Cancer peptide vaccine therapy is a cancer treatment method that aims to prevent or treat cancer by activating and proliferating a specific immune response against a cancer antigen expressed in cancer cells in the patient’s body, in which an antigen consisting of a peptide that is a part of the cancer antigen protein is administered as a vaccine.
Among such research, research on many cancer antigens, and development of vaccines targeting them have been conducted since the 1990s. Some cancer peptide vaccines have been reported to have an effect of inducing immunity specific to cancer at the laboratory level (Non Patent Literature 1: Fifis T. et al., Vaccine. 2004 Nov 25; 23 (2): 258-66). Further, some cancer vaccines have been reported to have exploratory efficacy at an early stage in clinical trials such as induction of immunity and prolongation of survival period (Non Patent Literature 2: Cancer Sci. 2018 Sep; 109 (9): 2660-2669 and Non Patent Literature 3: Cancer Sci. 2017 Dec; 108 (12): 2430-2437).
However, none of these cancer vaccines have been proved to have sufficient efficacy (therapeutic effect) in a confirmatory clinical trial, and none have been approved yet. One of the reasons why the efficacy has not been obtained is considered to be that the delivery of the vaccines to immune cells or activation of immune cells are insufficient.
It is an object of the present invention to provide a peptide vaccine that are complexed so that the peptide vaccine can be delivered specifically to the surface of specific immune cells. It is another object of the present invention to provide a method for delivering a peptide vaccine specifically to the surface of specific immune cells.
As a result of diligent studies, the inventors of the present invention have shown that the aforementioned objects can be achieved by providing a peptide vaccine combined with an IgG binding peptide that is capable of binding to an IgG that is an antibody against molecules on the surface of specific immune cells (e.g., dendritic cells).
More specifically, the present application provides the following aspects in order to achieve the aforementioned objects:
wherein
wherein
wherein
The peptide vaccine combined with an IgG binding peptide disclosed in the present invention enables the peptide vaccine to be efficiently delivered to the surface of specific immune cells (e.g., dendritic cells) and enhances the activation to improve the effects of the peptide vaccine.
The terms to be used in the present invention will be defined as follows:
According to one embodiment, the present invention discloses an invention relating to a peptide vaccine combined with an IgG binding peptide (
It is known that peptide vaccines, when administered alone, often fail to sufficiently induce immunity. The reason for this is thought to be the inability to deliver the peptide vaccines to the target immune cells. The peptide vaccine in the present invention provides a peptide vaccine combined with an IgG binding peptide in order to use IgG for delivering the peptide vaccine to the target immune cells for the purpose of compensating for such a commonly seen disadvantage of peptide vaccines.
As the IgG binding peptide to be used in the present invention, a known peptide can be used which is capable of binding IgG to the peptide of the peptide vaccine. Examples thereof can include, but not limited to, the IgG binding peptide described in WO2016/186206 (Patent Literature 1) (type 1) and the IgG binding peptide described in WO2018/230257 (Patent Literature 2) (type 2). The present invention can provide a peptide vaccine combined with such an IgG binding peptide. The IgG binding peptide that can be used in one embodiment of the present invention is selected from the group consisting of:
More specifically, the IgG binding peptide that can be used in one embodiment of the present invention is selected from the group consisting of:
Among these, the IgG binding peptide in which Xaa6 is Arg, Lys, or Leu is particularly useful in the present invention, and specific examples of the amino acid sequence of the useful IgG binding peptide can include
The IgG binding peptide of the present invention is preferably intended for binding to human IgG (e.g., any of the IgG subclasses of human IgG1, IgG2, IgG3, and IgG4) or humanized IgG, but can be used for IgG in animals other than human, such as rats, mice, and rabbits.
The method for combining the IgG binding peptide with a peptide vaccine may be any method. It can be produced by a method of combining the IgG binding peptide and the peptide of the peptide vaccine to produce one continuous peptide using a peptide synthesis method such as a conventional liquid-phase synthesis method and a solid-phase synthesis method, peptide synthesis by an automatic peptide synthesizer, and the like, or a method of combining the IgG binding peptide and the peptide of the peptide vaccine synthesized separately via a linker. Alternatively, the peptide may be produced by a gene recombination method using a nucleic acid encoding the peptide of the present invention. For example, the target peptide can be produced by incorporating a DNA encoding the amino acid sequence of one contiguous peptide with the peptide of the IgG binding peptide of the present invention and the peptide of the peptide vaccine into an expression vector and introducing it into the host cells, followed by culture.
In consideration of the ease of synthesis or the like, the combination method is preferably synthesis of the IgG binding peptide and the peptide of the peptide vaccine as one contiguous peptide. In the case of employing such a method, a desired sequence of the peptide vaccine combined with an IgG binding peptide can be designed, and the peptide can be synthesized using a peptide synthesizer (e.g., Symphony (R) X, available from Gyros Protein Technologies AB). The contaminants contained when synthesizing the peptide can be removed, for example, by chromatography such as gel filtration chromatography, ion-exchange column chromatography, affinity chromatography, and reverse phase column chromatography, ammonium sulfate fractionation, ultrafiltration, and the immunoadsorption method, to purify the peptide. For the purification by chromatography, equipment such as high-performance liquid chromatography (HPLC) can be used.
The present invention provides a conjugate in which the peptide vaccine combined with an IgG binding peptide is bound to IgG via an IgG binding peptide moiety (
In the case of using vaccines against pathogens as peptide vaccines and in the case of using cancer vaccines as peptide vaccines, delivery to immune cells such as dendritic cells or B cells is effective. Accordingly, the IgG to be used in the present invention for delivery to such cells can be those known as IgGs against a target substance characteristic to the immune cells which delivers the peptide vaccine, and thus an antibody (clinically developed by J&J, Roche, Seattle Genetics, etc.) against CD40 as a target substance, an antibody (clinically developed by Celldex) against DEC205 as a target substance, an antibody (clinically developed by Ascend Biopharmaceuticals) against DCIR as a target substance, and the like can be used for delivery to dendritic cells.
The IgG binding peptide moiety and the IgG that can be used for forming the conjugate in the present invention can be made by modifying specific amino acid residues of the IgG binding peptide moiety of the present invention by a crosslinking agent and forming a crosslinked structure with the specific amino acid residues of IgG Fc via the crosslinking agent. The method for modifying the IgG binding peptide moiety of the peptide vaccine combined with an IgG binding peptide of the present invention by a crosslinking agent can be performed by peptide synthesis of the peptide vaccine combined with an IgG binding peptide using the amino acid residues modified by the crosslinking agent or synthesis of the peptide vaccine combined with an IgG binding peptide and subsequent modification specific to the side chain of specific amino acid residues in the IgG binding peptide moiety.
The crosslinking agent that can be used for forming the conjugate in the present invention can be appropriately selected by those skilled in the art, and a compound having at least two sites capable of binding to the specific amino acid residues of the IgG binding peptide moiety can be used therefor. Examples thereof can include, but not limited to, a crosslinking agent containing at least one, preferably, two or more succinimidyl groups such as disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS), a crosslinking agent containing at least one, preferably, two or more imide acid moieties such as dimethyl adipimidate dihydrochloride (dimethyl adipimidate-2HCl, DMA), dimethyl pimelimidate dihydrochloride (dimethyl pimelimidate-2HCl, DMP), or dimethyl suberimidate dihydrochloride (dimethyl suberimidate-2HCl, DMS), and a crosslinking agent having an SS bond in addition to the aforementioned functional group such as dimethyl 3,3′-dithio-bis(propionimidate) dihydrochloride (dimethyl 3,3′-dithiobispropionimidate-2HCl, DTBP) or dithiobis(succinimidylpropionate) (dithiobis(succinimidyl propionate), DSP) (WO2016/186206 (Patent Literature 1) and WO2018/230257 (Patent Literature 2)).
The specific amino acid residues by the crosslinking agent can be modified by selecting a combination of a crosslinking agent and specific amino acid residues (WO2016/186206 (Patent Literature 1) and WO2018/230257 (Patent Literature 2)). For example, in the case of using a crosslinking agent containing a succinimidyl group such as DSS or DSG, only the side chain of lysine residues disposed as the specific amino acid residues of the IgG binding peptide moiety can be specifically modified with DSS or DSG by blocking the N-terminal of the IgG binding peptide moiety and subsequent reaction with DSS or DSG, since the crosslinking agent reacts with the amine (ε amino group) in the side chain of lysine residues and the primary amine (alpha amino group) present at the N-terminal of the polypeptide. Such a combination of amino acid residues and a crosslinking agent can be appropriately selected by those skilled in the art. Alternatively, in the case of using DSS or DSG, in a peptide free from lysine residues, the primary amine (alpha amino group) present at the N-terminal of a polypeptide can be specifically modified by unmodifying the alpha amino group.
In the present invention, the peptide vaccine of the present invention in which the IgG binding peptide moiety is modified by a crosslinking agent can form a conjugate by mixing with the IgG, to bind to the IgG. The conditions for the mixing step are not specifically limited, as long as they are conditions in which a crosslinking reaction occurs between the IgG binding peptide moiety of the present invention modified by a crosslinking agent and the IgG. For example, the crosslinking reaction can be performed by mixing the peptide vaccine of the present invention containing the IgG binding peptide moiety modified by a crosslinking agent and the IgG in a suitable buffer at room temperature (e.g., about 15° C. to 30° C.). In the mixing step, an appropriate amount of a catalyst that promotes the crosslinking reaction may be added, as required.
At that time, in order to enhance the binding properties between the IgG binding peptide moiety and the IgG, other reaction conditions in the mixing step may be appropriately adjusted in consideration of the peptide vaccine of the present invention containing the IgG binding peptide moiety modified by a crosslinking agent and the type of the IgG. For example, conditions such as pH 4.5 to 6.5 (e.g., pH 5.0 to 6.0, pH 5.2 to 5.8, pH 5.4 to 5.6, or about pH 5.5) or pH 6.5 to 8.5 (e.g., pH 6.9 to 7.9, pH 7.2 to 7.7, pH 7.3 to 7.5, or about pH 7.4) can be selected as the pH at the time of reaction, the mixing ratio of the peptide vaccine of the present invention containing the IgG binding peptide moiety modified by a crosslinking agent to the IgG can be, for example, 1:1 to 20:1, 2:1 to 20:1, or 5:1 to 10:1, as a molar ratio, and the mixing time (reaction time) can be, for example, 1 minute to 5 hours, 10 minutes to 2 hours, or 15 minutes to 1 hour.
According to another embodiment, the present invention can provide a method for delivering a peptide vaccine specifically to cells having target molecules targeted by the IgG on the cell surface using a conjugate of the peptide vaccine of the present invention combined with the aforementioned IgG binding peptide moiety and the IgG. Immune cells are selected as such desired cells, and the peptide vaccine is specifically delivered to the immune cells, thereby enabling an antibody against the peptide vaccine to be generated efficiently and cell-mediated immunity against the peptide vaccine such as activation of T cells against the peptide vaccine to be induced.
According to still another embodiment, the present invention can provide a vaccine composition for treating or preventing a target disease, comprising a conjugate of the peptide vaccine of the present invention combined with the aforementioned IgG binding peptide moiety and the IgG. The target disease to be treated or prevented by the vaccine composition of the present invention means the target from which the antigen of the peptide vaccine of the present invention is derived, and examples thereof can include infectious diseases against pathogens and cancers. Further, selection of an IgG depending on the immune cells which delivers the peptide vaccine enables an immune response to the immune cells to be induced. Accordingly, the vaccine composition of the present invention can be a vaccine composition for efficiently inducing generation of an antibody against the peptide vaccine or a vaccine composition for inducing cell-mediated immunity against the peptide vaccine such as activation of T cells against the peptide vaccine.
When used for treating a target disease, the vaccine composition of the present invention can comprise a peptide vaccine against the target disease and an IgG depending on the immune cells that induce the immune response intended to be induced in the patient who has developed the target disease. The target disease can be treated in the body of the patient by administering the vaccine composition of the present invention to the patient who has developed the target disease.
When used for preventing the target disease, the vaccine composition of the present invention can comprise a peptide vaccine against the target disease and an IgG depending on the immune cells that induce a preferred immune response intended to be induced against the target disease. An immune response when the cause of the target disease invades or occurs in the body of the subject can be induced in the body by administering the vaccine composition of the present invention to a subject who has not developed the target disease.
Since the delivery of the peptide vaccine of the present invention using an IgG to the immune cells is intended, the vaccine composition of the present invention can be parenterally administered by a route (such as intravenous injection, muscle injection, subcutaneous administration, or intraperitoneal administration) and can be prepared as various formulations in appropriate dosage forms depending on the administration route. The administration method and dosage form can be appropriately selected by those skilled in the art depending on the gender, age, body weight, symptoms, and the like of the patient.
The vaccine composition of the present invention can be formulated according to general knowledge in the art, including pharmaceutically acceptable carriers or additives. For example, when used as an injectable formulation, the conjugate of the present invention, for example, dissolved in a solution containing saline, a buffer, a glucose solution or the like and supplemented with a container adsorption inhibitor such as Tween 80, Tween 20, gelatin, human serum albumin can be used. Alternatively, it may be freeze-dried to form a dosage form to be dissolved for reconstruction before use, and examples of a stabilizer that can be used for freeze-drying include sugar alcohols such as mannitol and glucose and/or saccharides.
Hereinafter, the present invention will be specifically described with reference to examples. The examples shown below do not limit the present invention by any way.
This example was performed to produce an agonist antibody specifically against CD40 as the surface antigen, for the purpose of selecting dendritic cells that induce vaccine-specific T cells as immune cells that deliver the peptide vaccine of the present invention and activating the cells.
The anti-mouse CD40 antibody was produced as a chimeric antibody that fuses a variable region of FGK45 clone known as an anti-mouse CD40 agonist antibody with a constant region of a human antibody. The sequence (heavy chain: AEI27236.1 and light chain: AEI27235.1) published in NCBI (https://www.ncbi.nlm.nih.gov/) was used as the sequence of the variable region of FGK45 clone. A plasmid in which a light chain expression element (an EF-lalpha promoter, a secretion signal, a light-chain variable region, and a light-chain constant region were connected in tandem) and a heavy chain expression element (an EF-lalpha promoter, a secretion signal, a heavy-chain variable region, and a heavy-chain constant region were connected in tandem) were connected to pCI-neo was constructed as an expression vector.
The DNA sequences of the heavy-chain variable region and the light-chain variable region were designed based on the codons optimized for the hamster expression system based on the sequences obtained from the above database. The DNA sequences defining known amino acid sequences of the heavy-chain constant region moiety and the light-chain constant region moiety of a human antibody or the variants thereof can be used as the amino acid sequences of the heavy-chain constant region and the light-chain constant region. In this example, the sequences of a mutant (IgG1-lala) and human IgG kappa having L4A and L5A substitutions in the CH2 region of human IgG1 were used as the amino acid sequences of the heavy-chain constant region moiety and the light-chain constant region moiety, respectively.
ExpiCHO cells (invitrogen A2910002) were used as cells for antibody expression. The cells were cultured using ExpiCHO Expression Medium (Gibco, A2910002), and the antibody expression plasmid was transfected using ExpiCHO™ Expression System (GIbco A29129), followed by culture for 14 days, to collect a culture supernatant. The antibody was obtained by purifying the culture supernatant using a protein A column (MonoSpin ProA, GL Science 7510-11314) .
The purity and binding properties of the purified antibody were confirmed based on SDS-PAGE and binding ability to mouse splenocytes, to confirm that the desired anti-mouse CD40 agonist antibody was obtained.
The anti-human CD40 antibody was produced as a chimeric antibody fusing a variable region of 21.4.1 clone known as an anti-human CD40 agonist antibody and a constant region of a human antibody. The sequence published in a Patent Literature (Japanese Patent No. 4616555) was used as the sequence of 21.4.1 clone.
The production of the vector for the expression of the anti-human CD40 antibody and the expression/purification of the antibody were carried out by the same method as that used for the anti-mouse CD40 antibody described in (1).
The purity and binding properties of the purified antibody were confirmed based on SDS-PAGE and the target activation ability in CD40 forced expressing cells, to confirm that the desired anti-human CD40 agonist antibody was obtained.
This example was performed to produce an antigenic peptide against each of various target substances as an example of the peptide vaccine of the present invention.
As the sequence of each peptide vaccine,
As the IgG binding peptide sequence, a sequence (GPDCAYHRGELVWCTFH [IgG BP (1), (SEQ ID NO: 25)] or GPDCAYHKGELVWCTFH [IgG BP (2), (SEQ ID NO: 26)]) reported in Patent Literature PCT/JP2016/065061, and a newly created sequence (GPDCAWHRGELVWCTFH [IgG BP (3), (SEQ ID NO: 39)] or GPDCAWHLGELVWCTFH [IgG BP (4), (SEQ ID NO: 40)]) were used. In these sequences, the Xaa6 of the peptide described in SEQ ID NO: 4 is Arg, Lys, or Leu. Further, the sequence of an IgG binding peptide with partial mutation (GPDCAYHRGEAAACTFH [IgG BP (NC), SEQ ID NO: 27]) was used as the sequence of the IgG non-binding peptide as a negative control. These IgG binding peptides bind to the IgG antibody in a non-covalent bond to form a conjugate.
As a peptide for forming a conjugate (antibody-peptide vaccine conjugate (non-covalently bonded)) of the peptide vaccine and the antibody by a non-covalent bond, each peptide in which the vaccine peptide was connected in tandem to the N-terminal side of the IgG binding peptide, and two cysteines present in the IgG binding peptide were crosslinked by an S-S bond was synthesized [peptide (3-1) (SEQ ID NO: 30), peptide (7) (SEQ ID NO: 34), and (10) (SEQ ID NO: 37)] . Further, each peptide in which the vaccine peptide was connected in tandem to the N-terminal side of the IgG non-binding peptide ([IgG BP (NC) (SEQ ID NO: 27)]), and two cysteines present in the sequence of the IgG non-binding peptide were crosslinked by an SS bond was synthesized as a control peptide [peptide (4) (SEQ ID NO: 31) and peptide (11) (SEQ ID NO: 38)].
These peptides were produced by chemical synthesis. Further, a DBCO derivative peptide [peptide (6D)] in which a DBCO (Dibenzylcyclooctyne) group was imparted to the N-terminal side of the antigen peptide, and an IgG binding peptide derivative (IgG binding peptide-PEG-N3 [peptide (2P)]) in which a PEG linker and an azido group were imparted to the N-terminal side of the IgG binding peptide, and two cysteines were crosslinked by an SS bond were each produced as a peptide for forming an antibody-peptide vaccine conjugate (covalently bonded).
This example was performed to produce a conjugate of the peptide vaccine and the antibody. For producing the conjugate, a method of non-covalently complexing the antibody and the peptide vaccine and a method of covalently complexing them were used.
A conjugate of the peptide vaccine and the antibody by a non-covalent bond (antibody-peptide vaccine conjugate (non-covalently bonded)) was produced by mixing each peptide vaccine combined with the IgG binding peptide produced in Example 2 (peptide (3-1) (SEQ ID NO: 30), peptide (3-2) (SEQ ID NO: 41), peptide (3-3) (SEQ ID NO: 42), peptide (3-4) (SEQ ID NO: 43), peptide (7) (SEQ ID NO: 34), or peptide (10) (SEQ ID NO: 37)) and the anti-mouse CD40 antibody or the anti-human CD40 antibody produced in Example 1, or a commercially available human PD-L1 antibody (MedChemExpress CAS NO: 1380723-44-3) under conditions at room temperature.
A conjugate of the peptide vaccine and the antibody by a covalent bond (antibody-peptide vaccine conjugate (covalently bonded)) was produced as follows. First, a DBCO derivative peptide [peptide (6D)] and an IgG binding peptide-PEG-N3 [IgG BP (2P)] were prepared to 10 mM and 10.8 mM, respectively, using DMSO, followed by incubation for 2 hours by mixing at room temperature, to produce a fusion peptide of the two peptides (CMV-IgG binding peptide). Then, DSG (glutaric acid disuccinimidyl) dissolved in acetonitrile to a molar ratio of 1.4 times that of the CMV-IgG binding peptide was added thereto, followed by incubation for 4 hours at 50° C. in the presence of pyridine, to produce a CMV-IgG binding peptide having the same sequence as peptide (7) and modified into succinimidyl glutarate.
The peptide was purified using HPLC and then mixed with the anti-human CD40 antibody or the isotype control antibody (human IgG1-lala antibody, autologously prepared) produced in Example 1 at a molar ratio of 10:1, followed by incubation for 3 hours at room temperature. Generation of an antibody-peptide vaccine conjugate (covalently bonded) was confirmed by electrophoresis, and then small molecules were removed by ultrafiltration.
This example was performed for the purpose of examining the affinity for IgG of the H-2Kb-restricted OVA-derived peptide having each of the various IgG binding peptides prepared in Example 3.
Peptide (3-1) (SEQ ID NO: 30), peptide (3-2) (SEQ ID NO: 41), peptide (3-3) (SEQ ID NO: 42), peptide (3-4) (SEQ ID NO: 43), and peptide (4) (negative control, SEQ ID NO: 31) were used as the peptides to be evaluated.
For the H-2Kb restricted OVA-derived peptides having the aforementioned four different IgG binding peptide sequences, the binding activities to the antibody were evaluated. The IgG for which the affinity was evaluated was immobilized to a CM5 sensor chip (GE Health care) using the anti-CD40 antibody created in Example 1.
Specifically, the sample solution of each peptide was prepared by 3-fold dilution series from 3 nM to 243 nM, measured and analyzed with respect to the anti-CD40 antibody immobilized onto the CM5 sensor chip using BIAcore 8K (GE Health care) in the single kinetic mode. The dissociation constant was calculated using Biacore Insight Evaluation software to analyze the observation results.
As a result, it was confirmed that peptide (3-3) had about the same dissociation rate constant (Kd) as peptide (3-1) and had a higher affinity than peptide (3-2). In contrast, it was confirmed that peptide (3-4) had a higher affinity than peptide (3-2) and peptide (3-3). Further, peptide (4) as the negative control had a low affinity for the anti-CD40 antibody, and the dissociation rate constant and the affinity could not be calculated (
This example was performed for the purpose of examining whether or not the antibody-peptide vaccine conjugate (covalently bonded or non-covalently bonded) using each peptide vaccine produced in Example 3 had an ability to induce immunity in vitro.
Peripheral blood mononuclear cells (PBMCs) obtained from healthy volunteers were prepared to 2 X 106 cells/mL using RPMI1640 medium (Thermo 61870-0362) containing 10% serum (BioWest, S4190) and seeded on a 24-well plate (FALCON, 353047) in 500 micro L each, followed by culture under conditions of 37° C. and 5% CO2.
The anti-CD40 antibody and peptide (7) (SEQ ID NO: 34) were respectively adjusted to 20 micro g/mL and 1 micro g/mL using RPMI1640 medium containing 10% serum and 200 U/mL IL-2 (NIPRO, 87890) and then added to the well plate on which the PBMCs had been seeded in 500 micro L each (to final concentrations of 10 micro g/mL and 0.5 micro g/mL, respectively) (group 2).
The antibody-peptide vaccine conjugate (covalently bonded) using the anti-CD40 antibody-CMV peptide was adjusted to 21 micro g/mL using RPMI1640 medium containing the same additives as group 2 above and then added to the well plate on which the PBMCs had been seeded in 500 micro L each (to a final concentration of 10.5 micro g/mL) (group 3).
As controls, the following groups were provided:
LPS (SIGMA, L2762), R848 (Invivogen, vac-r848), and poly I:C (Invivogen, tlrl-pic) were respectively adjusted to 800 ng/mL, 2 mM, and 4 micro g/mL using RPMI1640 medium containing 10% serum and 200 U/mL IL-2 and then added to the well plate on which the PBMCs had been seeded in 330 micro L each.
After culture under conditions of 37° C. and 5% CO2 for 7 days, the cells were collected and stained using APC-CMV-tetramer (MBL, TS-0020-2C), BV421-anti-human CD3 antibody (Biolegend,300434), and FITC-anti-human CD8 antibody (Biolegend, 300906), followed by FACS analysis.
This example was performed for the purpose of examining whether or not the antibody-peptide vaccine conjugate (covalently bonded or non-covalently bonded) using each peptide vaccine produced in Example 3 had an ability to induce immunity in vitro by another method from that in Example 5.
CD14-positive monocytes were purified from PBMCs obtained from healthy volunteers using CD14 MACS beads (Miltenyl Biotec, 130-050-20110). CD14-negative cells were separately cryopreserved using a cell cryopreservation solution (Takara, CB011).
The CD14-positive monocytes purified were prepared to 1 × 106 cells/mL using RPMI1640 medium (Thermo, 61870-036) containing 10% FBS (SIGMA, 172072-500ML), 10 mM HEPES (nacalai, 17557-94), 50 micro M 2-mercapto ethanol (2-ME; nacalai, 21438-82), 6.5 ng/mL IL-4 (peprotech, 200-04), and 100 ng/mL GM-CSF (R&D, 215-GM-500), seeded on a 24-well plate (FALCON, 353047) in 1 mL each, followed by culture under conditions of 37° C. and 5% CO2.
The cells were collected 6 days after the start of culture, adjusted to 6 X 104 cells/mL using RPMI1640 containing 10% FBS (SIGMA, 172072-500 ML), non-essential amino acid (nacalai, 06344-56), 1 mM Sodium Pyruvate (nacalai, 06977-34), 10 mM HEPES (nacalai, 17557-94), and 50 micro M 2ME (nacalai, 21438-82), and seeded on a 96-well U-bottom plate (FALCON, 353077) in 50 micro L each.
The anti-human CD40 antibody and peptide (7) (SEQ ID NO: 34) were respectively adjusted to 40 micro g/mL and 2 micro g/mL using the same medium as used above for culturing the monocytes and then added to the 96 well plate on which the CD14-positive monocytes had been seeded in 50 micro L each (to final concentrations of respectively 10 micro g/mL and 0.5 micro g/mL) (group 2).
The antibody-peptide vaccine conjugate (covalently bonded) by the anti-CD40 antibody-CMV peptide was adjusted to 42 micro g/mL using RPMI1640 medium containing the same additives as group 2 above and then added to the 96 well plate on which the CD14-positive monocytes had been seeded in 50 micro L each (to a final concentration of 10.5 micro g/mL) (group 3).
As controls, the following groups were prepared:
Further, LPS, R848, poly I:C, and IL-2 were respectively adjusted to 800 ng/mL, 2 mM, 4 micro g/mL, and 80 U/mL using the medium and then added in 50 micro L each, in the same manner as in Example 5.
7 days from the start of culture under conditions of 37° C. and 5% CO2, the frozen CD14-negative cells were recovered and purified into CD8-positive cells using CD8 MACS beads (Miltenyl Biotec, 130-045-201). The cells were adjusted to 1.8 × 106 cells/mL using RPMI1640 containing 10% FBS, non-essential amino acid, 1 mM Sodium Pyruvate, 10 mM HEPES and 50 micro M 2ME and added in 50 micro L each, followed by culture at 37° C. The cells were collected 14 days from the start of the test and stained using APC-CMV-tetramer (MBL, TS-0020-2C), BV421-anti-CD3 antibody (Biolegend,300434), and FITC-anti-CD8 antibody (Biolegend, 300906), followed by FACS analysis.
This example was performed for the purpose of examining whether or not the antibody-peptide conjugate using each peptide vaccine produced in Example 3, when subcutaneously administered, was taken up by the dendritic cells (classical dendritic cell ½, (cDC1/cDC2)) present in the lymph nodes in the vicinity of the administration site, in vivo.
In this example, IgG BP (1) peptide (SEQ ID NO: 25) (the entire sequence: K*RRK*RRK*RRGPDCAYHRGELVWCTFH) supplemented with a peptide (K*RRK*RRK*RR) (“K*” indicating FAM-binding lysine) labeled with the fluorescent dye FAM (6-Carboxyfluorescein) was used as a test peptide.
7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) were divided into three groups, with three mice per group, and experiments were performed with the group composition in Table 3. Specifically, to each group, the following was subcutaneously administered to the inguinal region:
The dosage of each ingredient is as shown in Table 3.
Administration was performed by administering a single dose of the drug to each group of mice. The inguinal lymph nodes in the vicinity of the administration site were collected 5 hours later, and the tissue was disrupted to prepare lymph node cells. Thereafter, the cells were stained with the anti-CD11c antibody, the anti-I-A/I-E antibody, the anti-XCR1 antibody, and the anti-CD172a antibody, followed by flow cytometry analysis.
The uptake of fluorescently labeled IgG BP (1) peptide was evaluated based on the proportion of the FAM-positive cells in cDC1 cells (CD11c+/I-A/I-E+/XCR1+/CD172-) and cDC2 cells (CD11c+/I-A/I-E+/XCR1-/CD172+) .
This example was performed for the purpose of examining whether or not the antibody-peptide vaccine conjugate (non-covalently bonded) using each peptide vaccine produced in Example 3 had an ability to induce immunity, in vivo.
7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) were divided into five groups, with six mice per group, and experiments were performed with the group composition in Table 4. Specifically, to each group, the following was administered:
The administration was performed according to the schedule shown in
This example was performed for the purpose of examining the effect of the mixing ratio of the antibody and each peptide vaccine produced in Example 3 in the antibody-peptide vaccine conjugate (non-covalently bonded) using the peptide vaccine on the ability to induce immunity, in vivo.
7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) were divided into groups each with four mice, and experiments were performed with the group composition in Table 5. Specifically, to each group, the following was administered:
The administration was performed according to the schedule shown in
This example was performed for the purpose of examining the dose dependency of the antibody-peptide vaccine conjugate (non-covalently bonded) using each peptide vaccine produced in Example 3, in vivo.
7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) were divided into groups each with four mice, and experiments were performed with the group composition in Table 6. Specifically, groups 1 to 4 had a high dose of the peptide vaccine (10.5 micro g of the peptide vaccine) administered, and groups 5 to 8 had a medium dose of the peptide vaccine (2.1 micro g of the peptide vaccine) administered. Specifically, to each group, the following was administered:
The administration was performed according to the schedule shown in
This example was performed for the purpose of examining whether or not the antibody-peptide vaccine conjugate (non-covalently bonded) produced using each peptide vaccine produced in Example 3 and combined with the anti-CD40 antibody had an anti-tumor effect, in vivo.
E.G7 (ATCC (R)CRL-2113) as a mouse T cell lymphoma cell line forced to express the Ova antigen was subcutaneously transplanted into 7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) at 1 × 106 cells per mouse. The transplantation day was taken as day 0, and the mice were grouped (six mice per group) based on the tumor volume on day 3. Experiments were performed on each group with the group composition in Table 7. Specifically, to each group, the following was subcutaneously administered:
The drug was subcutaneously administered to the mice of each group twice in total on day 3 and day 10, and the tumor volume and the body weight were observed at a frequency of twice a week from the start of the drug administration.
As the survival period of each individual mouse was investigated, it was confirmed that the antibody: Ova-IgG binding peptide group (group 4) showed a more remarkable prolonging effect on the survival period than the Ova-IgG binding peptide group (group 3), the Ova-IgG non-binding peptide group (group 5), and the antibody: Ova-IgG non-binding peptide group (group 6) (
This example was performed for the purpose of evaluating whether or not the antibody-peptide vaccine conjugate (non-covalently bonded) using each peptide vaccine produced in Example 3 had an anti-tumor effect in a test system with higher clinical extrapolation, specifically, a test system employing a peptide sequence determined based on the mutation specific to cancer as a vaccine sequence.
7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) were divided into groups each with four mice, and experiments were performed with the group composition in Table 8. Specifically, to each group, the following was administered:
The administration was performed according to the schedule shown in
The spleen was collected on day 15, then 3 X 106 cells were suspended in 100 micro L of Splenocyte growth medium (RPMI-1640 (Thermo 61870-0362) containing 10% FBS (BioWest, S4190), 1 mM sodium pyruvate (Wako, 190-14881), 0.05 mM 2-ME (Wako, 131-14572), 20 mM HEPES (Wako, 345-06681), and 1% penicillin streptomycin (nakarai, 26253-84)) and seeded, and 100 micro L of a solution in which peptide (8) (SEQ ID NO: 35) was dissolved was added thereto to a final concentration of 10 micro g/mL, followed by culture under conditions of 37° C. and 5% CO2 for 15 hours.
22 micro L of 10 X Brefeldin A (Biolegend 420601) was added, followed by incubation for 3 to 4 hours and subsequent centrifugation at 2000 rpm, and the cells were collected and stained with Zombie Violet (BioLegend 423114), APC anti-mouse CD4 (BioLegend 100412), FITC anti-mouse CD8a (BioLegend 100706), and PE anti-mouse IFN-g (BioLegend 505807), followed by flow cytometry analysis. Immunity activation against Adpgk peptide was evaluated based on the proportion of the IFNgamma-positive cells in CD8-positive cells.
This example was performed for the purpose of examining whether or not the antibody-peptide vaccine conjugate (non-covalently bonded) using each peptide vaccine produced in Example 3 and combined with the CD40 antibody or the PD-L1 antibody had an ability to induce immunity, in vivo.
In this example, 7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) were first divided into four groups with five mice per group, and experiments were performed with the group composition in Table 9. Specifically,
The administration was performed according to the schedule shown in
This example was performed, as in Example 13, for the purpose of examining whether or not the antibody-peptide vaccine conjugate (non-covalently bonded) using each peptide vaccine produced in Example 3 and combined with the CD40 antibody or the PD-L1 antibody, when administered with a peptide vaccine and an antibody that do not form a conjugate, had an ability to promote immunity induction, in vivo.
7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) were divided into five groups, with six mice per group, and experiments were performed with the group composition in Table 10. Specifically,
The administration was performed according to the schedule shown in
This example was performed for the purpose of examining whether or not the antibody-peptide vaccine conjugate (non-covalently bonded) produced using each peptide vaccine produced in Example 3 and combined with an anti-PD-L1 antibody had an anti-tumor effect, in vivo.
E.G7 (ATCC (R)CRL-2113) as a mouse T cell lymphoma cell line forced to express the Ova antigen was subcutaneously transplanted into 7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) at 1 X 106 cells per mouse. The transplantation day was taken as day 0, and the mice were grouped (six mice per group) based on the tumor volume on day 4. Experiments were performed on each group with the group composition in Table 11. Specifically,
The drug was subcutaneously administered to the mice of each group twice in total on day 4 and day 11, and the tumor volume and the body weight were observed at a frequency of twice a week from the start of the drug administration.
As the survival period of each individual mouse was investigated, it was confirmed that the antibody: Ova-IgG binding peptide group (group 4) showed a more remarkable prolonging effect on the survival period than the Ova-IgG binding peptide group (group 2) and the anti-PD-L1 antibody alone group (group 3) (
This example was performed for the purpose of examining whether or not the antibody-peptide vaccine conjugate (non-covalently bonded) containing the non-synonymous somatic mutation sequence of a mouse tumor cell line had an anti-tumor effect, in vivo.
First, peptides each containing a non-synonymous somatic mutation sequence (each 27-mer, 30 peptides of peptide (101) to peptide (130)) were created as the non-synonymous somatic mutation sequence of a mouse tumor cell line, as shown in Table 12 below, based on the analysis results of the genome sequence obtained from the mouse tumor cell line MC-38.
Thereafter, the IgG BP (1) (SEQ ID NO: 25) was added to the aforementioned peptides (27-mer, peptide (101) to peptide (130)), to create peptides (each 44mer, 30 peptides of peptide (101BP) to peptide (130BP)) as shown in Table 13 below.
7 week-old, female, wild-type C57BL/6 mice (CLEA Japan, Inc.) were divided into two groups, and experiments were performed with the group composition in Table 14. Specifically, to two mice per pool, the following was subcutaneously administered:
The administration was performed according to the schedule shown in
The splenocytes and each peptide added were removed by aspiration, the filter plate was further washed, and then 100 micro L of the biotin-labeled mouse IFN-gamma antibody (MabTech) was added thereto, followed by incubation at 37° C. for 2 hours. The filter plate was washed, and 100 micro L of Streptavidin-ALP (MabTech) was added thereto, followed by further incubation for 1 hour. The filter plate was washed again, 100 micro L of a substrate solution (MabTech; BCIP/NBT-plus) was added thereto, and IFNgamma secreted by the cells was detected.
The peptide vaccine combined with an IgG binding peptide disclosed in the present invention enables the peptide vaccine to be efficiently delivered to the surface of specific immune cells (e.g., dendritic cells) and enhance the activation to enhance the effects of the peptide vaccine.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-076785 | Apr 2020 | JP | national |
This application is the U.S. National Phase under 35 U.S.C. § 371 of International Application PCT/JP2021/016133, filed Apr. 21, 2021, designating the U.S., and published in Japanese as WO 2021/215462 on Oct. 28, 2021 which claims priority to Japanese Patent Application No. 2020-076785 filed Apr. 23, 2020, the entire content of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/016133 | 4/21/2021 | WO |
