WO2025248564A1 - Nouvelle plateforme de particules pseudovirales basée sur une bioconjugaison à médiation par l'intéine avec un antigène cible et encapsulée avec des facteurs immunomodulateurs - Google Patents

Nouvelle plateforme de particules pseudovirales basée sur une bioconjugaison à médiation par l'intéine avec un antigène cible et encapsulée avec des facteurs immunomodulateurs

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Publication number
WO2025248564A1
WO2025248564A1 PCT/IN2025/050817 IN2025050817W WO2025248564A1 WO 2025248564 A1 WO2025248564 A1 WO 2025248564A1 IN 2025050817 W IN2025050817 W IN 2025050817W WO 2025248564 A1 WO2025248564 A1 WO 2025248564A1
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vlp
intein
platform
antigen
antigens
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Subhash Singh
Susheel Singh
Shalini Singh
Vandana Singh
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Protextent Biosolutions Private Ltd
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Protextent Biosolutions Private Ltd
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/765—Reovirus; Rotavirus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10141—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10142—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18141—Use of virus, viral particle or viral elements as a vector
    • C12N2795/18142—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule

Definitions

  • the present invention relates to the field of recombinant vaccines, specifically to the design and development of a novel Virus-Like Particle (VLP) platform capable of encapsulating various immunomodulatory factors within its core and the VLP surface can be efficiently bioconjugated with different antigens of choice (target antigens) through specific intein-mediated bioconjugation, and enables site-specific delivery of encapsulated immunomodulatory factors to generate tailored immune response against target antigens.
  • VLP Virus-Like Particle
  • VLPs Virus-like particles
  • HPV human papillomavirus
  • VLP-based vaccines rely on genetic fusion or chemical conjugation techniques to display the target antigens on the VLP surface.
  • Genetic fusion methods involve inserting the gene encoding the target antigen into the capsid protein sequence of the VLP. This approach often disrupts the capsid protein’s ability to self-assemble into VLPs due to structural alterations, necessitating extensive optimization for each specific antigen.
  • GSK’s RTS,S/AS01 Mosquirix
  • the first licensed malaria vaccine took over 20 years of development. A significant portion of this time was spent optimizing the production of chimeric VLPs, as inserting the malaria antigen into the hepatitis B surface antigen capsid protein backbone required complex adjustments to maintain the VLP structure.
  • Such lengthy development timelines and the need for extensive optimization pose substantial barriers to the rapid deployment of vaccines, especially during outbreaks of emerging infectious diseases besides adding to the costs.
  • Chemical conjugation techniques involve chemically linking antigens to the VLP surface. While this method avoids the structural disruptions associated with genetic fusion, it often lacks site specificity, which can result in heterogeneous products with variable immunogenicity. Moreover, chemical conjugation may leave behind undesirable chemical residues, and the harsh conditions required for the reactions can damage sensitive antigens, reducing their immunogenicity. The unpredictability and complexity of these processes increase the cost and time required for vaccine development.
  • VLP-based vaccines primarily focus on eliciting antibody responses.
  • infectious diseases including those caused by intracellular pathogens like viruses and certain bacteria
  • effective protection also requires a robust cellular immune response, particularly the activation of T-cells.
  • Existing VLP platforms often struggle to induce strong T-cell responses, limiting their efficacy against such pathogens.
  • VLPs have shown promise in presenting antigens for cancer immunotherapy, the capacity to modulate immune responses specifically to target tumors or autoimmune conditions remains limited.
  • SpyBiotech employs the SpyCatcher-SpyTag system for antigen conjugation.
  • This technology utilizes a peptide-protein pair that spontaneously forms a covalent bond, allowing antigens to be "plugged” onto the VLP surface. While this method provides a rapid and flexible way to display antigens, it leaves behind a significant residual scar—approximately 120 amino acids—from the conjugation tags. This residual sequence can be immunogenic itself, potentially interfering with the immune response against the target antigen and complicating the vaccine formulation.
  • VLP-based vaccine technologies there is a pressing need for a versatile and efficient platform that can rapidly accommodate a wide array of antigens with minimal optimization.
  • Such an improved VLP platform would significantly reduce the time and cost associated with vaccine development and enable the production of vaccines that elicit comprehensive immune responses, including both humoral and cellular immunity.
  • a VLP platform that allows for the targeted delivery of encapsulated immunomodulatory factors could specifically modulate priming of the immune response through influencing the cross-talk between the Antigen Presenting Cells (APCs) and the T-cells to generate a tailored immune response towards the target antigen, is highly desirable, and could potentially improve the vaccine efficacy against both infectious and non-communicable diseases.
  • APCs Antigen Presenting Cells
  • the present invention aims to provide a novel VLP platform that leverages intein-mediated bioconjugation of the target antigen as a promising solution.
  • Inteins are protein segments that can excise themselves from a host protein sequence and facilitate the ligation of the flanking protein sequences, effectively "splicing" them together.
  • This intein-mediated bioconjugation allows for site-specific and efficient linkage of antigens to VLPs under mild physiological conditions, preserving antigen integrity and VLP assembly. The result is a "plug-and-display" approach that requires minimal optimization for each new antigen, drastically reducing the pre-clinical development phase.
  • VLP platform can prime and enhance the desired immune response against the target antigen. This capability is particularly advantageous for designing vaccines against pathogens that require a balanced immune response, including strong T-cell activation with a possibility of generating long term immune memory against the target antigen, also applicable for conditions like cancer and autoimmune disorders, where modulating immune responses is crucial for therapeutic success.
  • the principal object of the present invention is to provide a VLP platform that utilizes intein-mediated bioconjugation for the efficient, site-specific, and near trace-less attachment of antigens.
  • Another object of the present invention is to enhance the immunogenicity of the VLP-surface bioconjugated antigens, facilitating the rapid and scalable production of vaccines.
  • Another object of the present invention is to provide a VLP platform capable of specific and efficient bioconjugation with a wide variety of antigens, including viral, bacterial, fungal, parasitic, cancer, and autoimmune disease-related antigens, without compromising the structural integrity of the VLP or the antigen.
  • Another object of the present invention is to significantly shorten the pre-clinical development phase of VLP-based vaccines by employing a “plug-and-display” approach using intein-mediated bioconjugation with various antigens of interest (AOI). This reduces the extensive optimization typically required in existing technologies, thus saving time and resources.
  • AOI antigens of interest
  • Yet another object of the present invention is to establish a bioconjugation method that leaves minimal to no residual tags or scars on the final VLP-antigen product, ensuring that the conjugation process does not interfere with the immunogenic properties of the antigen or the functionality of the VLP.
  • Yet another object of the present invention is to encapsulate the VLP core with immunomodulatory (small molecules: metabolites, adjuvants / cytokines) factors capable of modulating the priming and outcome of the adaptive immune response of a specific type (Th1: humoral, Th1: cell-mediated, Tfh, Th17, Th2 or Treg) against the target antigen through influencing the cross-talk between the APCs and the T-cells.
  • immunomodulatory small molecules: metabolites, adjuvants / cytokines
  • Th1 humoral
  • Th1 cell-mediated, Tfh, Th17, Th2 or Treg
  • This invention relates to the field of recombinant vaccines, particularly the development of a novel and versatile VLP platform designed to improve the immunogenicity of a wide range of antigens.
  • This innovative VLP platform employs intein-mediated bioconjugation to efficiently and site-specifically bioconjugate with antigens of choice to the surface of pre-assembled and purified VLPs. The approach enables a “plug-and-display” mode, facilitating the rapid screening and prioritization of vaccine candidates, thereby accelerating the pre-clinical development of vaccines.
  • VLP platform addresses critical limitations in current VLP-based vaccine technologies, such as the extensive optimization required for antigen insertion within capsid proteins, which often disrupts the VLP structure and significantly extends development timelines.
  • Traditional methods like those used in GSK's RTS,S/AS01 malaria vaccine and other dengue vaccines, involve complex processes to ensure proper VLP assembly, often necessitating dilution with wild-type backbones or employing chemical conjugation techniques that leave undesirable remnants on the final product.
  • VLP platform of the present disclosure utilizes intein-based protein bioconjugation, which is highly efficient and nearly trace-less, leaving minimal or no residual amino acids from the conjugation tags. This not only preserves the structural integrity and immunogenicity of the VLP-antigen complex but also significantly reduces the time and cost involved in pre-clinical development. Additionally, this novel VLP platform can be employed for the encapsulation of various immunomodulatory factors, including cytokines, adjuvants, and small molecule metabolites, within the VLP core. This feature allows for precise site-specific delivery of these factors to the APCs, enhancing generation of tailored immune response against the target antigen and thereby improving vaccine efficacy.
  • This novel VLP platform extends to its application across a broad spectrum of infectious diseases where antibodies play a primary protective role. Furthermore, it can be adapted for use in non-infectious diseases, such as cancer and autoimmune disorders, by priming effective modulation of immune response through delivery of the encapsulated immunomodulators for generating specific T-cell responses (Th1: humoral, Th1: cell-mediated, Tfh, Th2, Th17 or Treg).
  • Th1 humoral
  • Th1 cell-mediated, Tfh, Th2, Th17 or Treg
  • This adaptability makes the platform a powerful tool for modulating immune responses, either by enhancing or selectively abrogating specific signaling pathways, for obtaining a tailored immune response effective against the target antigen and as per the therapeutic needs of various cancers and autoimmune disorders.
  • VLP platform of the present disclosure offers several distinct advantages.
  • the SpyCatcher-SpyTag system used by some competitors while similar in its plug-and-display approach, leaves a substantial scar of about 120 amino acids from the conjugation tags, which can potentially interfere with the desired immune response against the target antigen or the functionality of the VLP.
  • the ntein-based method of the present disclosure overcomes this limitation by providing a near scar-free bioconjugation process with trace-less or minimal (few amino acids) carryover from the conjugation-tag sequences, allowing for a cleaner and more efficient conjugation of target antigen with the VLP for vaccine development.
  • Another significant advantage of the novel VLP platform is its ability to encapsulate and deliver immunomodulatory factors (including cytokines, adjuvants, and small molecule metabolites) alongside priming the immune system against the target antigen.
  • immunomodulatory factors including cytokines, adjuvants, and small molecule metabolites
  • This capability provides a dual mechanism of action—one that primes APCs and another that modulates the ensuing immune response towards promoting a tailored, robust and specific immune response against the target antigen for enhancing the vaccine efficacy.
  • This approach not only improves the initial priming of specific immune response but could also support the induction of strong memory of immune responses, crucial for long-term protection.
  • this design of novel and versatile VLP platform of the present disclosure supports the development of combination multivalent vaccines by enabling the presentation of multiple distinct antigens on the VLP surfaces. Batches of VLPs each could be easily bioconjugated with target antigens from multiple strains of a pathogen or a range of related pathogens. The desired combinations of such VLPs could then be co-administered, as well as for creating combination vaccines that can protect against multiple diseases simultaneously.
  • the commercial potential of this invention is significant, given the estimated market size of over USD 20 billion by 2030 for vaccines targeting both infectious and non-infectious diseases.
  • the scalability and versatility of the novel VLP platform makes it suitable for large-scale commercial production, meeting the high demand for effective vaccines in global markets. Additionally, the modularity of this novel VLP platform allows for rapid adaptation and customization, which is particularly valuable in responding to emerging infectious threats or tailoring vaccines to individual patient needs in personalized medicine.
  • this invention offers a transformative approach to VLP-based vaccine development.
  • the novel VLP platform provides a powerful, flexible, and efficient tool for enhancing vaccine efficacy across a wide range of applications.
  • This innovative technology not only addresses the shortcomings of existing methods but also opens new avenues for the development of next-generation vaccines that are faster, more effective, and more adaptable to diverse medical challenges.
  • FIG. 1 shows a schematic representation of the novel VLP platform along with all steps and major components involved therein.
  • Figure 4 shows immunomodulator encapsulation efficiency within the VLP core as a function of the various concentrations of the immunodulator used during the encapsulation process.
  • the word “may” is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must).
  • the words “a” or “an” mean “at least one” and the word “plurality” means “one or more” unless otherwise mentioned.
  • the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as “including,” “comprising,” “having,” “containing,” or “involving,” and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps.
  • compositions or an element or a group of elements are preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.
  • the present invention pertains to a novel and versatile VLP platform designed to enhance the immunogenicity of various antigens for vaccine development.
  • This VLP platform leverages intein-mediated bioconjugation to achieve efficient, site-specific, and trace-less conjugation of target antigens to the surface of pre-assembled and purified VLPs.
  • the VLP core encapsulated with immunomodulatory factors could stimulate generation of tailored, robust and specific type of immune response against the target antigens for enhancing the vaccine efficacy.
  • This invention thus, represents a significant advancement in the field of recombinant vaccines, offering a streamlined, adaptable and scalable approach to developing vaccines with enhanced efficacy against a wide range of infectious and non-infectious diseases.
  • the VLP platform of the present disclosure consists of pre-assembled virus-like particles that serve as a scaffold for antigen presentation and encapsulated immunomodulatory factors within its core.
  • VLPs are non-infectious, virus-mimicking structures that retain the immunogenic properties of viruses, making them ideal candidates for vaccine development.
  • the novel aspect of this platform consists of the following two attributes: i) use of intein-mediated protein conjugation, a bioconjugation method that allows for the precise and efficient attachment of antigens to the VLP surface without leaving undesirable residual tags or scars; and ii) encapsulation of different small molecule immunomodulators (including cytokines, adjuvants, and small molecule metabolites) for specific priming of tailored immune response through influencing the outcome of interactions between the APCs and the T-cells towards eliciting a specific type of immune response (Th1: humoral, Th1: cell mediated, Tfh, Th2, Th17, or Treg).
  • intein-mediated protein conjugation a bioconjugation method that allows for the precise and efficient attachment of antigens to the VLP surface without leaving undesirable residual tags or scars
  • encapsulation of different small molecule immunomodulators including cytokines, adjuvants, and small molecule metabolites
  • the disclosure relates to a VLP platform for vaccine development, comprising a VLP structure capable of encapsulating immunomodulatory factors within its core and presenting antigens on its surface; and an intein-mediated bioconjugation system for the site-specific attachment of a target antigen to the VLP structure.
  • the intein-mediated bioconjugation occurs under mild physiological conditions, preserving the structural integrity of the VLP-backbone and the target antigen.
  • the intein-mediated bioconjugation system comprises of short intein sequences, flanked by the VLP capsid protein, and its cognate intein-partner with the target antigen sequence, enabling self-excision of the intein-tag and its cognate partner, and surface ligation of the VLP capsid protein to the target antigen sequence.
  • Intein-mediated bioconjugation is a protein splicing mechanism that enables the covalent linkage of proteins or peptides under mild physiological conditions.
  • short cognate tags are engineered on both the backbone capsid protein of the VLP and the target antigen. Upon mixing, these tags undergo intein-mediated splicing, resulting in a covalent and site-specific bioconjugation of the target antigen to the VLP. This process is highly efficient and nearly traceless, leaving minimal (a few amino acids) or no residual amino acids from the tag sequences, thereby maintaining the integrity and functionality of both the VLP and the target antigen.
  • This novel VLP platform consists of a unique combination of two key capabilities: 1. Encapsulation of different immunomodulators within the VLP core capable of generating tailored immune response against the antigen of interest (AOI); and 2. Short split intein (In) fusion tag designed to be exposed on the VLP-surface capable of trans-splicing mediated bioconjugation with cognate intein (cIn) fusion tag expressed with AOI.
  • AOI antigen of interest
  • This novel VLP platform could be customized to display variety of antigens from different infectious diseases, cancer and autoimmune disorders, as well as specific priming and generation of tailored immune response against the AOI which expedites pre-clinical vaccine development and rapid selection of best combination of target antigen and immunomodulators with improved vaccine efficacy outcomes.
  • a method of producing a VLP-based vaccine comprising:
  • the method further comprises a step of encapsulating immunomodulatory factors (metabolites, small molecules, pharmacologically active compounds, peptides, proteins, cytokines, hormones, signalling molecules, or adjuvants) within the VLP core to enhance the immune response.
  • immunomodulatory factors metabolic substances, small molecules, pharmacologically active compounds, peptides, proteins, cytokines, hormones, signalling molecules, or adjuvants
  • the intein-mediated bioconjugation is performed such that minimal to no residual amino acids (near trace-less) from the intein-tags remain on the final VLP-antigen bioconjugated product.
  • the target antigen or antigen of interest can be selected from viral proteins, bacterial proteins, cancer antigens, and autoimmune disease-related antigens.
  • Table 1 shows the list of antigens of interest for infectious diseases.
  • Table 1 Infectious Diseases Antigen(s) of Interest (AOI) Human Papillomavirus (HPV) L1, L2, E1, E2, E3, E4, E5, E6, E7, and subdomains thereof Influenza Hemagglutinin (HA), Neuramimidase (NA), Nucleoprotein (NP), Matrix proteins (M1& M2), NS1, and strain transcendent protective antigenic subdomains from HA, NA and M2 individually or any combinations thereof Coronaviruses Beta-CoV (SARS-CoV, SARS-CoV2, MERS-CoV) Spike protein (S1 and S2) and subdomains thereof including the RBD, Nucleocapsid protein (NP) from each of the respective coronaviruses, and subdomains thereof Human Immunodeficiency Virus (HIV) Envelop proteins gp120, gp41, gp160, and subdomains thereof Human adenovirus (HAdv) Hexon protein, penton base protein, fiber-knob (FK
  • Bordetella pertussis Pertusis exotoxin (S1, S2, S3, S4 and S5 subunits), filamentous haemaglutinin (FHA), fimbrial antigens (FIM2/3), pertactin (PRN), and subdomains thereof Legionella pneumophila Lpg0127 (AcsB), Lpg0199 (CydA), Lpg0533 (SucB), Lpg0534 (SucC), Lpg1594 (LdsB), Lpg1595, Lpg1596 (YfcX), Lpg2271, Lpg2311, Lpg2312, Lpg2272, Lpg2273 (UgpB), Lpg2217, Lpg0688, Lpg2025, and subdomains thereof Mycobacterium tuberculosis Ag85A/B, ESAT-6, CFP10, TB10.4, MPT64, MPT70, MPT83, PPE18, PPE68
  • stolonifer FTR1, Cell wall proteins, and subdomains thereof Cancers Cervical, Anal & Oropharyngeal Human papilloma virus L1 and L2 proteins from different HPV types, E1, E2, E3, E4, E5, E6, E7, and subdomains thereof Hepatocellular Carcinoma Hepatitis B surface antigen, Hepatitis B core antigen, and subdomains thereof Non-Small Cell Lung Cancer Epidermal growth factor (EGF), tumor-associated antigens (TAAs): MAGE-A3, NY-ESO1, CEA, WT1, MUC1; Neoantigens: KRAS G12C, Tp53 mutations / variants, and subdomains thereof Melanoma TAAs: MART1, gp100 (PMEL), Tyrosinase and tyrosinase related proteins (TRP1 / TRP2), NY-ESO1, MAGE-A1, A3, A4, PRAME, melanoma specific n
  • Clostridium difficile derived proteins /virulence factors Clostridium difficile derived proteins /virulence factors, Mycobacterium avium subsp. paratuberculosis (MAP), HSPs, IBD specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereof
  • VLP-based vaccine technologies rely on either direct insertion of antigens into the capsid protein backbone or chemical conjugation methods, such as those employed by GSK for their RTS,S/AS01 malaria vaccine or similar approach used for dengue vaccine by other competitors. These approaches often require extensive optimization to ensure proper VLP assembly, as any insertion within the backbone protein can disrupt the overall three-dimensional structure, leading to prolonged pre-clinical development times and increased costs.
  • the SpyCatcher-SpyTag system results in a large residual scar of approximately 120 amino acids, which can negatively impact or interfere with the target antigen's immunogenicity or may even interfere with the VLP's structure and stability.
  • the intein-based bioconjugation process of the present disclosure is nearly scar-free, providing a cleaner and more efficient alternative that preserves the desired properties of the VLP-antigen complex.
  • a unique feature of the novel VLP platform of the present disclosure is its ability to encapsulate various immunomodulatory factors within the VLP core. These factors can include metabolites, small molecules, pharmacologically active compounds, peptides, proteins, cytokines, hormones, signaling molecules, or adjuvants. Encapsulation allows for the precise delivery of these immunomodulatory agents to APCs, which could influence the priming of T-cell for generating a tailored and robust specific type of immune response against the conjugated antigen and thereby enhancing the vaccine efficacy.
  • VLP serves both as an antigen presentation scaffold and as a delivery vehicle for immunomodulatory factors—enables the modulation of immune responses in a controlled manner capable of generating tailored, robust and specific immune response against the target antigen for enhancing the vaccine efficacy.
  • encapsulated immunomodulators can boost the activation of APCs, while cytokines can steer the immune response towards a desired pathway, such as enhancing T-cell activation or promoting antibody production or cell mediated immunity, as well as development of desired long-term immune memory. This capability is particularly valuable for developing vaccines against complex diseases, including cancer and autoimmune disorders, where tailored immune modulation is crucial.
  • AP205 VLP backbone SEQ ID 3
  • NpuDnaE C N-terminal intein tag
  • the In-tag AP205 is dialysed extensively through 3.5kDa membrane to remove denaturing urea in presence of Resiquimod to enable encapsulation within the assembling AP205-VLP core.
  • FIG. 1 shows split HBcAg-VLP bioconjugated with another AOI (JEV-EDIII, a subdomain from Japanese Encephalitis Envelop protein) and encapsulated with Kynurenine.
  • JEV-EDIII a subdomain from Japanese Encephalitis Envelop protein
  • Recombinant HBcAg VLP backbone (SEQ ID 5) is expressed as split VLP encoding a N-term fragment (Nf) with a stop codon and internal ribosomal binding site (IRBS) followed by N-terminal intein (In) tag ( NpuDnaE C ) (SEQ ID 1) in fusion with the C-term VLP fragment (Cf). Both Nf and Cf VLP fragments were co-purified under denaturing conditions from E. coli extracts. In-tag HBcAg fragments were dialysed extensively through 3.5kDa membrane to remove denaturing urea in presence of Kynurenine to enable encapsulation within the assembling AP205-VLP core.
  • Purified AP205 (SEQ ID 3) or HBcAg (Nf + Cf) (SEQ ID 5) proteins under denaturing condition were adjusted to a concentration of about 1mg/ml and dialysed extensively through 3.5 kDa membrane to remove urea and favor VLP assembly in presence of varying concentrations of imunomodulator (0.1 to 10 mM).
  • the VLP-suspension with encapsulated immunomodulator was separated from free immunomodulator molecules using extensive dialysis using 300kDa membrane.
  • the amount of encapsulated immunomodulator from about 5 x 10 14 VLPs was collected upon VLP denaturation, and the amount of encapsulated immunomodulator was estimated using UV-Visible spectrophotometry; Resiquimod (260 nm) and Kynurenine (360 nm).
  • the encapsulation efficiency (%) profile for Resiquimod in AP205 VLP and Kynurenine in HBcAg VLP, respectively is plotted as a function of immunomodulator concentration in Figure 4.
  • the VLP platform's versatility allows it to be adapted for a wide range of applications. It is particularly suited for the development of vaccines against various infectious diseases where T-cell mediated antibody responses are critical for protection, such as viral, bacterial, and parasitic infections, as well as generation of long-term memory (Tfh) of protective humoral immune response. Additionally, this VLP platform can be used to effectively prime specifically tailored T-cell responses, making it applicable to several viral diseases (such as CD8 + T-cell mediated immunity) and non-infectious diseases like cancer and autoimmune conditions through modulating the balance between T conv and T reg mediated immune responses against different target antigens.
  • viral diseases such as CD8 + T-cell mediated immunity
  • non-infectious diseases like cancer and autoimmune conditions through modulating the balance between T conv and T reg mediated immune responses against different target antigens.
  • this novel VLP platform supports the development of multivalent vaccines, where multiple distinct antigens could be easily bioconjugated with target antigens from multiple strains of a pathogen or a range of related pathogens, to be displayed on surface of different batches of VLPs. The desired combinations of such VLPs could then be co-administered, as well as for creating combination vaccines that provide broad protection against various diseases simultaneously.
  • This novel VLP platform is designed with scalability in mind, making it suitable for large-scale commercial production.
  • the intein-mediated conjugation process is highly efficient and straightforward not requiring the extensive optimization typical of other VLP technologies, reducing both the time and cost of vaccine development.
  • the encapsulation of the VLP core with immunomodulatory factors is a straightforward and scalable process easily achieved during the step of VLP assembly. Such ease of scalability ensures that this novel VLP-platform can meet the demands of high-volume vaccine production, addressing global health needs efficiently.
  • the novel VLP-platform of the present disclosure offers distinct advantages in terms of efficiency, flexibility, and cost-effectiveness. For instance, the need for extensive structural optimization is eliminated, as the intein-mediated conjugation preserves the VLP’s architecture without requiring antigen insertion into the capsid protein backbone. Additionally, minimal or near trace-less nature of the bioconjugation process ensures that the immunogenicity of the antigen remains unaltered, resulting in a more effective immune response.
  • the novel VLP-platform of the present disclosure also has the capacity to encapsulate and deliver immunomodulatory factors alongside target antigen to further differentiate it from competitors, providing a unique mechanism to enhance vaccine efficacy through targeted immune modulation. This feature not only improves initial immune priming but also supports generation of tailored immune response such as the development of robust memory responses, which are essential for long-lasting protection.
  • the novel VLP platform of the present disclosure represents a transformative approach to vaccine development. By leveraging intein-mediated bioconjugation and incorporating immunomodulatory encapsulation, this platform addresses critical limitations of existing VLP technologies and offers a highly adaptable, efficient, and scalable solution for enhancing vaccine efficacy.
  • This innovative VLP-technology has the potential to revolutionize the field of recombinant vaccines, providing a powerful tool for combating a wide array of diseases and advancing public health on a global scale.

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Abstract

La présente invention concerne une nouvelle plateforme de particules pseudovirales (VLP) pour le développement de vaccins améliorés. Cette nouvelle plateforme VLP est constituée de deux attributs uniques : i) la bioconjugaison de protéines à médiation par l'intéine, un procédé qui permet une fixation précise et efficace d'antigènes cibles à la surface des VLP sans laisser d'étiquettes ou de cicatrices résiduelles indésirables ; et ii) l'encapsulation de différents immunomodulateurs à petites molécules (y compris des cytokines, des adjuvants et des métabolites à petites molécules) pour l'amorçage spécifique de la réponse immunitaire personnalisée en influençant le résultat d'interactions entre les cellules présentatrices d'antigène (CPA) et les lymphocytes T pour déclencher un type spécifique de réponse immunitaire (Th1 : humoral, Th1 : à médiation cellulaire, Tfh, Th2, Th17 ou Treg). Cette plateforme de VLP basée sur une bioconjugaison à médiation par l'intéine facilite une approche de type « brancher et afficher », réduisant significativement le temps et les ressources nécessaires au développement de vaccins appropriés pour une large gamme d'applications, y compris des vaccins contre des maladies infectieuses, des immunothérapies anticancéreuses et des traitements pour des troubles auto-immuns. De plus, l'encapsulation de facteurs immunomodulateurs à l'intérieur du noyau des VLP permet leur distribution aux CPA, générant ainsi un type de réponse immunitaire robuste et spécifique pour une efficacité de vaccin améliorée. La présente invention concerne une solution polyvalente, évolutive et rentable pour le développement rapide de vaccins qui induisent des réponses immunitaires complètes et personnalisées, comprenant à la fois une immunité humorale et cellulaire avec une mémoire à long terme.
PCT/IN2025/050817 2024-05-30 2025-05-30 Nouvelle plateforme de particules pseudovirales basée sur une bioconjugaison à médiation par l'intéine avec un antigène cible et encapsulée avec des facteurs immunomodulateurs Pending WO2025248564A1 (fr)

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