Virus-like particle conjugates

Abstract

To provide new immunogenic compositions, methods of manufacturing immunogenic compositions, and methods of treating and preventing infections with the immunogenic compositions.SOLUTION: An immunogenic composition comprises virus-like particles (VLPs) obtained or derived from L1 and / or L2 proteins of Human papilloma virus (HPV), conjugated with a spacer arm and a carrier protein.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to immunogenic compositions, conjugates, virus-like particle (VLP) compositions, vaccines, and methods directed to the treatment and / or prevention of infection with human papillomavirus.

[0002] <Reference to Related Applications> This application claims priority to U.S. Non-provisional Application No. 16 / 435,502, filed June 9, 2019, which claims priority to U.S. Provisional Application No. 62 / 683,787, filed June 12, 2018, and U.S. Provisional Application No. 62 / 683,543, filed June 11, 2018, each of which is incorporated by reference in its entirety. [Background technology]

[0003] Human papillomavirus (HPV) is a double-stranded DNA virus that targets the basal cells of squamous epithelium for infection. The circular DNA genome of HPV consists of two major oncogenes, E6 and E7, and two major structural protein genes, L1 and L2. Most of the conventional vaccines are developed based on these components. The L1 protein of HPV expressed recombinantly in vitro self-assembles into virus-like particles (VLPs). The VLPs carry HPV type-specific conformational neutralizing epitopes and are used to develop VLP-based vaccine products. Usually, VLPs are expressed recombinantly in yeast, bacterial, or insect cell expression systems.

[0004] Currently, two HPV vaccines are commercially available: Gardasil (Merck and Co. Inc.) and Cervarix (GSK). The composition and dosage of the Gardasil vaccine includes HPV VLP1 proteins with 6, 11, 16, 18, 1, 33, 45, 52, and 58, a total of nine serotypes, and an aluminum adjuvant. The VLPs are present in an amount of 20-40 μg per dose. The vaccine is administered as a three-dose regimen following a 0, 2, and 6 month schedule. The Cervarix vaccine includes HPV VLP16 and 18L1 proteins and is adjuvanted with aluminum hydroxide and MPLA (3D-MPL). The VLPs are present in an amount of 20 μg per dose. The vaccine is also administered as a three-dose regimen following a 0, 2, and 6 month schedule.

[0005] The cost and number of doses of vaccines remain major obstacles to immunizing populations in developing countries, therefore lower cost and lower dose vaccines are greatly needed worldwide. Summary of the Invention

[0006] The present invention overcomes the problems and drawbacks associated with current approaches and designs and provides new immunogenic compositions, methods for producing immunogenic compositions, and methods for treating and preventing infectious diseases with immunogenic compositions.

[0007] One embodiment of the present invention relates to an immunogenic composition, such as a vaccine, comprising a virus-like particle (VLP) obtained or derived from the L1 and / or L2 proteins of human papillomavirus (HPV) conjugated to a spacer arm and a carrier protein. Preferably, the HPV comprises serotypes 6, 11, 16, 18, 31, 33, 45, 52, and / or 58. Preferably, the spacer arm comprises a heterofunctional, homobifunctional, or multifunctional spacer arm, or in particular NH2-PEG-NH2 / NHS, NHS / NH2-PEG-COOH, Mal-PEG-NH2, Mal-PEG-NHS, CHO-PEG-CHO, SH-PEG-NH2, ADH, HZ-PEG-HZ, SMPH, SMCC, 4-Arm-PEG-NH2. Preferably, the carrier protein comprises tetanus toxoid, diphtheria toxoid, CRM197, tetanus toxoid fragment (TTHc), meningococcal protein PorB, RSV viral protein, B. pertussis protein, pertussis toxoid (PT), adenylate cyclase toxin (ACT), 69KDa protein, human papillomavirus protein antigen, human papillomavirus VLP form, hepatitis B virus core antigen, hepatitis B virus VLP form, derivatives of HBsAg, and / or combinations thereof. Preferably, the immunogenic composition comprises an adjuvant, preferably the adjuvant comprises an aluminum salt, calcium phosphate, liposomes of monophosphoryl lipid A (MPLA), saponin QS-21, TLR ligands, and / or potent TLR4 / 7 / 8 / 9 agonists. Preferred aluminum salts include one or more of aluminum phosphate, aluminum sulfate, and / or aluminum hydroxide. Preferably, the immunogenic composition enhances the efficacy of a conventional vaccine when administered to a patient.

[0008] Another related embodiment of the invention includes an immunogenic composition as described herein, wherein the VLP is obtained or derived from an HPV L1 protein or an HPV L2 protein, and the carrier protein is CRM197.

[0009] Another related embodiment of the invention includes an immunogenic composition as described herein, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 16 and / or 18, and the carrier protein is CRM197.

[0010] Another related embodiment of the invention includes an immunogenic composition as described herein, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 6 and / or 11, and the carrier protein is CRM197.

[0011] Another related embodiment of the invention includes an immunogenic composition as described herein, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 31 and / or 33, and the carrier protein is CRM197.

[0012] Another related embodiment of the invention includes an immunogenic composition as described herein, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 45 and / or 52, and the carrier protein is CRM197.

[0013] Another related embodiment of the invention includes an immunogenic composition as described herein, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 58, and the carrier protein is CRM197.

[0014] Another related embodiment of the invention includes an immunogenic composition as described herein, wherein the VLP comprises an L1 protein conjugated to an L2 protein, and the carrier protein is CRM197.

[0015] Another related embodiment of the invention includes processes for making and using the immunogenic compositions of the present disclosure.

[0016] Preferably, the immunogenic compositions are stable and provide protection against infection at lower doses or less frequently than are available using conventional immunogenic compositions.

[0017] Other embodiments and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. [Brief description of the drawings]

[0018] [Figure 1] Bifunctional spacer arms for activation of CRM197; Mal-Maleimide, NHS-Succinimide, PEG-Polyethylene glycol derivatives, ADH-Adipic acid dihydrazide, HZ-Hydrazide, 1k and 3K-Mn1000 and 3500. [Diagram 2] Schematic diagram of the mechanism of action of VLPs. [Diagram 3] Schematic diagram of the conjugation of the carrier protein CRM197 with a spacer arm to VLPs. [Figure 4] Schematic diagram of VLP-CRM-VLP conjugates in which HPV VLP L116 type is the first conjugated according to scheme 2, followed by VLP L118 type conjugated with VLP L1-CRM197 conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Surprisingly, it was discovered that an HPV immunity conjugate equivalent to the conventional HPV vaccine could be made in just two doses with reduced antigen per dose, resulting in a fifth of the price of the conventional vaccine and requiring fewer vaccine doses, making the vaccine more practical and widely available.

[0020] The present disclosure relates to pharmaceutical conjugate vaccine compositions for human cervical cancer, comprising a conjugate vaccine and one L2-HPV VLP, preferably derived from an L1 HPV clone and conjugated to a carrier protein, preferably CRM197 and CRM197-like proteins, using a spacer arm, and a virus-like particle used in a pharma- ceutically acceptable aluminum adjuvant (or non-aluminum adjuvant) and a suitable buffer. The conjugate combination includes a combination of L1 and L2 VLPs from areas where common infections of these serotypes exist.

[0021] The present disclosure relates to immunogenic compositions comprising virus-like particles (VLPs) obtained or derived from the L1 and / or L2 proteins of human papillomavirus (HPV) conjugated with a spacer arm and a carrier protein. Preferably, the HPV comprises serotypes 6, 11, 16, 18, 31, 33, 45, 52, and / or 58. Preferably, the spacer arm comprises a heterofunctional, homobifunctional, or multifunctional spacer arm, or in particular NH2-PEG-NH2 / NHS, NHS / NH2-PEG-COOH, Mal-PEG-NH2, Mal-PEG-NHS, CHO-PEG-CHO, SH-PEG-NH2, ADH, HZ-PEG-HZ, SMPH, SMCC, 4-Arm-PEG-NH2. Preferably, the carrier protein comprises tetanus toxoid, diphtheria toxoid, CRM197, tetanus toxoid fragment (TTHc), meningococcal protein PorB, RSV viral protein, B. pertussis protein, pertussis toxoid (PT), adenylate cyclase toxin (ACT), 69KDa protein, human papillomavirus protein antigen, human papillomavirus VLP form, hepatitis B virus core antigen, hepatitis B virus VLP form, derivatives of HBsAg, and / or combinations thereof. Preferably, the immunogenic composition comprises an adjuvant, preferably the adjuvant comprises an aluminum salt, calcium phosphate, liposomes of monophosphoryl lipid A (MPLA), saponin QS-21, TLR ligands, and / or potent TLR4 / 7 / 8 / 9 agonists. Preferred aluminum salts include one or more of aluminum phosphate, aluminum sulfate, and / or aluminum hydroxide. Preferably, the immunogenic composition enhances the efficacy of a conventional vaccine when administered to a patient.

[0022] The present disclosure provides immunogenic compositions comprising VLPs obtained from or derived from HPV L1 or L2 proteins and having a carrier protein of CRM197; VLPs that are bivalent L1 VLP conjugates, where the HPV is serotype 16 and / or 18 and the carrier protein is CRM197; VLPs that are bivalent L1 VLP conjugates, where the HPV is serotype 6 and / or 11 and the carrier protein is CRM197; VLPs that are bivalent L1 VLP conjugates, where the HPV is serotype 31 and / or 33 and the carrier protein is CRM197; VLPs that are bivalent L1 VLP conjugates, where the HPV is serotype 45 and / or 52 and the carrier protein is CRM197; VLPs that are bivalent L1 VLP conjugates, where the HPV is serotype 58 and the carrier protein is CRM197; and VLPs that comprise L1 protein conjugated to L2 protein and have a carrier protein of CRM197.

[0023] The present disclosure includes formulations of VLP conjugate vaccines having at least 9, 10, or more valences that reduce the required dosage (8 - 10 μg / dose of individual VLPs compared to the 20 - 40 μg / dose used in Gardasil, Merck or Cervarix, GSK) adsorbed to aluminum phosphate or aluminum hydroxide, or other suitable TLR7 / 8 / 9 or TLR4 adjuvants.

[0024] <Examples of VLP - protein conjugate formulations> VLP1 - CRM197 - VLP2 (4 - and 9 - valent L1, 10 - valent L2); HPV - VLP / CRM197 (monovalent conjugate); and VLP1 - CRM197 - VLP2 (4 - and 9 - valent L1, 10 - valent L2). Conjugates include conjugation with HBsAg, such as HBsAg - VLP / CRM197.

[0025] The present disclosure is directed to a 9-valent vaccine candidate based on L1 VLPs, as well as the addition of a 10-valent vaccine based on L2-VLPs. Four sets of two L1 VLPs are chemically conjugated to a carrier protein (such as CRM197 in E. coli) using a bifunctional spacer arm, and one L1-VLP is conjugated to an L2 VLP. The L1 and L2-based VLP conjugates can generally be structurally represented as follows: L1-XX-VLP-Spacer Arm-CRM197L1-XX-VLP L1-XX-VLP-Spacer Arm-CRM197L2-VLP XX can be any L1 VLP Examples included are: L1VLP16-Spacer Arm-CRM197-L1VLP18 L1VLP6-Spacer Arm-CRM197-L1VLP11 L1VLP31-Spacer Arm-CRM197-L1VLP33 L1VLP45-Spacer Arm-CRM197-L1VLP52 L1VLP58-Spacer Arm-CRM197-L2VLP

[0026] The following examples illustrate embodiments of the present invention but should not be construed as limiting the scope of the invention. EXAMPLES

[0027] Example 1: Expression and purification of recombinant HPV L1 and L2V LPs in yeast. L1 protein forms all nine serotypes and L2 protein is expressed in the yeast Hansenula polymorpha. Fermentation cycle: ~120 hours. Expression induced by methanol using the HP promoter. L1 and L2 proteins were extracted using the yeast cell wall lytic enzyme Zymolase, followed by benzonase treatment for nucleic acid removal. The extraction step was performed in TRIS buffer, 20-50 mM, pH 8.5-9.5. The clarified extract (concentration 1mg / ml, L1 protein 150-200ug / ml) was applied to cation exchange chromatography followed by gel filtration chromatography. In this step, di-assembly was utilized and assembly step ensured that VLPs were assembled in the correct configuration and stable molecules. This is confirmed using TEM. VLPs were stabilized using appropriate buffers. Finally, VLPS are concentrated using ultrafiltration.

[0028] All serotype L1 and L2 VLP purification yields are in the region of 7-15% of the cell extract, and the purified VLPs are 98-99% homogeneous in purity. Figure 2 depicts a process flow chart showing the experimental procedure used to purify L1 and L2 HPV serotype VLPs, where the amino acid sequence of all purified VLPs is checked and free thiols and lysines are used for stability and site availability for chemical conjugation to carrier proteins.

[0029] Example 2. Chemical conjugation procedure of L1 and L2 VLPs with CRM197. Chemical conjugation of VLPs is achieved by the use of chemical cross-linking agents as well as a variety of conjugation modalities employed, e.g., PEGylated homo- or heterobifunctional conjugation reagents that have the same or two distinct reactive groups that can bind to different, distinct functional targets, one on the antigen and the other on the VLP (usually an amine or sulfhydryl residue).

[0030] Example 3. Activation of CRM197 using a bifunctional spacer arm. Activation of the carrier protein CRM197 using PEGylated non-homo- or hetero-bifunctional spacer arms. CRM-197 (10 mg / ml) is dissolved in activation buffer followed by addition of the bifunctional spacer arm (see Figure 1) in the presence of PB or MES buffer, 80 mM-200 mM, pH 5.8-6.2. Functionalized CRM197 was purified using a 10-30 KD TFF cassette (Figure 2).

[0031] Example 4. Conjugation process of functionalized CRM197 to L1 VLP. The basic method steps for conjugation are as follows: 1. L1 or L2--Chemical conjugation between the VLP and the carrier protein CRM197 (Figure 3). 2. Assessment of HPV VLP integrity following conjugation with CRM197. 3. Analytical characterization of HPV L1 and L2 VLPs and chemically conjugated bivalent unimolecular or bivalent VLPS. 4. Formulation of VLP-protein conjugates. 5. Comparison of immunogenicity with Gardasil-9. 6. Stability testing of VLP conjugates. Examples of conjugates that are formed are as follows: Conjugation process of I.L116 HPV-VLPs to functionalized CRM197. II. Conjugation process of L1 HPV 16 VLP-CRM to L1-HPV 18 VLP (Figure 4). III.L1 58 HPV-VLP-CRM197-L2 Conjugation process of HPV VLPs.

[0032] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. It is intended that the specification and examples be considered exemplary only, with a true scope and spirit of the invention being indicated by the following claims. Furthermore, the term "consisting of" includes the terms "consisting of" and "consisting essentially of."

Claims

1. A method for producing an immunogenic composition comprising a virus-like particle (VLP), comprising: Attaching a heterobifunctional, homobifunctional, or multifunctional spacer arm assembly to a carrier protein; conjugating an assembly of human papillomavirus (HPV) L1 or L2 proteins to a heterobifunctional, homobifunctional, or multifunctional spacer arm bound to a carrier protein; conjugating the carrier protein bound to the spacer arms to another set of heterobifunctional, homobifunctional, or multifunctional spacer arms; linking a separate collection of human papillomavirus (HPV) L1 or L2 proteins to said separate collection of heterobifunctional, homobifunctional, or multifunctional spacer arms; forming a composition comprising an L1 or L2 protein of a human papillomavirus bound to another spacer arm bound to a carrier protein bound to a spacer arm bound to an L1 or L2 protein of a human papillomavirus; The method comprising:

2. 2. The method of claim 1, wherein the HPV comprises serotypes 6, 11, 16, 18, 31, 33, 45, 52, and / or 58.

3. The spacer arm is NH ２ -PEG-NH ２ / NHS, NHS / NH ２ -PEG-COOH, Mal-PEG-NH ２ , Mal-PEG-NHS, CHO-PEG-CHO, SH-PEG-NH ２ , ADH, HZ-PEG-HZ, SMPH, SMCC, and / or 4-Arm-PEG-NH ２ The method of claim 1 or 2, comprising:

4. 4. The method of any one of claims 1 to 3, wherein the carrier protein comprises tetanus toxoid, diphtheria toxoid, CRM197, tetanus toxoid fragment (TTHc), meningococcal protein PorB, RSV viral protein, B. pertussis protein, pertussis toxoid (PT), adenylate cyclase toxin (ACT), 69KDa protein, human papillomavirus protein antigens, human papillomavirus VLP forms, hepatitis B virus core antigen, hepatitis B virus VLP forms, derivatives of HBsAg, and / or combinations thereof.

5. The method of claim 1, wherein the composition further comprises an adjuvant.

6. The method of claim 5, wherein the adjuvant comprises an aluminum salt, calcium phosphate, liposomes of monophosphoryl lipid A (MPLA), saponin QS-21, a TLR ligand, and / or a potent TLR4 / 7 / 8 / 9 agonist.

7. 7. The method of claim 6, wherein the aluminum salt is selected from the group consisting of aluminum phosphate, aluminum sulfate and / or aluminum hydroxide.

8. The method of any one of claims 1 to 7, wherein the VLPs are HPV L1 and / or HPV L2 proteins and the carrier protein comprises CRM197.

9. 9. The method of any one of claims 1 to 8, wherein the VLP comprises a bivalent L1 VLP conjugate, the HPV is serotype 16 and / or 18, and the carrier protein comprises CRM197.

10. The method of any one of claims 1 to 8, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 6 and / or 11, and the carrier protein comprises CRM197.

11. The method of any one of claims 1 to 8, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 31 and / or 33, and the carrier protein comprises CRM197.

12. The method of any one of claims 1 to 8, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 45 and / or 52, and the carrier protein comprises CRM197.

13. The method of any one of claims 1 to 8, wherein the VLP is a bivalent L1 VLP conjugate, the HPV is serotype 58, and the carrier protein comprises CRM197.

14. The method of any one of claims 1 to 8, wherein the VLP comprises an L1 protein conjugated to an L2 protein and the carrier protein comprises CRM197.

15. The method of any one of claims 1 to 14, wherein administration of the composition to a patient enhances the efficacy of a conventional vaccine.

16. A method for producing an immunogenic composition comprising a virus-like particle (VLP), comprising: conjugating an assembly of L1 proteins of human papillomavirus (HPV) to a heterobifunctional, homobifunctional, or multifunctional spacer arm bound to a carrier protein; conjugating a carrier protein already conjugated to a heterobifunctional, homobifunctional, or multifunctional spacer arm to another collection of heterobifunctional, homobifunctional, or multifunctional spacer arms that are conjugated to a collection of L1 or L2 proteins of a human papillomavirus (HPV), the HPV comprising serotypes 6, 11, 16, 18, 31, 33, 45, 52, and / or 58, at least one spacer arm comprising a heterobifunctional spacer arm, and the carrier protein comprising tetanus toxoid or diphtheria toxoid; and forming a composition comprising an L1 protein of a human papillomavirus bound to another spacer arm bound to a carrier protein bound to a spacer arm bound to an L1 or L2 protein of the human papillomavirus; The method comprising:

17. The method of claim 16, wherein the composition further comprises an adjuvant.

18. A method for producing an immunogenic composition comprising a virus-like particle (VLP), comprising: conjugating an assembly of L2 proteins of human papillomavirus (HPV) to a heterobifunctional, homobifunctional, or multifunctional spacer arm bound to a carrier protein; conjugating a carrier protein already conjugated to a heterobifunctional, homobifunctional, or multifunctional spacer arm to another collection of heterobifunctional, homobifunctional, or multifunctional spacer arms that are conjugated to a collection of L1 or L2 proteins of a human papillomavirus (HPV), the HPV comprising serotypes 6, 11, 16, 18, 31, 33, 45, 52, and / or 58, at least one spacer arm comprising a heterobifunctional spacer arm, and the carrier protein comprising tetanus toxoid or diphtheria toxoid; and forming a composition comprising an L2 protein of a human papillomavirus bound to another spacer arm bound to a carrier protein bound to a spacer arm bound to an L1 or L2 protein of the human papillomavirus; The method comprising:

19. The method of claim 18, wherein the composition further comprises an adjuvant.

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