Novel vaccines against HPV and HPV-related diseases

Abstract

Embodiments relate to novel vaccines against human papilloma virus (HPV) and HPV-related diseases, including various cancer types. The HPV vaccine is composed of anti-human dendritic cell (DC) surface receptor antibodies, including E6 / 7 proteins of CD40 and HPV 16 and 18. The described technology is not limited to the preparation of vaccines against HPV 16-and HPV 18-related diseases, and can be suitable for the preparation of vaccines carrying E6 / 7 from any type of HPV. The described HPV vaccines can target DC, major and professional antigen presenting cells (APCs), and are capable of inducing and activating potent HPV E6 / 7-specificity and potent CD4 + and CD8 + T cell responses. The HPV vaccines may be used for the prevention of HPV infections and HPV-related diseases, as well as for the treatment of HPV-related diseases, including cancer.

Description

[0001] This application is a divisional application of PCT international application PCT / US2015 / 011236 filed on January 13, 2015, which entered the Chinese national phase on September 12, 2016, with application number 201580013480.8 and invention title "A New Vaccine Against HPV and HPV-Related Diseases". Background of the Invention

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 926,821, filed January 13, 2014, and U.S. Provisional Patent Application No. 62 / 002,718, filed May 23, 2014, both of which are incorporated herein by reference in their entirety.

[0003] This invention was made with government support, granted by the National Institutes of Health and the National Institute of Allergy and Infectious Diseases under approval number U19AI057234. The government holds certain rights to this invention.

[0004] 1. Field of Invention

[0005] This invention relates generally to the medical field. More specifically, it relates to novel and innovative vaccines against human papillomavirus (HPV) and HPV-related diseases, including various types of cancer.

[0006] 2. Description of related technologies

[0007] Human papillomavirus (HPV) is one of the most common sexually transmitted pathogens. Current HPV prophylactic vaccines have shown significant clinical efficacy in preventing HPV infection, but they have not shown effectiveness in treating infected patients or HPV-related cancers. HPV infection causes almost all cervical cancers, as well as many anal, vaginal, vulvar, penile, and oropharyngeal (throat) cancers. Therefore, there is a great need to develop safe and effective vaccines for patients infected with HPV and those with HPV-related cancers. HPV infection also causes HIV-related malignancies and cancers.

[0008] Current HPV vaccines are recombinant virus-like particles prepared from the capsid (L1) proteins of HPV 6, 11, 16, and 18. These vaccines elicit a strong antibody response and thus prevent HPV infection. To suppress viral replication and eliminate HPV-associated cancers, vaccines need to stimulate a strong T-cell response, particularly cytotoxic CD8+ lymphocytes (CTLs) capable of killing virus-infected cells, thereby inhibiting HPV replication and HPV-associated tumor cells.

[0009] Several types of vaccine models have been tested (including peptide, protein, and DNA-based vaccines, as well as vaccines carried by attenuated live vectors), but these vaccines have drawbacks in efficacy or safety, particularly in immunocompromised patients. This deficiency necessitates the development of safe and potent immunotherapeutic vaccines against HPV-associated cancers.

[0010] Extensive evidence concludes that almost all cervical cancer cases are caused by persistent infection with a group of HPV types, particularly HPV 16 and HPV 18. These HPV types also cause a significant proportion of other mucosal cancers, including vulvar, vaginal, anal, penile, and oropharyngeal cancers. HPV 16 is the predominant type of squamous cell carcinoma of the cervix, while HPV 18 is the second most common type, with a prevalence ranging from 12.6% in Central / South America to 25.7% in South Asia. Furthermore, HPV 18 is associated with rapidly developing and potentially more aggressive cervical cancers. However, subclinical infection is the most common manifestation of HPV infection. Different studies report subclinical infection rates ranging from 15% to 36% in sexually active adults. Invention Overview

[0011] This invention discloses a fusion protein comprising an anti-CD40 antibody or a fragment thereof, comprising at least three complementarity-determining regions derived from the anti-CD40 antibody, at least one peptide linker, and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises at least a variable region derived from the anti-CD40 antibody. The variable region may be derived from the light chain or the heavy chain. In some embodiments, the anti-CD40 antibody or fragment thereof comprises at least a variable region derived from the light chain of the anti-CD40 antibody and at least a variable region derived from the heavy chain of the anti-CD40 antibody. In some embodiments, the anti-CD40 antibody or fragment thereof comprises six CDRs derived from the anti-CD40 antibody. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, the peptide linker or multiple peptide linkers are flexible linkers. In some embodiments, the peptide linker or multiple peptide linkers comprises one or more glycosylation sites. In some embodiments, the peptide linker or multiple peptide linkers are Flexv1 (SEQ ID NO:5) and / or f1 (SEQ ID NO:6). In some embodiments, the HPV antigens are E6 and E7. In some embodiments, the fusion protein includes the sequence of SEQ ID NO:19. In some embodiments, the fusion protein includes the sequence of SEQ ID NO:21. In some embodiments, at least one HPV E6 antigen is an HPV16 antigen and at least one HPV E6 antigen is an HPV18 antigen. In some embodiments, at least one HPV E7 antigen is an HPV16 antigen and at least one HPV E7 antigen is an HPV18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV16 antigen and at least one HPV E6 antigen is an HPV18 antigen, and at least one HPV E7 antigen is an HPV16 antigen and at least one HPV E7 antigen is an HPV18 antigen.

[0012] This invention also discloses a fusion protein comprising at least the amino acid sequence of SEQ ID NO: 11-13 or SEQ ID NO: 14-16 and at least one human papillomavirus (HPV) E6 or E7 antigen. In some embodiments, the fusion protein comprises SEQ ID NO: 11-13 and SEQ ID NO: 14-16. In some embodiments, the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the fusion protein further comprises a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the peptide linker comprises one or more glycosylation sites. In some embodiments, the peptide linker is Flexv1 (SEQ ID NO: 5) and / or f1 (SEQ ID NO: 6).

[0013] The present invention also discloses pharmaceutical compositions comprising any of the above-described fusion proteins.

[0014] The present invention also discloses a method for preparing any of the above-described fusion proteins, comprising isolating the fusion protein from a recombinant host cell expressing the fusion protein.

[0015] This invention also discloses compositions comprising a dendritic cell targeting complex, the dendritic cell targeting complex comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least three complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises at least a variable region derived from the anti-CD40 antibody. The variable region may be derived from the light chain or the heavy chain. In some embodiments, the anti-CD40 antibody or fragment thereof comprises at least a variable region derived from the light chain of the anti-CD40 antibody and at least a variable region derived from the heavy chain of the anti-CD40 antibody. In some embodiments, the anti-CD40 antibody or fragment thereof comprises six CDRs derived from the anti-CD40 antibody. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, the peptide linker or multiple peptide linkers are flexible linkers. In some embodiments, the peptide linker or multiple peptide linkers includes one or more glycosylation sites. In some embodiments, the peptide linker or multiple peptide linkers are selected from Flexv1 (SEQ ID NO:5) or f1 (SEQ ID NO:6). In some embodiments, the HPV antigen is E6 and E7. In some embodiments, the fusion protein includes the sequence of SEQ ID NO:19. In some embodiments, the fusion protein includes the sequence of SEQ ID NO:21. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen and at least one HPV E6 antigen is an HPV 18 antigen. In some embodiments, at least one HPV E7 antigen is an HPV 16 antigen and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen and at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 16 antigen and at least one HPV E7 antigen is an HPV 18 antigen.

[0016] In some embodiments, the HPV E6 and E7 antigens are selected from SEQ ID NO: 1-4. In other embodiments, the HPV E6 and E7 antigens have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or any range thereof, with any combination of SEQ ID NO: 1-4. In other embodiments, the HPV E6 and E7 antigens have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or any range thereof, with any combination of SEQ ID NO: 1-4. In other embodiments, the HPV E6 antigen is a subset of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 consecutive amino acids of any range deduced from SEQ ID NO: 1 and / or 3, and the HPV E7 antigen is a subset of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 15, or 120 consecutive amino acids of any range deduced from SEQ ID NO: 2 and / or 4, and the HPV E7 antigen is a subset of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 15, or 120 consecutive amino acids of any range deduced from SEQ ID NO: 2 and / or 4.

[0017] The present invention also discloses a vector comprising a polynucleotide sequence encoding a fusion protein, the fusion protein comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least three complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody light chain variable region. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody heavy chain variable region. In some embodiments, the polynucleotide sequence encodes at least one HPV type 16 E6 antigen, at least one HPV type 16 E7 antigen, or at least one HPV type 18 E6 antigen and at least one HPV type 18 E7 antigen. In some embodiments, the polynucleotide sequence encodes a polypeptide comprising SEQ ID NO:19. In some embodiments, the polynucleotide sequence encodes a polypeptide comprising SEQ ID NO:21. In some embodiments, the polynucleotide sequence encodes at least one HPV 16 E6 antigen, at least one HPV 16 E7 antigen, at least one HPV 18 E6 antigen, and at least one HPV 18 E7 antigen.

[0018] This invention also discloses a method for preventing human papillomavirus (HPV) infection, comprising administering to a patient a composition comprising a dendritic cell targeting complex, the dendritic cell targeting complex comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least six complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody light chain variable region and an anti-CD40 antibody heavy chain variable region. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 16 antigen. In some embodiments, at least one HPV E6 antigen is an HPV type 18 antigen and at least one HPV E7 antigen is an HPV type 18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen, at least one HPV E7 antigen is an HPV 16 antigen, at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:19. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:21. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the method further comprises administering different HPV vaccines to the patient. In some embodiments, the different HPV vaccines are Gardasil™ or Cervarix™.

[0019] This invention also discloses a method for treating human papillomavirus (HPV) infection, comprising administering to a patient a composition comprising a dendritic cell targeting complex, the dendritic cell targeting complex comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least six complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody light chain variable region and an anti-CD40 antibody heavy chain variable region. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 16 antigen. In some embodiments, at least one HPV E6 antigen is an HPV type 18 antigen and at least one HPV E7 antigen is an HPV type 18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen, at least one HPV E7 antigen is an HPV 16 antigen, at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:19. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:21. In some embodiments, the method further includes administering different HPV treatments to the patient.

[0020] The present invention also discloses a method for inducing an immune response against at least one HPV epitope, comprising administering to a patient a composition comprising a dendritic cell targeting complex, the dendritic cell targeting complex comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least six complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody light chain variable region and an anti-CD40 antibody heavy chain variable region. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 16 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 18 antigen and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen, at least one HPV E7 antigen is an HPV 16 antigen, at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:19. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:21. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the method further comprises administering different HPV vaccines to the patient. In some embodiments, the different HPV vaccines are Gardasil™ or Cervarix™.

[0021] The present invention also discloses a method for enhancing an immune response against at least one HPV epitope, comprising administering to a patient a composition comprising a dendritic cell targeting complex, the dendritic cell targeting complex comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least six complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody light chain variable region and an anti-CD40 antibody heavy chain variable region. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 16 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 18 antigen and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen, at least one HPV E7 antigen is an HPV 16 antigen, at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:19. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:21. In some embodiments, enhancing the immune response involves enhancing or increasing or strengthening memory T cells. In some embodiments, the method further includes administering different HPV treatments to the patient.

[0022] This invention also discloses a method for preventing human papillomavirus (HPV)-related diseases, comprising administering to a patient a composition comprising a dendritic cell targeting complex, the dendritic cell targeting complex comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least six complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody light chain variable region and an anti-CD40 antibody heavy chain variable region. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 16 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 18 antigen and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen, at least one HPV E7 antigen is an HPV 16 antigen, at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO: 19. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO: 21. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the HPV-related disease is a dysplasia, a benign tumor, a precancerous tumor, or cancer. In some embodiments, the HPV-related disease is cancer. In some embodiments, the cancer is cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, pharyngeal cancer, or lung cancer. In some embodiments, the method further comprises administering different HPV vaccines to the patient. In some implementations, the different HPV vaccines are Gardasil™ or Cervarix™.

[0023] This invention also discloses a method for treating human papillomavirus (HPV)-related diseases, the method comprising administering to a patient a composition comprising a dendritic cell targeting complex, the dendritic cell targeting complex comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least six complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody light chain variable region and an anti-CD40 antibody heavy chain variable region. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 16 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 18 antigen and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen, at least one HPV E7 antigen is an HPV 16 antigen, at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:19. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:21. In some embodiments, the HPV-related disease is a dysplasia, a benign tumor, a precancerous tumor, or cancer. In some embodiments, the HPV-related disease is cancer. In some embodiments, the cancer is cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, pharyngeal cancer, or lung cancer. In yet another embodiment, the cancer is head and neck cancer. In some embodiments, the method further includes administering different treatments to the patient. In some embodiments, the method further includes administering cancer treatment to the patient.

[0024] The present invention also discloses a method for inhibiting HPV-infected cells in a patient, comprising administering to the patient an effective amount of a composition comprising any and more than one fusion protein or carrier. In some embodiments, the HPV-infected cells are located in a tumor.

[0025] This invention also discloses a method for reducing the size or mass of tumors or tumors comprising HPV-infected cells in patients with HPV infection, comprising administering to the patient an effective amount of a composition comprising any and more fusion proteins or carriers. During or after treatment, the percentage reduction in tumor size or mass, or the percentage of tumor regression, can be at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% to 100% or any range thereof. The percentage reduction in tumor size or mass, or the percentage of tumor regression, can be such that it reduces tumor size to alleviate discomfort and improves the patient's quality of life or leads to or is associated with a clinically favorable outcome.

[0026] This invention discloses a method for prolonging the survival of patients or subjects suffering from HPV-related diseases, comprising administering to the patient a composition comprising a dendritic cell targeting complex, the dendritic cell targeting complex comprising: an anti-CD40 antibody or a fragment thereof, the anti-CD40 antibody or fragment thereof comprising at least six complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody or fragment thereof comprises an anti-CD40 antibody light chain variable region and an anti-CD40 antibody heavy chain variable region. In some embodiments, the anti-CD40 antibody or fragment thereof is humanized. In some embodiments, at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 16 antigen. In some embodiments, at least one HPV E6 antigen is an HPV type 18 antigen and at least one HPV E7 antigen is an HPV type 18 antigen. In some embodiments, at least one HPV E6 antigen is an HPV 16 antigen, at least one HPV E7 antigen is an HPV 16 antigen, at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 18 antigen. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:19. In some embodiments, the dendritic cell targeting complex comprises SEQ ID NO:21. In some embodiments, the HPV-related disease is a dysplasia, a benign tumor, a precancerous tumor, or cancer. In some embodiments, the HPV-related disease is cancer. In some embodiments, the cancer is cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, pharyngeal cancer, or lung cancer. In yet another embodiment, the cancer is head and neck cancer. In some embodiments, the method further includes administering different treatments to the patient. In some embodiments, the method further includes administering cancer treatment to the patient. In some aspects, the survival extension period is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or any range thereof. In some respects, the period of extended survival is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or any range thereof. In some respects, the period of extended survival is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months, or any range thereof. In some respects, the period of extended survival is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 years, or any range thereof.

[0027] Any of the methods disclosed above can be implemented using any of the fusion proteins, compositions and / or vectors disclosed above.

[0028] As used herein, “a” or “an” may mean one or more. As used herein in the claims (multiple claims), when used with the word “comprising”, the word “a” or “an” may mean one or more.

[0029] The term “or” is used in the claims to mean “and / or” unless expressly specified to refer only to alternatives or the alternatives are mutually exclusive; however, this disclosure supports the definition of “and / or” referring only to alternatives. As used herein, “another” may mean at least a second or more.

[0030] Throughout this application, the term "about" is used to refer to values ​​that include variations inherent in the means or methods used to determine the value, or variations present in the subjects of the study.

[0031] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific embodiments show preferred embodiments of the invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. Brief description of the attached diagram

[0032] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of specific embodiments provided herein.

[0033] Figure 1 : A plan to develop a recombinant anti-DC receptor-E6 / E7 vaccine.

[0034] Figure 2 SDS-PAGE analysis of anti-DC receptor antibodies fused to HPV 16 E6 and E7 antigens after affinity purification of protein A.

[0035] Figure 3 Anti-CD40-HPV16.E6 / 7 can effectively bind to human blood DCs.

[0036] Figure 4 Anti-CD40-HPV16.E6 / 7 can induce HPV16.E6 / 7-specific CD4+ and CD8+ T-cell responses.

[0037] Figure 5 Anti-CD40-HPV16.E6 / 7 can effectively induce HPV16.E6 / 7-specific CD4+ and CD8+ T-cell responses.

[0038] Figure 6 In human CD40Tg mice, anti-CD40-HPV16.E6 / 7 can inhibit the development of TC-1 tumors.

[0039] Figure 7 The combined effect of adjuvant and anti-CD40-HPV16.E6 / 7.

[0040] Figure 8: In hCD40 transgenic animals, CD40HVac polymerase-IC induces E6 / 7-specific CD8+ T cells.

[0041] Figure 9: CD40HVac plus polymeric IC-induced therapeutic immunity in hCD40Tg mice. (a) Survival curves. 10 mice per group. (b) TC-1 tumor development. 10 mice per group.

[0042] Figure 10 The percentage of E7-specific tetramer+CD8+ T cells in the tumor but not in the blood is negatively correlated with tumor volume.

[0043] Figure 11: When inducing E6 / 7-specific CD8+ T cell responses, CD40HVac prepared using anti-CD40 (clone 12E12) is more effective than that prepared using anti-CD40 (clone 12B6).

[0044] Description of the illustrative implementation scheme

[0045] As described in the examples, anti-CD40 antibodies fused with HPV E6 and E7 antigens (hereinafter referred to as "anti-CD40-HPV16.E6 / 7") have been shown to induce strong immune responses (including strong T-cell responses) against said antigens. This provides a highly effective method for inducing and enhancing immune responses against HPV antigens. Furthermore, anti-CD40-HPV16.E6 / 7 has been combined with polyIC adjuvants for a prime-boost strategy to suppress TC-1 tumor development in human CD40 transgenic mice. Therefore, it has been demonstrated that said anti-CD40-HPV16.E6 / 7 can elicit E6 / E7-specific CD8+ cytotoxic T lymphocytes and, when administered with polyIC adjuvants, acts as an effective vaccine.

[0046] I. Nucleic Acids

[0047] In some embodiments, a recombinant nucleic acid encoding the protein, polypeptide, or peptide described herein is present. Polynucleotides considered for use in methods and compositions include those encoding antibodies, HPV antigens, linker regions, or adjuvants that encode DC receptors or their binding moieties.

[0048] As used in this application, the term "polynucleotide" refers to a recombinant or isolated nucleic acid molecule that does not contain total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, granules, bacteriophages, viruses, etc. In some respects, a polynucleotide includes a regulatory sequence substantially separate from its naturally occurring gene or protein-coding sequence. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA, or synthetic), analogues thereof, or combinations thereof. Additional coding or non-coding sequences may, but are not required, be present within the polynucleotide.

[0049] In this regard, the terms “gene,” “polynucleotide,” or “nucleic acid” are used to refer to nucleic acids (including any sequence required for appropriate transcription, post-translational modification, or localization) that encode proteins, polypeptides, or peptides. As understood by those skilled in the art, the term includes genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid fragments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. Nucleic acids encoding all or part of a polypeptide may comprise a continuous nucleic acid sequence encoding all or part of such a polypeptide. It is also contemplated that the particular polypeptide may be encoded by variations of nucleic acids comprising slightly different nucleic acid sequences but also encoding the same or substantially similar proteins (see above).

[0050] In specific embodiments, there are isolated nucleic acid fragments and recombinant vectors, HPV antigens, a linker region or a fusion protein containing a DC receptor antibody or multiple DC receptor antibodies or fragments thereof, an HPV antigen (e.g., E6 or E7 from any HPV type), and a linker region, wherein the recombinant vector incorporates a nucleic acid sequence encoding a polypeptide (e.g., an antibody or fragment thereof) that binds to a DC receptor. The term “recombinant” may be used in conjunction with the name of a polypeptide or a specific polypeptide, and this generally refers to a polypeptide generated from a nucleic acid molecule manipulated in vitro or a polypeptide generated from a nucleic acid molecule that is a replication product of such a molecule.

[0051] Regardless of the length of the coding sequence itself, nucleic acid fragments can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, and other coding fragments, allowing for significant variations in their full length. Therefore, nucleic acid fragments of virtually any length can be used, with the total length preferably limited by the ease of preparation and intended use in the intended recombinant nucleic acid protocol. In some cases, nucleic acid sequences can encode polypeptide sequences with additional heterologous coding sequences, for example, to achieve polypeptide purification, transport, secretion, post-translational modification, or therapeutic benefits such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides can be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide different from the modified polypeptide.

[0052] In some embodiments, there are polynucleotide variants with high identity to the sequences disclosed herein; there are polynucleotide variants that, when used with the methods described herein (e.g., BLAST analysis using standard parameters), have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity to the polynucleotide sequences provided herein, including all values ​​and ranges therebetween. In some aspects, the isolated polynucleotide will comprise: a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95%, or more identity to the amino acid sequence described herein over its full length; or a nucleotide sequence complementary to the isolated polynucleotide.

[0053] carrier

[0054] Polypeptides can be encoded by nucleic acid molecules. Nucleic acid molecules can be in the form of nucleic acid vectors. The term "vector" is used to refer to a vector nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted to introduce it into a cell where it can replicate and be expressed. The nucleic acid sequence can be "heterologous," meaning it is in an environment different from the cell into which the vector is introduced or from the nucleic acid into which it is incorporated. The nucleic acid sequence includes sequences homologous to sequences in the cell or nucleic acid but located in a position within the host cell or nucleic acid where they are not normally found. Vectors include DNA, RNA, plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). Those skilled in the art will have well-equipped facilities to construct vectors using standard recombination techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both of which are incorporated herein by reference). The vector can be used in host cells to produce an antibody or fragment thereof that binds to a dendritic cell receptor, an HPV antigen or multiple HPV antigens (e.g., E6 and / or E7 from one or more HPV types), a linker or multiple linker regions, an adjuvant or multiple adjuvants, any combination of the above proteins, or a fusion protein or multiple fusion proteins including any combination of the above proteins.

[0055] The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. Expression vectors can contain various "control sequences," which are nucleic acid sequences operatively linked in a particular host organism that are essential for transcription and, possibly, translation of the coding sequence. In addition to control sequences governing transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions and are described herein.

[0056] host cells

[0057] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably. All these terms also include their progeny, which are any and all descendants. It should be understood that all progeny can be different due to intentional or unintentional mutations. In the context of expressing heterologous nucleic acid sequences, “host cell” refers to a prokaryotic or eukaryotic cell, and includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by a vector. Host cells can, and have been, used as recipients of vectors or viruses. Host cells can be “transfected” or “transformed,” which refers to the method by which exogenous nucleic acids, such as recombinant protein-coding sequences, are transferred to or introduced into host cells. Transformed cells include primary test cells and their progeny.

[0058] Some vectors may employ control sequences that enable them to replicate and / or be expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand conditions under which all the aforementioned host cells are incubated to maintain them and allow vector replication. It is also understood and known that techniques and conditions will enable the large-scale preparation of vectors, as well as the preparation of nucleic acids encoded by the vectors and their homologous polypeptides, proteins, or peptides.

[0059] Expression System

[0060] Numerous expression systems exist that include at least some or all of the compositions discussed above. Prokaryotic and / or eukaryotic systems can be used to implement embodiments to generate nucleic acid sequences or their homologous polypeptides, proteins, and peptides. Many such systems are commercially available and widely distributed.

[0061] Insect cell / baculovirus systems can produce high levels of protein expression of heterologous nucleic acid fragments, as described in U.S. Patents 5,871,986 and 4,879,236, both of which are incorporated herein by reference and are available, for example, from INVITROGEN® under the name MAXBAC® 2.0 and from CLONTECH® under the name BACPACK™ Baculovirus Expression System.

[0062] In addition to publicly available expression systems, other examples of expression systems include STRATAGENE®'s fully controlled inducible mammalian expression system, which involves the synthetic ecdysone inducible receptor or its pET expression system, and an E. coli (E. coli) expression system. Another example of an inducible expression system can be obtained from INVITROGEN. ® It carries the T-REX™ (tetracycline-regulated expression) system, an inducible mammalian expression system using a full-length CMV promoter. INVITROGEN ® It also provides what is known as Pichia pastoris (methanol). Pichia methanolica This is a yeast expression system designed to produce recombinant proteins at high levels in the methanol-nutritive yeast *Pichia pastoris*. Those skilled in the art will understand how expression vectors, such as expression constructs, can be used to produce nucleic acid sequences or their homologous polypeptides, proteins, or peptides.

[0063] II. Protein Composition

[0064] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein and can be programmed to modulate one or more properties of a polypeptide with or without loss of other functions or properties. Substitutions can be conserved, meaning that one amino acid is replaced by an amino acid having a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following variations: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamate; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions can be non-conservative, affecting the function or activity of the peptide. Non-conservative changes typically involve replacing residues with chemically different residues, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa.

[0065] The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also contemplated that bacteria containing such variants can be used in the composition and method. Therefore, the protein does not need to be isolated.

[0066] The term “functionally equivalent codons” as used in this article refers to codons that encode the same amino acid, such as the six codons for arginine or serine, and also to codons that encode biologically equivalent amino acids (see the table below).

[0067] Cipher Subtable

[0068]

[0069] It should also be understood that amino acid and nucleic acid sequences may each include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and as long as the sequences meet the criteria set forth above, including maintaining the biological protein activity involved in protein expression, they remain substantially as described in one of the sequences disclosed herein. The addition of terminal sequences is particularly suitable for nucleic acid sequences that may, for example, include various non-coding sequences flanking the 5' or 3' portion of a coding region.

[0070] The following discussion focuses on altering the amino acids of a protein to produce equivalent or even improved second-generation molecules. For example, certain amino acids can replace other amino acids in the protein structure without significantly losing their binding ability to interact with structures such as antigen-binding regions of antibodies or binding sites on substrate molecules, or losing antigenicity in antigenic peptides or proteins. Since the ability to interact and the properties of a protein define its biological functional activity, certain amino acid substitutions can be made in the protein sequence and in its underlying DNA coding sequence to produce proteins with similar properties. Therefore, the inventors have considered that various alterations can be made to the DNA sequence of a gene without significantly losing its biological efficacy or activity.

[0071] When making such changes, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilic amino acid index in conferring biological functions of protein interactions is generally understood in the art (Kyte and Doolittle, 1982). It is acknowledged that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn limits the protein's interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0072] It is also understood in the art that similar amino acid substitutions can be performed efficiently based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, describes how the maximum local average hydrophilicity of a protein, governed by the hydrophilicity of its neighboring amino acids, is correlated with the protein's biological properties. It is believed that an amino acid can be substituted for another amino acid with a similar hydrophilicity value and still produce a biologically and immunologically equivalent protein.

[0073] As described above, amino acid substitutions are generally based on the relative similarity of the substituents in the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering various of the above-mentioned characteristics are well known and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0074] Consider a total polypeptide, peptide, and / or protein of about 0.001 mg to about 10 mg per milliliter in the composition. Therefore, the concentration of protein (including fusion protein) in the composition may be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or higher (or any range thereof). Among them, approximately, at least approximately or at most approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% can be antibodies that bind to the DC receptor or fusion proteins containing antibodies that bind to the DC receptor, and can be used in combination with other HPV antigens, adapter regions, or adjuvants described herein.

[0075] Peptides and Peptide Preparation

[0076] The implementation schemes involve polypeptides, peptides, and proteins, and their immunogenic fragments, used in the various aspects described herein. For example, analyzing the binding of a specific antibody to a DC receptor and the presentation of an HPV antigen, or using a specific antibody to bind to a DC receptor and present an HPV antigen. In specific implementation schemes, all or some of the proteins described herein may also be synthesized in solution or on a solid carrier according to conventional techniques. Various automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference. Alternatively, recombinant DNA techniques may be employed, wherein the nucleotide sequence encoding a peptide or polypeptide is inserted into an expression vector, transformed or transfected into suitable host cells, and cultured under suitable expression conditions.

[0077] One implementation involves using gene-to-cell transfer, including microorganisms, for the preparation and / or presentation of proteins. The gene for the protein of interest can be transferred to a suitable host cell, followed by cell culture under suitable conditions. Nucleic acids encoding virtually any polypeptide can be used. This paper discusses the generation of recombinant expression vectors and the elements included therein. Alternatively, the protein to be produced can be an endogenous protein typically synthesized by cells used for protein production.

[0078] In some respects, the DC receptor or receptor fragment comprises substantially all of the extracellular domains of the protein, which have at least 85%, at least 90%, at least 95%, or at least 97-99% identity with a sequence selected in terms of fragment sequence length, including all values ​​and ranges therein.

[0079] The immunogenic composition also includes a fusion protein composed of an HPV antigen or an immunogenic fragment of an HPV antigen (such as E6 or E7). The HPV antigen can be from any type (such as type 16 or type 18). For example, the HPV antigen can be selected from a single HPV type or a combination of HPV types. The HPV antigen or combination of HPV antigens can be derived from HPV... 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, Models 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. Alternatively, the embodiments may also include a single fusion protein of HPV protein or an immunogenic fragment thereof or an anti-DC receptor antibody or a fragment thereof, as a fusion protein with a heterologous sequence, such as a provider of a T-cell epitope or a purified tag, for example β-galactosidase, glutathione S-transferase, green fluorescent protein (GFP), an epitope tag such as FLAG, myc tag, a multihistidine, or a viral surface protein such as influenza virus hemagglutinin, or a bacterial protein such as tetanus toxoid, diphtheria toxoid, or CRM197.

[0080] Antibodies and antibody-like molecules

[0081] In some respects, one or more antibodies or antibody-like molecules (e.g., peptides containing an antibody CDR domain) specific to the DC receptor can be obtained or generated. These antibodies can be used in the various diagnostic or therapeutic applications described herein.

[0082] As used herein, the term "antibody" is intended to refer broadly to any immune binding agent, such as IgG, IgM, IgA, IgD, and IgE, as well as polypeptides containing antibody CDR domains that retain antigen-binding activity. Therefore, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single-domain antibodies (DAB), Fv, scFv (single-chain Fv), and polypeptides (e.g., anticalin) or nanobodies having antibody CDRs, scaffolding domains displaying CDRs, or CDR-displaying frameworks. For example, the nanobodies may be antigen-specific VHHs (e.g., recombinant VHHs) derived from camelid IgG2 or IgG3, or CDR-displaying frameworks derived from these camelid Igs. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. The methods used to prepare and characterize antibodies are well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

[0083] “Mini-antibodies” or “minibodies” have also been considered for implementation. Minibodies are sFv polypeptide chains comprising an oligomeric domain at their C-terminus separated from the sFv by a hinge region (Pack et al., 1992). The oligomeric domain includes self-associative... - Helices, such as leucine zippers, can be further stabilized by additional disulfide bonds. Oligomeric domains are designed to be compatible with transmembrane vector folding, a process considered to facilitate the folding of peptides into functional binding proteins in vivo. Generally, microantibodies are prepared using recombinant methods well-known in the art. See, for example, Pack et al. (1992); Cumber et al. (1992).

[0084] Antibody-like binding mimic peptides are also considered in the implementation plan. Liu et al. (2003) described “antibody-like binding mimic peptides” (ABiP), which are peptides that serve as simplified antibodies and have the specific advantages of a longer serum half-life and a more convenient synthetic method.

[0085] Alternative backbones for antigen-binding peptides, such as CDRs, are also available and can be used to generate DC receptor-binding molecules according to the implementation scheme. Generally, those skilled in the art know how to determine the type of protein backbone to which at least one CDR generated from the original antibody is grafted. More specifically, it is known that the selection of such a backbone must meet a maximum number of the following criteria (Skerra, 2000): good phylogenetic conservation; known three-dimensional structure (e.g., by crystallography, NMR spectroscopy, or any other technique known to those skilled in the art); small size; little or no post-transcriptional modification; and / or ease of preparation, expression, and purification.

[0086] Such a protein backbone can be derived from, but is not limited to, structures selected from: fibronectin and preferably fibronectin type III domain 10, lipid transport proteins, anticalin (Skerra, 2001), and structures derived from Staphylococcus aureus (…). Staphylococcus aureus Protein Z, thioredoxin A, or proteins with repetitive motifs such as "anchor repeats" (Kohl et al., 2003), "armadillo repeats," "leucine-rich repeats," and "thropopeptide repeats." For example, anticalin or lipid transporter derivatives are a class of binding proteins with affinity and specificity for a variety of target molecules and can be used as DC receptor-binding molecules. Such proteins are described in U.S. Patent Publications Nos. 20100285564, 20060058510, 20060088908, and 20050106660, and PCT Publication No. WO2006 / 056464, which are incorporated herein by reference.

[0087] In some respects, a framework derived from toxins and protein inhibitors of neuronal NO synthase (PIN) can also be used, such as toxins from scorpions, insects, plants, mollusks, etc.

[0088] Monoclonal antibodies (MAbs) are recognized to have certain advantages, such as reproducibility and large-scale production. Implementation methods include monoclonal antibodies derived from humans, mice, monkeys, rats, hamsters, rabbits, and chickens.

[0089] Also considered are “humanized” antibodies, such as chimeric antibodies, bispecific antibodies, recombinant and engineered antibodies and fragments thereof from mice, rats or other species carrying human constant and / or variable domains. As used herein, the term “humanized” immunoglobulin refers to an immunoglobulin containing a human framework region and one or more CDRs derived from non-human (typically mouse or rat) immunoglobulins. The non-human immunoglobulin providing the CDR is referred to as the “donor”, ​​and the human immunoglobulin providing the framework is referred to as the “recipient.” A “humanized antibody” is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. To describe antibodies in some embodiments, the strength of the antibody molecule’s binding epitope can be measured, which is called affinity. The affinity of an antibody can be determined by measuring the association constant (Ka) or dissociation constant (Kd). Antibodies considered useful in some embodiments may have an association constant of about, at least about, or at most about about 10e6, 10e7, 10e8, 10e9 or 10e10 M or any range thereof. Similarly, in some embodiments, the antibody has a dissociation constant of about, at least about, or at most about 10e-6, 10e-7, 10e-8, 10e-9, or 10e-10 M, or any range thereof. These values ​​are reported for the antibodies discussed herein, and the same analysis can be used to evaluate the binding properties of these antibodies.

[0090] In some embodiments, peptides that specifically bind to DC receptors can bind to DC receptors on the cell surface and present HPV antigens, which can generate a strong immune response. Furthermore, in some embodiments, the peptides used can provide protective immunity against HPV or provide a treatment for HPV by generating a strong immune response.

[0091] 1. Methods for generating antibodies

[0092] Methods for generating antibodies (e.g., monoclonal antibodies and / or monoclonal antibodies) are known in the art. In short, polyclonal antibodies are prepared by immunizing animals with a DC receptor polypeptide or a portion thereof according to an embodiment and collecting antiserum from the immunized animals.

[0093] Many animal species can be used to prepare antiserum. Typically, animals used for antiserum preparation include rabbits, mice, rats, hamsters, guinea pigs, or goats. The choice of animal can be based on ease of operation, cost, or the required serum volume, as will be known to those skilled in the art. It should be understood that antibodies can also be genetically prepared by generating transgenic mammals or plants that produce antibodies against the heavy and light chain sequences of the target immunoglobulin and in a recyclable form. In the case of transgenic production in mammals, antibodies can be produced in and recovered from the milk of goats, cows, or other mammals. See, for example, U.S. Patents 5,827,690, 5,756,687, 5,750,172, and 5,741,957.

[0094] As is also well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by using a nonspecific stimulant of the immune response, referred to as an adjuvant. Suitable adjuvants include any acceptable immunostimulatory compound, such as cytokines, chemokines, cofactors, toxins, protoplasmic groups, synthetic compositions, or carriers encoding the adjuvant.

[0095] Adjuvants that may be used depending on the implementation scheme include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, polyIC, montaninde, GMCSF, and monophospholipid A (MPL). RIBI containing three bacterial-derived components—MPL, trehalose dimethicone ester (TDM), and cell wall skeleton (CWS)—in a 2% squalene / Tween 80 emulsion is also considered. Even MHC antigens may be used. Exemplary adjuvants may include complete Freund's adjuvant (containing a nonspecific stimulant for an immune response to killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and / or aluminum hydroxide adjuvant.

[0096] In addition to adjuvants, co-administration of biological response modulators (BRMs) that have been shown to upregulate T-cell immunity or downregulate the activity of suppressor cells may be ideal. Such BRMs include, but are not limited to, cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); low-dose cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ); cytokines such as interferon, IL-2 or IL-12; or genes encoding proteins involved in immune assist functions, such as B-7.

[0097] The amount of immunogen composition used to generate antibodies is based on the nature of the immunogen and the changes in the animal used for immunization. Various routes can be used to administer the immunogen, including but not limited to subcutaneous, intramuscular, intradermal, intraepidermal, intravenous, intratumoral, and intraperitoneal administration. Antibody production can be monitored by collecting blood samples from the immunized animal at different points after immunization.

[0098] Mab can be readily prepared using well-known techniques, such as those exemplified in U.S. Patent 4,196,265, which is incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogenic composition, such as a purified or partially purified protein, polypeptide, peptide, or domain composition, whether wild-type or mutant. The immunizing composition is administered in a manner that effectively stimulates antibody-producing cells.

[0099] The method for generating monoclonal antibodies (MAbs) generally begins with the same route as the preparation of polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to generate monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used to generate monoclonal antibodies. The use of rats is well known and can offer certain advantages (Goding, 1986, pp. 60-61). Mice (e.g., BALB / c mice) are routinely used and generally yield a high percentage of stable fusions.

[0100] If necessary, the MAb produced by any means can be further purified using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Monoclonal antibody fragments can be obtained from monoclonal antibodies prepared by methods including digestion with enzymes such as pepsin or papain, and / or by chemical reduction cleavage of disulfide bonds. Alternatively, monoclonal antibody fragments can be synthesized using an automated peptide synthesizer.

[0101] Molecular cloning methods are also considered for the production of monoclonal antibodies. In one embodiment, a combined immunoglobulin phage library is prepared from RNA isolated from the spleen of an immunized animal, and phages expressing suitable antibodies are selected through screening using cells expressing the antigen and control cells. The advantage of this method over conventional hybridoma techniques is that it can generate and screen up to approximately 10⁴ times more antibodies in a single cycle, and novel specificity is generated by the combination of H and L chains, further increasing the chances of discovering suitable antibodies.

[0102] Another embodiment relates to the preparation of antibodies, for example, as described in U.S. Patent No. 6,091,001, which describes a method for generating antibody-expressing cells from a genomic sequence of a cell including a modified immunoglobulin locus using disclosed Cre-mediated site-specific recombination. The method involves first transfecting antibody-producing cells with a homologous targeting vector containing a lox site and a targeting sequence homologous to a first DNA sequence of a region of an immunoglobulin locus to be converted into a modified region, thus inserting the first lox site into the genomic sequence via site-specific homologous recombination. Cells are then transfected with a lox targeting vector containing a second lox site adapted for Cre-mediated recombination with an integrated lox site and the modified sequence to convert the region of the immunoglobulin locus into a modified region. This conversion is performed in vivo by interacting the lox site with Cre to allow the modified sequence to be inserted into the genomic sequence via Cre-mediated site-specific recombination of the lox site.

[0103] Alternatively, monoclonal antibody fragments can be synthesized using an automated peptide synthesizer or by expressing a full-length gene or gene fragment in E. coli.

[0104] It is also considered that monoclonal antibodies can be further screened or optimized for properties related to specificity, affinity, half-life, immunogenicity, binding association, binding dissociation, or overall functional properties related to the expected therapeutic or protective effect. Therefore, it is considered that monoclonal antibodies may have 1, 2, 3, 4, 5, or 6 or more variations in the amino acid sequence of the 1st, 2nd, 3rd, 4th, 5th, 6th, or 6th CDRs of the monoclonal antibodies or humanized antibodies provided herein. It is considered that amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of CDR1, CDR2, CDR3, CDR4, CDR5, or CDR6 in the VJ or VDJ region of the variable region of the antibody's light or heavy chain may have insertions, deletions, or substitutions using conserved or non-conserved amino acids. Those amino acids that can be substituted or constitute substitutions are disclosed above.

[0105] In some implementations, fragments of the whole antibody can function as antigen-binding fragments. Examples of binding fragments include (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of either VH or VL domains (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003); (v) isolated CDR regions; (vi) F(ab')2 fragments, bivalent fragments containing two linked Fab fragments; and (vii) single-chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that enables the two domains to associate to form an antigen-binding site (Bird et al., 1988; Huston et al., 1989). (viii) Bispecific single-chain Fv dimers (PCT / US92 / 09965) and (ix) “bifunctional antibodies”, multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; Holliger et al., 1993). Fv, scFv, or bifunctional antibody molecules can be stabilized by adding a disulfide bridge connecting the VH and VL domains (Reiter et al., 1996). Microantibodies containing scFv bound to the CH3 domain can also be prepared (Hu et al., 1996). All citations in this paragraph are incorporated by reference.

[0106] Antibodies also include bispecific antibodies. Bispecific or bifunctional antibodies form second-generation monoclonal antibodies, in which two distinct variable regions are combined in the same molecule (Holliger & Winter, 1999). Their use has been demonstrated in the diagnostic and therapeutic fields by their ability to recruit novel effector functions or target several molecules on the surface of tumor cells. When bispecific antibodies are to be used, these can be conventional bispecific antibodies, which can be manufactured in various ways (Holliger et al., 1993), for example, prepared by chemical methods or by hybridomas, or can be any of the aforementioned bispecific antibody fragments. These antibodies can be obtained by chemical methods (Glennie et al., 1987; Repp et al., 1995) or by somatic cell methods (Staerz & Bevan, 1986), but also by genetic engineering techniques that enable heterodimerization, thereby facilitating the purification process of the discovered antibodies (Merchand et al., 1998). Examples of bispecific antibodies include those using BiTE™ technology, where binding domains of two antibodies with different specificities can be used and directly linked via a flexible short peptide. This allows the two antibodies to be combined on a short, single polypeptide chain. Bifunctional antibodies and scFvs can be constructed using only the variable domains, without the Fc region, potentially reducing the effectiveness against idiotype reactions. All citations in this paragraph are incorporated by reference.

[0107] Bispecific antibodies can be constructed as full IgG, bispecific Fab′2, ​​Fab′PEG, bifunctional antibodies, or alternatively, bispecific scFv. Furthermore, two bispecific antibodies can be linked together using conventional methods known in the art to form a tetravalent antibody.

[0108] Bispecific bifunctional antibodies differ from bispecific whole antibodies in that they can be particularly useful because they can be readily constructed and expressed in *E. coli*. Bifunctional antibodies (and many other peptides such as antibody fragments) with suitable binding specificity can be readily selected from a library using phage display (WO94 / 13804). If one arm of the bifunctional antibody remains constant, for example, having specificity against the DC receptor, then a library can be formed in which the other arm is varied and antibodies with suitable specificity are selected. Bispecific whole antibodies can be prepared by alternative engineering methods, as described in Ridgeway et al., 1996, which is incorporated herein by reference.

[0109] Antibody and peptide conjugate

[0110] The implementation scheme provides antibodies and antibody-like molecules targeting the DC receptor, and polypeptides and peptides linked to at least one pharmaceutical agent to form antibody conjugates or payloads. To enhance the efficacy of antibody molecules as diagnostic or therapeutic agents, at least one desired molecule or moiety is typically linked, covalently bound, or conjugated. Such molecules or moiety can be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activities, such as cytotoxic or immunostimulatory activities. Non-limiting examples of effector molecules linked to antibodies include adjuvants, toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, etc. In contrast, reporter molecules are defined as any part that can be detected using analytical methods. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabeled molecules, haptens, fluorescently labeled molecules, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands such as biotin. The following U.S. patent applications are incorporated herein by reference in such extent as to disclose antibodies, antibody moieties, antigens, adapters, specific sequences of such antibodies, antigens and adapters, adjuvants, fusion proteins or other components of therapeutic compositions, host cells or compositions, dendritic cell sources and dendritic cell culture / activation, and dendritic cell derivatives and derivatives derived from dendritic cells, and methods of using such fusion proteins: 12 / 024,036; 12 / 024,897; 12 / 025,010; 12 / 026,095; 12 / 036,138; 12 / 036,158; 12 / 504,463; 12 / 717. 778; 12 / 717,789; 12 / 717,804; 12 / 718,365; 12 / 882,052; 12 / 882,052; 13 / 100,684; 13 / 208,993; 13 / 269,951; 13 / 282,112; 13 / 415,564; 13 / 424,582; 13 / 430,206; 13 / 594,397; 13 / 596,526; WO2010 / 104749; 13 / 465,371; 13 / 397,932; PCT / US13 / 72217 and PCT / US2013 / 05839.

[0111] Certain examples of antibody conjugates are those in which an antibody is attached to a detectable label. A “detectable label” is a compound and / or element that can be detected due to its specific functional properties and / or chemical characteristics, the use of which enables the antibody to which it is attached to to be detected and / or further quantified when necessary.

[0112] Antibody conjugates include those primarily intended for in vitro use, wherein the antibody is linked to a second binding ligand and / or enzyme (enzyme tag) that will produce a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) catalase, or glucose oxidase. Preferred second binding ligands are biotin and / or avidin and streptavidin compounds. The use of these tags is well known to those skilled in the art and described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241; each incorporated herein by reference.

[0113] Molecules containing azido groups can also be used to form covalent bonds with proteins via reactive daene intermediates generated by low-density ultraviolet light (Potter & Haley, 1983). Specifically, 2- and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azidonucleotides have been used to locate the nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et al., 1989) and can be used as antibody binders.

[0114] Several methods for linking or conjugating antibodies to their conjugated portions are known in the art. Some linking methods involve the use of metal chelating complexes, employing, for example, organic chelating agents linked to the antibody, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3,6-diphenylglyuron-3 (U.S. Patents 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies can also be reacted with enzymes in the presence of conjugating agents such as glutaraldehyde or periodate. Conjugates with fluorescein labels are prepared in the presence of these conjugating agents or by reaction with isothiocyanates. In U.S. Patent 4,938,948 (incorporated herein by reference), imaging of breast tumors is achieved using a monoclonal antibody, and the detectable imaging portion is bound to the antibody using a linker such as methylparaben or 3-(4-hydroxyphenyl)propionic acid-N-succinimide.

[0115] In some embodiments, immunoglobulin derivatization by selectively introducing thiol groups into the Fc region of the immunoglobulin is considered, without altering the reaction conditions at the antibody conjugation site. Antibody conjugates prepared according to this method are disclosed to exhibit improved lifetime, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific linkages of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to sugar residues in the Fc region, have also been disclosed in the literature (O'Shannessy et al., 1987, incorporated herein by reference). This method has been reported to produce promising antibodies for diagnostic and therapeutic purposes, which are currently under clinical evaluation.

[0116] 2. Dendritic cell-specific antibodies

[0117] In some respects, antibodies used to target HPV antigens to dendritic cells are dendritic cell-specific antibodies that bind to dendritic cell receptors or receptors expressed by dendritic cells. Several antibodies that can be used for this purpose are known in the art.

[0118] In some embodiments, anti-DCIR antibodies are used to target HPV antigens to dendritic cells. One example includes an anti-dendritic cell immune receptor monoclonal antibody conjugate, wherein the conjugate comprises an antigenic peptide loaded or chemically conjugated to the antibody. Such antibodies are described in U.S. Application No. 61 / 332,465, which is incorporated herein by reference.

[0119] In other embodiments, anti-CD40 antibodies are used to target HPV antigens to dendritic cells. Compositions and methods for expressing, secreting, and using anti-CD40 antibodies as vaccines and antigen delivery vectors having a linked antigenic peptide are described in WO 2010 / 104761; all methods disclosed herein are incorporated herein by reference. In some embodiments, the anti-CD40 antibody includes heavy and light chain variable regions from monoclonal antibodies 12E12, 11B6, or 12B4. In other embodiments, the anti-CD40 antibody includes heavy and light chain CDRs from monoclonal antibodies 12E12, 11B6, or 12B4.

[0120] In some respects, anti-LOX-1 antibodies are used to target HPV antigens to dendritic cells. One example of such an antibody can be used to target the LOX-1 receptor on immune cells and enhance antigen presentation by antigen-presenting cells expressing LOX-1. An example of such a LOX-1 antibody is described in WO 2008 / 103953, the contents of which are incorporated herein by reference.

[0121] In other respects, anti-CLEC-6 antibodies are used to target HPV antigens to dendritic cells. An example of such antibodies includes anti-CLEC-6 antibodies used to enhance antigen presentation via antigen-presenting cells expressing CLEC-6. Such antibodies are described in WO 2008 / 103947, the methods and contents of which are incorporated herein by reference.

[0122] In yet another embodiment, an anti-Dectin-1 antibody is used to target HPV antigens to dendritic cells. Anti-Dectin-1 antibodies that enhance antigen presentation by antigen-presenting cells expressing Dectin-1 are described in WO 2008 / 118587, the contents of which are incorporated herein by reference.

[0123] In some respects, peptide linkers are used to connect dendritic cell-specific antibodies to HPV antigens to be presented. Peptide linkers can incorporate glycosylation sites or introduce secondary structures. Furthermore, these linkers improve the expression efficiency or stability of fusion proteins, and thus enhance the efficiency of antigen presentation to dendritic cells. Such linkers may include: SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO: 6), PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO: 29), TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO: 30), QTPTNTISVTPTNNSTPTNNSNPKPNP (SEQ ID NO: 5), or TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO: 31). These examples and others are discussed in WO2010 / 104747, the contents of which are incorporated herein by reference.

[0124] In other implementations, the immune adjuvant is directly fused to dendritic cell-specific antibodies to enhance vaccine efficacy. In some aspects, the immune adjuvant can be a Toll-like receptor (TLR) agonist. TLR agonists include those derived from gut Salmonella (… Salmonella enterica ) or Vibrio cholerae ( Vibrio cholerae TLR agonists are flagellated proteins. They can be specific to certain TLR types (i.e., TLR2, TLR5, TLR7, or TLR9 agonists) and can exist in any combination or as any variant. Examples of such immune adjuvants are described in U.S. Applications No. 13 / 208,993, No. 13 / 415,564, and WO 2012 / 021834, the entire contents of which are incorporated herein by reference. U.S. Patent Publications 2012 / 0039,916 and 2012 / 023,102 are also incorporated by reference to their respective disclosures of different TLR agonists.

[0125] In some embodiments, compositions and fusion proteins comprising dendritic cell antibodies and HPV antigens are used to treat HPV-related diseases or HPV-related pathologies. In some embodiments, HPV-related diseases are dysplasia, benign tumors, precancerous tumors, or cancer (malignant tumors). In some embodiments, the tissues or organs affected by dysplasia, benign tumors, precancerous tumors, or cancer are the cervix, vulva, vagina, penis, anus, oropharynx, head and neck, pharynx, or lungs. In some specific embodiments, HPV-related diseases or HPV-related pathologies are cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and / or anal intraepithelial neoplasia (AIN). In yet another embodiment, compositions and fusion proteins comprising dendritic cell antibodies and HPV antigens are used to treat HPV-related warts, plantar warts, flat warts, anogenital warts, anal lesions, genital cancer, verrucous epidermal dysplasia, localized epithelial hyperplasia (oral), oral papilloma, oropharyngeal carcinoma, verrucous cysts, or laryngeal papilloma.

[0126] III. Treatment Methods

[0127] As described above, the compositions and methods of using the compositions can treat subjects who have, are suspected of having, or are at risk of developing HPV-related infections or diseases (e.g., prevent HPV infection or HPV-related diseases, or elicit a strong immune response to or enhance an immune response to HPV or HPV-related diseases).

[0128] As used herein, the phrase "immune response" or its equivalent "immunological response" refers to a humoral (antibody-mediated), cellular (mediated by antigen-specific T cells or their secreted products), or both humoral and cellular responses in a recipient patient against the proteins, peptides, or polypeptides of the present invention. Treatment or therapy can be an active immune response induced by the administration of an immunogen or a passive treatment achieved by the administration of a fusion protein composition, an immunogenic composition or protein composition comprising an antibody / antigen fusion protein, material comprising an antibody / antigen fusion protein, or sensitized T cells.

[0129] For the purposes of this specification and the appended claims, the terms “epitope” and “antigenic determinant” are used interchangeably to refer to a site on an antigen that B cells and / or T cells respond to or recognize. B cell epitopes can be formed from consecutive amino acids or from discontinuous amino acids that are close together by ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by ternary folding are generally lost upon treatment with denaturing solvents. In a distinctive spatial conformation, an epitope typically comprises at least three and more, usually at least five or eight to ten amino acids. Methods for determining the spatial conformation of an epitope include those described in the Epitope Localization Scheme (1996). For CD8 cells, T cells recognize consecutive epitopes of about nine amino acids, and for CD4 cells, consecutive epitopes of 13 to 15 amino acids are recognized. T cells that recognize epitopes can be identified by in vitro assays measuring antigen-dependent proliferation, such as by incorporation of 3H-thymidine into sensitized T cells in response to epitopes (Bruke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assays, Tigges et al., 1996, cited in citation), or by cytokine secretion.

[0130] The presence of a cell-mediated immune response can be determined by proliferation analysis (CD4(+) T cells) or CTL (cytotoxic T lymphocyte) analysis. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of the immunogen can be distinguished by isolating IgG and T cells from an immunized syngeneic animal, respectively, and measuring the protective or therapeutic effect in a second subject. As used herein and in the claims, the terms “antibody” or “immunoglobulin” are used interchangeably.

[0131] Optionally, the antibody, or preferably the immune portion of an antibody, may be chemically conjugated to a fusion protein with other proteins or expressed as a fusion protein with other proteins. For the purposes of this specification and the appended claims, all such fusion proteins are included in the definition of an antibody or the immune portion of an antibody.

[0132] In one implementation, the method includes treating a disease or condition caused or suspected to be caused by an HPV pathogen. In some aspects, the implementation includes treatment for HPV infection, such as infection acquired from an HPV-positive individual. In some implementations, treatment is administered in the presence of HPV antigens. Furthermore, in some instances, treatment includes administering other agents commonly used to combat viral infections.

[0133] The therapeutic composition is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount to be administered depends on the subject being treated. The precise amount of active ingredient to be administered depends on the judgment of the practitioner. Suitable regimens for initial and follow-up administration are also variable, but initial administration followed by subsequent administration is typical.

[0134] The methods of application can vary considerably. Any conventional method of administering peptide therapeutic agents is applicable. These are considered to include oral administration on a physiologically acceptable solid matrix or in a physiologically acceptable dispersion, parenteral administration by injection, etc. The dosage of the composition will depend on the route of administration and will vary according to the size and health condition of the subject.

[0135] In some cases, it may be desirable to administer the composition multiple times, for example, 2, 3, 4, 5, 6 or more times. It can be administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11, 12 weeks, including all ranges in between.

[0136] Combination therapy

[0137] The compositions and related methods, particularly those that bind to DC receptors and deliver antibodies that bind to HPV antigens or multiple antigens or peptides or multiple peptides to patients / subjects, may also be used in combination with conventional antiviral or anticancer therapies or drugs. These include, but are not limited to, entry inhibitors, CCR5 receptor antagonists, nucleoside reverse transcriptase inhibitors, nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and maturation inhibitors. Anticancer therapies include, but are not limited to, chemotherapy, radiotherapy, or radiation therapy.

[0138] The compositions and related methods, particularly those for administering antibodies that bind to DC receptors and deliver HPV antigens or multiple antigens or peptides or multiple peptides to patients / subjects, may also be used in combination with the administration of one or more anticancer drugs, including but not limited to abiraterone acetate, abitrexate (methotrexate), abraxane (paclitaxel albumin-stabilized nanoparticles), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, ado-trastuzumab Emtansine, adriamycin (doxamycin hydrochloride), adrucil (fluorouracil), afatinib maleate, and A Finitor (evermus), Idalor (imiquimod), Aldehyde interleukin, Alemumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), Ambochlorin (chlorambucil), Amboclorin (chlorambucil), Aminolevulinic acid, Anastrozole, Aprepitant, Acada ​​(pamir phosphate disodium), Renin (anastrozole), Anoxin (exemestane), Arranon (nerapine), Arsenic trioxide, Arzerra (orfamumab), Erwinia asparaginase, Avastin (bevacizumab), Axitinib, Azacitidine, BEACOPP, Bendamustine hydrochloride, BEP, Bevacizumab, Bexarotin, Tosimomumab and I 131 Iodotosimomab), Bleomycin, Bortezomib, Bosulif (bosutinib), Bosutinib, Brentuximab Vedotin, Cabazitaxel, Cabozantinib S-malate, CAF, Campaz (alemumab), Captopril (irinotecan hydrochloride), Capecitabine, CAPOX, Carboplatin, Carboplatin-paclitaxel, Carfilzomib, CeeNU (cyclohexanonitrosourea), Cerubidine (daunorubicin hydrochloride), Cervarix (including HPV) Recombinant bivalent HPV vaccines containing recombinant L1 protein of types 16 and 18, cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cyclophosphamide, clorapine, clofarex, clolar, CMF, Cometriq (cazozidinib-S-malate), COPP, COPP-ABV, Cosmegen (actinomycin D), crizotinib, CVP, cyclophosphamide, cyfos (ifosfamide), cytarabine, liposomes, cytarabine-U, cyclophosphamide, dabrafenib, dazometazidine, decitabine D, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denifedipine, denosumab, depoCyt (cytarabine liposomes).DepoFoam (cytarabine liposome), dexrazoxane hydrochloride, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dazometamide), Efudex (fluorouracil), Elitek (raburicase), Ellence (epirarubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag ethanolamine, Emend (arepimetamide), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Ervedg e (Vimodexa), Erlotinib Hydrochloride, Erwinaze (Erwinia asparaginase), Etopophos (Etoposide phosphate), Etoposide, Etoposide phosphate, Evacet (Doxorubicin hydrochloride liposome), Everolimus, Evista (Raloxifene hydrochloride), Exemestane, Fareston (Toremifene), Faslodex (Flushexant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fluoradarabine phosphate), Fluoradarabine phosphate, Fluoroplex (Fluorouracil), Fluorouracil, Foles (Methotrexate), Foles PFS (Methotrexate), FOLFIRI, FOLFIRI-Bevacizumab, FOLFIRI-Cetuximab, FOLFIRINOX, FOLFOX, Folotyn (Platrouxa), FU-LV, Fulvestrant, Gardasil (a recombinant quadrivalent HPV vaccine containing recombinant L1 protein for HPV types 6, 11, 16, and 18), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemcitabine-Ozolpidem, Gemzar (Gemcitabine Hydrochloride), Afatinib (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Glucapeptide, Goserelin Acetate, Halaven (Erebulin Mesylate), Hercepti Trastuzumab, Bivalent HPV Vaccine (Recombinant), Quadrivalent HPV Vaccine (Recombinant), Hycamtin (Topotecan Hydrochloride), Ibrutinib, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A (Recombinant Interferon Alpha-2b), 131I-Tosimomab and Tosimomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidesin), Ixapipron, Ixempra (Ixapipron), Jakafi (Ruxotinib Phosphate), Jevtana (Cabbathaxanthin).Kadcyla (Ado-trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Recombinant Keratinocyte Growth Factor), Kyprolis (Carfilzomib), Lapatinib Toluenesulfonate, Lenalidomide, Letrozole, Calcium Leucovorin, Leukeran (Chlorimuric Acid), Leucylproline Acetate, Levulan (Aminolevulinic Acid), Linfolizin (Chlorimuric Acid), LipoDox (Oxytocin Hydrochloride Liposome), Cytarabine Liposome, Cyclohexanonitrosourea, Lupron (Leucylproline Acetate), LupronDepo t (leucylproline acetate), Lupron Depot-Ped (leucylproline acetate), Lupron Depot-3Month (leucylproline acetate), Lupron Depot-4Month (leucylproline acetate), Marqibo (vincristine sulfate liposome), Matulane (isopropylamine benzylhydrazine hydrochloride), nitrogen mustard hydrochloride, megestrol acetate, megestrol acetate, Mekinist (trametinib), mercaptopurine, sodium thiosulfate, Mesnex (sodium thiosulfate), Methazolastone (temozolomide), methotrexate, methotrexate Methotrexate (LPF), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitozytrex (Mitomycin C), MOPP, Mozobil (Mozobil), Mustargen (Nitrogen Mustard Hydrochloride), Mutamycin (Mitomycin C), Mylosar (Azacitidine), Mylotarg (Gilotumab), Paclitaxel Nanoparticles (Paclitaxel Albumin-Stabilized Nanoparticles), Navelbine (Vinorelbine Tartrate), Nerapine, Neosar (Cyclophosphamide), Neupogen (Figrintin), Nexavar (Toluene) Sorafenib sulfonate, nilotinib, Novadex (tamoxifen citrate), Nplate (romilastine), oxaliplatin, oxamumab, homoharringtonine, Oncaspar (pegapascalase), Ontak (denisil interleukin), OEPA, OPPA, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, recombinant keratinocyte growth factor, palonosetron hydrochloride, pamidronate disodium, panitumumab, Paraplatin (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, pegapamase, pegylated interferon α-2b, PEG-intron (Peginterferon e-2b), pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), praxavila, pomalidomidePomalyst (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone, Isopropylbenzylhydrazine Hydrochloride, Proleukin (Adeleukin), Prolia (Denosema), Promacta (Eltrombopag Ethanolamine), Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Radium-223 Dichloride, Raloxifene Hydrochloride, Raburicase, R-CHOP, R-CVP, Recombinant Bivalent HPV Vaccine, Recombinant Quadrivalent HPV Vaccine, Recombinant Interferon Alpha-2b, Regorafenib, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), Rituximab, Romidixin, Romistachycin, Rubidomycin (Daunorubicin Hydrochloride), Ruxotinib Phosphate, Sclerosol Intrapleural Aerosol (talc), Sipuleucel-T, Sorafenib Tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc Powder (talc), Steritalc (talc), Stivarga (regorafenib), Sunitinib Malate, Sutent (sunitinib Malate), Sylatron (pegylated interferon alpha-2b), Synovir (thalidomide), Synribo (homoharringtonine), TAC, Tafinlar (dabrafenib), Talc, Tamoxifen Citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotin), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), Temozolomide, tesimolimus, thalidomide, Thalomid, Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (tesimolimus), tosimomab and I 131I-Tosimomab, Totect (dexrazoxan hydrochloride), Trametinib, Trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib dimethylbenzenesulfonate), Vandetanib, VAMP, Vectibix (panitumumab), VeIP, Velban (vincrine sulfate), Velcade (bortezomib), Velsar (vincrine sulfate), Vemurafenib, VePesid (etoposide), Viadur (leucylproline acetate), Vidaza (azacitidine), Vincrine sulfate, Vincasar PFS (vincristine sulfate), Vincristine sulfate, Vincristine sulfate liposomes, Vinorelbine tartrate, Vemodiga, Voraxaze (glutapeptide), VorinostatVotrient (pazopanib hydrochloride), Wellcovorin (calcium leucovorate), Xalkori (crizotinib), Xeloda (capecitabine), XELOX, Xgeva (denosema), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (teimomab), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zoledronic acid, Zolinza (vorinostat), Zytiga (zoledronic acid), and Zytiga (abiraterone acetate).

[0139] The compositions and related methods, particularly the administration of antibodies that bind to DC receptors and deliver HPV antigens or multiple antigens or peptides or multiple peptides to patients / subjects, can also be used in conjunction with the administration of radiotherapy, including but not limited to X-rays, gamma rays, and charged particles. This radiation can be delivered by an external instrument (external beam radiotherapy) or it can originate from radioactive material placed within the body near cancer cells (internal or short-range radiotherapy). Internal radiotherapy can be systemic (e.g., radioactive iodine). External beam radiotherapy can include, but is not limited to, three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT), image-guided radiotherapy (IGRT), helical tomotherapy, stereotactic radiosurgery (SRS), body stereotactic radiotherapy (SBRT), proton therapy, or other charged particle beams (such as electron beams). Internal or short-range radiotherapy can include intrastitial short-range therapy, which uses a radiation source placed within the tumor tissue and can be used to deliver a dose higher than external beam radiotherapy with less damage to normal tissue. Short-range radiotherapy can be administered at low or high dose rates. In another embodiment, short-range radiation therapy can be permanent or temporary. Radiation therapy can include whole-body radiation therapy. Whole-body radiation therapy can include swallowing or injecting radioactive materials, including but not limited to any single dose, multiple dose, or combined dose of radioactive iodine (…). 131I), teimimozumab (Zevalin®), 131-tosimozumab (Bexxar®), samarium-153-lecithin (Quadramet®), and strontium chloride-89 (Metastron®), or any monoclonal antibody bound to a radioactive material. Depending on the implementation, the radiation dose may be adjusted based on the specific disease, condition, or cancer being treated. In some implementations, the single dose or total dose may be 1-10 Gy, 10-20 Gy, 20-40 Gy, 40-60 Gy, or 60-80 Gy, or any value or range derived therefrom. In some implementations, the radiation therapy or dose may be graded. In one implementation, the total dose may be graded daily or weekly. In some implementations, the daily graded dose may be 1.8-2 Gy. Consider grading the total dose into daily or weekly doses ranging from 0.1 Gy to 10 Gy.

[0140] In one aspect, the treatment is considered for use in combination with antiviral or anticancer therapy. Alternatively, the treatment may be administered minutes to weeks before or after other pharmaceutical treatments. In embodiments where other pharmaceuticals and / or proteins or polynucleotides are administered separately, it is generally ensured that the effective period does not terminate between each delivery so that the therapeutic composition can still exert a beneficial combined effect on the subject. In such cases, it is considered that the two forms may be administered separately over approximately 12-24 hours, more preferably over approximately 6-12 hours. However, in some cases, a significantly prolonged period of administration may be desired, where interruptions between the respective administrations range from several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8).

[0141] In another aspect, the vaccine can be administered as part of a primary / booster immunization strategy. In any of the embodiments described herein, the primary vaccine dose can be administered using a DC-specific antibody fused to an HPV antigen. The booster vaccine can be administered using a second vaccine of the same type or from a different type. Examples of other HPV vaccines include Gardasil™ (a recombinant quadrivalent HPV vaccine containing recombinant L1 proteins of HPV types 6, 11, 16, and 18) or Cervarix™ (a recombinant bivalent HPV vaccine containing recombinant L1 proteins of HPV types 16 and 18). Further examples of such different vaccines include naked DNA vaccines or recombinant viruses. The second vaccine may include additional HPV antigens besides the E6 or E7 antigen used in the first vaccine. It is also contemplated that the second vaccine may include HPV proteins, such as E6 or E7 proteins, plus an adjuvant administered directly or independently.

[0142] Various combinations of treatments can be used, such as antiviral or anticancer ("A"), and antibody vaccines ("B") that include antibodies that bind to DC receptors and deliver HPV antigens or peptides or their consensus peptides.

[0143]

[0144] The antibody composition will be administered to the patient / subject in accordance with the general protocol for administering these compounds, taking into account the toxicity of the composition, if any. Treatment cycles are expected to be repeated as needed. Various standard therapies, such as hydration therapy, may also be considered and may be used in combination with the described treatments.

[0145] Universal pharmaceutical compositions

[0146] In some embodiments, the pharmaceutical composition is administered to a subject. Different aspects may involve administering an effective amount of the composition to the subject. In some embodiments, an antibody that binds to a DC receptor and delivers an HPV antigen or peptide or a concomitant peptide thereof may be given to a patient to protect against or treat infection with one or more HPV types, or to protect against or treat one or more HPV-related diseases such as cancer. Alternatively, an expression vector encoding one or more such antibodies or peptides may be given to a patient as a prophylactic treatment. Additionally, such compositions may be administered in combination with antibiotics, antivirals, or anticancer agents. Such compositions are generally dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0147] The terms "pharmaceuticalally acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, anaphylactic, or other adverse reactions when administered to animals or humans. As used herein, "pharmaceuticalally acceptable carriers" include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where any conventional media or agent is incompatible with the active ingredient, their use in immunogenic and therapeutic compositions is considered. Supplemental active ingredients, such as other anti-infective agents and vaccines, may also be incorporated into the composition.

[0148] The active compound can be formulated for parenteral administration, for example, for injection via mucosal, intravenous, intramuscular, subcutaneous, intratumoral, or even intraperitoneal routes. Typically, such compositions can be prepared as liquid solutions or suspensions; they can also be prepared in solid forms suitable for addition to liquids prior to injection to prepare solutions or suspensions; and the formulations can also be emulsified.

[0149] Suitable drug forms for injectable applications include sterile aqueous solutions or dispersions; preparations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid enough to be easily injected. It must also be stable under manufacturing and storage conditions and must be protected against microbial contamination such as bacteria and fungi.

[0150] Protein compositions can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of proteins) and pharmaceutically acceptable salts formed with: inorganic acids, such as hydrochloric acid or phosphoric acid; or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed from free carboxyl groups can also be derived from: inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0151] Pharmaceutical compositions may contain solvents or dispersion media, such as water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Suitable flowability can be maintained, for example, by using coatings such as lecithin, in the case of dispersions by maintaining a desired particle size, and by using surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, will be preferred. Prolonged absorption of injectable compositions can be achieved by using absorbents such as aluminum monostearate and gelatin in the composition.

[0152] The preparation of sterile injection solutions is as follows: The active compound is added in the required amount to a suitable solvent containing the various other components listed above, followed by filtration sterilization or an equivalent step. Generally, dispersions are prepared by adding various sterilized active ingredients to a sterile solvent containing a base dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injection solutions, preferred methods include vacuum drying and freeze-drying techniques, which produce powders of the active ingredients, plus any additional desired components from the preceding sterile filtration solution.

[0153] The composition will generally be administered via any conventional route. This includes, but is not limited to, oral, nasal, or buccal administration. Alternatively, administration may be by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, intratumoral, intranasal, or intravenous injection. In some embodiments, the vaccine composition may be inhaled (e.g., U.S. Patent 6,651,655, which is specifically incorporated herein by reference). Such compositions will generally be administered as pharmaceutically acceptable compositions comprising physiologically acceptable carriers, buffers, or other excipients.

[0154] The effective amount of the therapeutic or preventative composition is determined based on the intended target. The term "unit dose" or "dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired response discussed above in relation to its administration, i.e., the appropriate route and regimen. The amount to be administered, depending on the number of treatments and the unit dose, depends on the desired protection.

[0155] The precise amount of the composition also depends on the judgment of the practitioner and is unique to each individual. Factors affecting dosage include the subject's physical and clinical condition, route of administration, intended therapeutic goal (symptom relief or cure), and the potency, stability, and toxicity of the specific composition.

[0156] During preparation, the solution will be administered in a manner compatible with the dosage form and in an amount effective for treatment or prevention. The formulation is readily administered in various dosage forms, such as the injectable solution types described above.

[0157] IV. Examples

[0158] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the embodiments, following representative techniques discovered by the inventors, function well in the implementation of the invention and can therefore be considered as preferred embodiments for its implementation. However, those skilled in the art will understand that many variations can be made in the disclosed specific embodiments without departing from the spirit and scope of the invention, and the same or similar results can still be obtained.

[0159] Example 1—Recombinant fusion protein of anti-DC receptor (DCR) and HPV E6 and E6 fusion proteins

[0160] Figure 1This invention presents a protocol for developing expression constructs that generate anti-DC receptor antibodies fused to E6 and E7 sequences from HPV 16 and 18. The protocol determines the order in which antigen cassettes encoding E6 and E7 from HPV 16 and 18 are efficiently secreted and intact when fused to the C-terminus of the H chain. There are 64 possible combinations of these four sequences alone, with even more when flexible adapter sequences are inserted. The inventors' strategy is a stepwise approach, starting with each antigen individually, with or without a preceding flexible adapter [8 initial constructs], and then selecting those vectors that express the most efficiently for adding additional cassettes. Each construction and testing cycle takes one week. The final establishment of the production CHO-S cell line takes an additional 8 weeks, including scaling up to a level suitable for vaccine production for clinical studies in humans and animals in vitro.

[0161] Transient expression vectors encoding antibody heavy chains have an in-frame Nhe I site at the C-terminus and insert an antigen or flexible adapter encoding a Spe I-Not I cassette between the Nhe I and Not I sites. In this connection, the Nhe I site is lost, but each cassette encodes a new C-terminal in-frame Nhe I site. Therefore, additional antigen or adapter cassettes can be added in a iterative manner. Each new construct was transiently transfected into 293F cells using a matched light chain vector, and the secreted vaccine was separated by protein A affinity at 72 hours and analyzed using SDS-PAGE. Well-expressed constructs are preferred vectors for adding new cassettes. Figure 1 ).

[0162] The inventors engineered expression constructs using HPV 16 E6 and E7 and HPV 18 E6 and E7 sequences fused to the C-terminus of the antibody heavy chain. When co-transfected into 293F cells using a matching light chain expression vector, constructs having HPV 16 or HPV 18 E6 or HPV 16 or HPV 18 E7 sequences directly fused to the C-terminal codons of the anti-dendritic cell receptor antibody heavy chain failed to secrete any detectable vaccine (not shown). However, similar vectors incorporating a flexible adapter sequence (Flex v1) secreted a vaccine (…). Figure 2 (Channels 1 and 3). Furthermore, constructs that added HPV E6 to the Flex v1 HPV E7 vector also secreted vaccines, and vice versa. Figure 2(Channels 2 and 4). Successful expression of such vaccines does not depend on the variable region sequence. Therefore, the Flex v1-HPV E6 / E7 sequence was transferred into an established vector for stable expression of anti-CD40-Flex v1-HPV 16 or 18E6-HPV 16E7, anti-Langerin-Flex v1-HPV 16 or 18E6-HPV 16E7, and control hIgG4-Flex-v1-HPV 16 or 18E6-HPV 16E7 in CHO-S cells.

[0163] In peripheral blood from healthy donors, anti-CD40-HPV16.E6 / 7 can efficiently bind to dendritic cells (DCs). Peripheral blood mononuclear cells (PBMCs) were obtained from the blood of healthy individuals. PBMCs were incubated on ice for 15 minutes in the presence of varying amounts of anti-CD40-HPV16.E6 / 7 or control IgG4-HPV16.E6 / 7 protein. Cells were vigorously washed and then stained with anti-E6 / 7 antibody to detect the protein bound to the cell surface. Figure 3 As shown, anti-CD40-HPV16.E6 / 7 can effectively bind to human blood dendritic cells (DCs) (CD3-CD19-CD14-HLA-DR+CD11c+). Conversely, IgG4-HPV16.E6 / 7 does not bind to the same DCs. In peripheral blood from healthy donors, anti-CD40-HPV 18.E6 / 7 also binds to DCs (data not shown).

[0164] Anti-CD40-HPV16.E6 / 7 effectively activated E6 / 7-specific memory CD4+ and CD8+ T cells from HPV-related cancer patients. The in vitro immunogenicity of anti-CD40-HPV16.E6 / 7 was evaluated using PBMCs from HPV-positive head and neck cancer patients. Patient PBMCs were loaded with peptide pools containing recombinant HPV16.E6 / 7 protein, anti-CD40-HPV16.E6 / 7, or HPV16.E6 / 7 protein. In this experiment, the same molar concentration of E6 / 7 was applied to each protein to compare the levels of response in CD4+ and CD8+ T cells expressing E6 / 7-specific IFNg. After 7 days of in vitro culture, PBMCs were restimulated for 5 hours using the E6 / 7 peptide pool in the presence of brevidin A. Cells were then stained for IFNg expression. Figure 4The results showed that anti-CD40-HPV16.E6 / 7 was more effective than HPV16.E6 / 7 in eliciting both IFNg+CD4+ and IFNg+CD8+ T cell responses. The level of HPV16.E6 / 7-specific IFNg+CD4+ T cell response induced by anti-CD40-HPV16.E6 / 7 was similar to that induced by a peptide pool used as a positive control. Therefore, our novel vaccine model, consisting of anti-DCR and HPV antigens, including E6 / 7, is effective in activating antigen-specific cellular immune responses in patients with HPV-related cancers. Furthermore, such HPV antigen (E6 and E7)-specific CD8+ CTLs are expected to effectively suppress tumor development and induce tumor rejection in patients.

[0165] In vivo, anti-CD40-HPV16.E6 / 7 can stimulate E6 / 7-specific CD4+ and CD8+ T cell responses. To test the in vivo immunogenicity of the anti-CD40-HPV16.E6 / 7 vaccine, human CD40 transgenic mice were used. On day 0, five animals were subcutaneously immunized with 30 μg of anti-CD40-HPV16.E6 / 7 additive IC, followed by two booster doses of the same vaccine. On day 7 after the second booster, CD4+ and CD8+ T cells were purified from the spleen and then restimulated with either one of the HPV16.E6 / 7 peptide clusters 1-5, none, a peptide pool containing prostate-specific antigen (PSA), or the HPV16.E6 / 7 peptide pool. Figure 5 The results showed that in human CD40 transgenic mice, anti-CD40-HPV16.E6 / 7 induced HPV 16 E6 / 7 peptide cluster 2 and 3-specific CD4+ and cluster 5-specific CD8+ T cell responses. Importantly, the level of E6 / 7-specific CD8+ T cell responses was greater than that of E6 / 7-specific CD4+ T cell responses. This indicates that the anti-CD40-HPV16.E6 / 7 vaccine is particularly effective in inducing CD8+ CTLs that can kill HPV-infected cells and tumor cells. Figure 5 Each point in the graph represents data generated using a single mouse.

[0166] In human CD40 transgenic mice, anti-CD40-HPV16.E6 / 7 inhibited TC-1 tumor development. The efficacy of the anti-CD40-HPV16.E6 / 7 polymeric IC vaccine was tested in TC-1 challenged human CD40 transgenic mice. On day 0, two groups of animals (human CD40+ and human CD40- mice, n=5 per group) were subcutaneously challenged with the TC-1 tumor cell line. On days 6 and 12, the animals were immunized with the anti-CD40-HPV.E6 / 7 polymeric IC. Tumor development was assessed and presented on… Figure 6In both human CD40+ and CD40- mice, tumors of similar size developed 14 days after TC-1 challenge. In human CD40- mice, TC-1 tumors developed rapidly after challenge, reaching 1000 mm on day 25. 3 However, in human CD40+ mice, TC-1 tumor development was significantly delayed. Our data demonstrate that the anti-CD40-HPV16.E6 / 7 vaccine targets human CD40 and thus induces E6 / 7-specific CD8+ CTLs, such as... Figure 5 As shown in the figure, it inhibits TC-1 tumor progression in animals.

[0167] In human CD40 transgenic mice, the effects of poly-IC, poly-IC plus montanide, GM-CSF plus montanide, and montanide alone on the immunogenicity of the anti-CD40-HPV.E6 / 7 vaccine were tested. Four animals in each group were subcutaneously immunized with 30 μg of either anti-CD40-HPV.E6 / 7 alone or anti-CD40-HPV16.E6 / 7 with the indicated adjuvant. Figure 7 Seven days later, blood was obtained from individual animals and stained using tetramer. As shown in the figure below, poly-IC effectively promoted anti-CD40-HPV16.E6 / 7-specific CD8+ T cell responses. Montanide alone or GM-CSF in Montanide did not significantly promote E6 / 7-specific CD8+ T cell responses.

[0168] The following sequence is based on the humanized 12E12 anti-human CD40VK2 VH2 antibody—the protein sequence is the expected mature secreted protein sequence and the DNA sequence includes the initiation ATG and leader peptide regions. Alternatively, the HPV 18 sequence can be grafted onto the C-terminus of the VK2 chain, and a broader-spectrum vaccine can be produced by combining it with the HPV 16 sequence on the VH2 H chain.

[0169]

[0170] HPV 16 E6

[0171] MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVGDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPE

[0172] (SEQ ID NO:1)

[0173] HPV 16 E7

[0174] MHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCK

[0175] (SEQ ID NO:2)

[0176] HPV 18 E6

[0177] MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVGEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNP

[0178] (SEQ ID NO:3)

[0179] HPV 18 E7

[0180] MHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCK

[0181] (SEQ ID NO:4)

[0182] Flexv1

[0183] QTPTNTISVTPTNNSTPTNNSNPKPNP

[0184] (SEQ ID NO:5)

[0185] f1

[0186] SSVSPTTSVHPTPTSVPPTPTKSSP

[0187] (SEQ ID NO:6)

[0188] h anti - CD40VK2 - LV - hIgGK - C

[0189] DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0190] (SEQ ID NO:7)

[0191] h anti-CD40 VH2-LV-hIgG4H-C

[0192] EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0193] (SEQ ID NO:8)

[0194] Anti-CD40 12E12 light chain variable region

[0195] DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIGNLEPEDIATYYCQQFNKLPPTFGGGTKLEIK

[0196] (SEQ ID NO:9)

[0197] Anti-CD4012E12 heavy chain variable region

[0198] CEVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVS

[0199] (SEQ ID NO:10)

[0200] h anti-CD40VK2-LV-hIgGK-C

[0201] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0202] (SEQ ID NO:17)

[0203] h anti-CD40VK2-LV-hIgGK-C DNA sequence (including the leader peptide region)

[0204]

[0205] (SEQ ID NO: 18)

[0206] h anti-CD40VH2-LV-hIgG4H-C-Flex-v1-HPV 16-E6-HPV 16-E7-f1

[0207] (The bold and italic single underline sequence is HPV 16 E6; the bold and italic double underline sequence is HPV 16 E7; the non-bold and non-italic single underline (Flexv1) and the non-bold and non-italic double underline (f1) sequence are flexible glycosylation linker sequences.)

[0208]

[0209] (SEQ ID NO: 19)

[0210] h anti-CD40VH2-LV-hIgG4H-C-Flex-v1-HPV 16-E6-HPV 16-E7-f1 DNA sequence

[0211] (Including the leader peptide region)

[0212] ATG TGA

[0213] (SEQ ID NO: 20)

[0214] h anti-CD40VH2-LV-hIgG4H-C-Flex-v1-HPV 18E6-HPV 18E7-f1

[0215] (The bold and italic single underline sequence is HPV 18 E6; the bold and italic double underline sequence is HPV 18 E7; the non-bold and non-italic single underline (Flexv1) and the non-bold and non-italic double underline (f1) sequence are flexible glycosylation linker sequences.)

[0216]

[0217] (SEQ ID NO:21)

[0218] h anti-CD40VH2-LV-hIgG4H-C-Flex-v1-HPV 18E6-HPV 18E7-f1 DNA sequence

[0219] (Including the leader peptide region)

[0220] ATG TGA

[0221] (SEQ ID NO:22)

[0222] Example 2—Recombinant fusion protein of anti-DC receptor (DCR), TLR ligand and HPV sequence

[0223] The following is an example of a TLR2 ligand (triacylated adhesion protein, expressed in E. coli) in which the C residue in the D1 leader domain is esterified, and when anti-CD40 has, for example, an anchoring protein domain fused to the C-terminus of the distal end of the HPV E6 / 7 sequence or the C-terminus of the L or H chain, it can be non-covalently attached to the anti-CD40-HPV vaccine.

[0224] D1-6His-cohemin-Nhe-Spe-Not (Note that other cancer antigen sequences can be added to the distal end of the cohemin domain)

[0225] MKKLLIAAMMAAALAACSQEAKQEVKEAVQAVESDVKDTAMGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGE LIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASLIKTSEF

[0226] (SEQ ID NO:23)

[0227] D1-6His-cohesin-Nhe-Spe-Not DNA sequence

[0228] ATGAAAAAACTGCTGATTGCCGCCATGATGGCTGCAGCTCTGGCCGCATGCAGCCAGGAAGCCAAACAGGAAGTGAAAGAAGCCGTGCAGCCGTGGAAAGCGATGTGAAAGATACCGCCATGGGCAGCAGCCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATAT GGCTAGTATGGATCTGGATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAATATACCTGTAAGATTCAGTGGTATACCATCCAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAAAACCGGGAGAAT TGATAGTTGACCCGAATCCTACCAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATGATAGTATTCCTGTTTGCGGAAGACAGCGGAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCTAACGGGCTCAGTGTAATC AAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACGGTGGAGTAAATGTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTAGATGCAGCTAGCTTAATTAAAACTAGTGAATTCTGA

[0229] (SEQ ID NO:24)

[0230] Below is an example (underlined) of an anti-CD40L chain with a preferred TLR5L flagellin domain. This will be co-transfected with a matching H chain carrying the HPV E6 / 7 antigen at the C-terminus.

[0231] h anti-CD40VK2-LV-hIgGK-C-Flgn1-Flgn2

[0232] (The underlined sequence represents the flagellin domain)

[0233] DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS IERLSSG LRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSI QAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQASQPELAEAAAKT TENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQ AS

[0234] (SEQ ID NO:25)

[0235] h anti-CD40 VK2-LV-hIgGK-C-Flgn1-Flgn2 DNA sequence

[0236]

[0237] (SEQ ID NO: 26)

[0238] The following are examples of HPV 18E6 / 7 sequences fused to the H chain of a DC-targeting antibody (in this case, the anti-Langerhans protein). Alternatively, it can be fused to the H chain of a preferred anti-CD40 antibody to replace the HPV 16 sequence, or fused downstream of the HPV 16 sequence, or fused to the anti-CD40L chain—in each case preparing a vaccine containing both HPV 16 and HPV 18 sequences.

[0239] Anti-Langerhans protein 15B10H-LV-hIgG4H-C-HPV 18E7-HPV 18E6-f1

[0240] (The bold and italic single-underline sequence is HPV 18 E6; the bold and italic double-underline sequence is HPV 18 E7; the non-bold and non-italic double-underline (f1) sequence is a flexible glycosylation linker sequence.)

[0241]

[0242] (SEQ ID NO:27)

[0243] Anti-Langerhans protein 15B10H-LV-hIgG4H-C-HPV 18E7-HPV 18E6-f1 DNA sequence

[0244]

[0245] (SEQ ID NO:28)

[0246] Example 3—CD40-targeted HPV vaccine (CD40 HVac)

[0247] In human CD40 transgenic B6 (hCD40Tg) mice, CD40HVac plus polyIC induced E6 / 7-specific CD8+ CTLs. hCD40Tg and wild-type (WT) animals (5 mice / group) were immunized subcutaneously (SC) with 30 μg CD40HVac plus 50 μg polyIC in PBS (100 μl) and boosted twice every two weeks. In separate experiments, the amounts of CD40HVac and polyIC were predetermined. Seven days after the second boost, IFN ELISPOT was performed using CD8+ T cells purified from the spleen (Fig. 8a). Compared with WT mice, hCD40Tg mice induced an increased number of CD8+IFN+ T cells. The inventors also observed increased E7-specific CD8+ T cells in the blood of hCD40Tg mice, as measured by tetramer staining (Fig. 8b). Furthermore, CD40HVac polymerized IC induced higher levels of E6 / 7-specific CD4+ T cell responses in hCD40Tg mice than in WT animals (not shown). In summary, the inventors conclude that CD40HVac targets human CD40 in vivo and is therefore capable of inducing E6 / 7-specific cellular responses. The inventors also found that, despite the agonistic properties of the humanized anti-CD40 antibody used in CD40HVac, CD40HVac polymerized IC (adjuvant) was more potent than CD40HVac alone in inducing E6 / 7-specific T cell responses in hCD40Tg mice (data not shown).

[0248] CD40HVac polymerized ICs can enhance therapeutic immunity in hCD40Tg animals. Using HPV E6 / 7-expressing TC-1 tumor cells (5x10⁻¹²), [the following data was obtained]. 410 hCD40Tg mice were subcutaneously attacked. The inventors demonstrated that animals developed palpable tumors on day 6 post-TC-1 challenge. Animals were then immunized subcutaneously, intramuscularly (IM), or intraperitoneally (IP) with 30 μg CD40HVac plus 50 μg polyIC on days 6, 12, and 24. Control groups remained unimmunized. Figure 9a shows that all animals receiving CD40HVac plus polyIC survived, while all control animals died. PolyIC injection alone did not improve survival (data not shown). In separate experiments, we measured TC-1 tumor development by assessing tumor volume (Figure 9b). All control animals (10 mice) developed tumors and died within 40 days of TC-1 challenge. Conversely, CD40HVac plus polyIC treatment inhibited tumor development. It was also noted that some treated animals developed large tumors (200–600 mm). 3 Furthermore, these tumors regressed over time during vaccination. In conclusion, the inventors find that CD40HVac induces therapeutic immunity in hCD40Tg mice. Moreover, the data suggest that the immune pathway is a significant factor potentially influencing the overall therapeutic efficacy of the CD40HVac regimen.

[0249] CD8+ CTL infiltration into the tumor is key to tumor regression. Using a large number of TC-1 tumor cells (2x10⁻¹²) is crucial. 5 Human CD40 transgenic (hCD40Tg) mice were subcutaneously attacked with 30 μg CD40HVac plus 50 μg polyIC on days 6 and 12. In the absence of immunization, all animals died within 25 days post-tumor challenge. On day 60, the percentage of H2-Db(RAHYNIVTF) tetramer+CD8+ T cells in the tumor and blood was assessed. Figure 10 The percentage of tetramer+CD8+ T cells in the tumor (left) is negatively correlated with tumor volume. No such correlation exists between the percentage of tetramer+CD8+ T cells in the blood (right) or spleen (not shown) and tumor volume. Therefore, antigen-specific CD8+ CTL infiltration into the tumor is crucial for tumor regression. Consequently, we anticipate enhancing the efficacy of CD40HVac by promoting effector cell infiltration into and retention within mucosal tumors.

[0250] In inducing HPV16.E6 / 7-specific CD8+ T cell responses, CD40HVac prepared from anti-CD40 (clone 12E12) was more effective than that prepared from anti-CD40 (clone 12B4). The inventors compared the ability of recombinant fusion proteins prepared from three different clones (12E12 and 12B4) of anti-CD40 mAb to stimulate HPV16.E6 / 7-specific CD8+ T cell responses. PolyIC was used as an adjuvant. hCD40Tg animals received three doses of the recombinant fusion protein (30 μg / dose) plus polyIC (50 μg / dose) via subcutaneous injection. Seven days after the third immunization, the percentage of E7-specific CD8+ T cells in the blood was determined by tetramer staining. As shown on the left side of Figure 11a, the recombinant fusion protein prepared from 12E12 was more effective than those prepared from the 12B4 clone in inducing E6 / 7-specific CD8+ T cell responses. Using ELISPOT analysis of spleen cells, the inventors also found that anti-CD40(12E12)-HPV16.E6 / 7 was more effective than anti-CD40(12B6)-HPV16.E6 / 7 in inducing IFNγ+CD8+ T cell responses (left side of Figure 11b). HPV16.E6 / 7 fused with both clones of anti-CD40 mAb produced similar levels of E6 / 7-specific IFN+CD4+ T cell responses (right side of Figure 11b).

[0251] All methods disclosed and claimed herein can be formulated and practiced in accordance with the present invention without excessive experimentation. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations may be made to the methods and steps or the order of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0252] References

[0253] The following references are specifically incorporated herein by reference, and may provide exemplary procedures or other details that supplement the exemplary procedures or other details described herein.

[0254] Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988

[0255] Atherton et al., 1985

[0256] Ausubel et al., 1996

[0257] Barany and Merrifield, 1979

[0258] Bird et al., 1988

[0259] Burke et al., 1994

[0260] Cumber et al., 1992

[0261] Dholakia et al., 1989

[0262] Epitope Mapping Protocols (1996)

[0263] Glennie et al., 1987

[0264] Goding, 1986, pp. 60-61

[0265] Holliger et al., 1993

[0266] Holliger & Winter, 1999

[0267] Holt et al., 2003

[0268] Hu et al. . 1996

[0269] Huston et al., 1988

[0270] Khatoon et al., 1989

[0271] King et al., 1989

[0272] Kohl et al., 2003

[0273] Kyte and Doolittle, 1982

[0274] Liu et al., 2003

[0275] McCafferty et al., 1990

[0276] Merchand et al., 1998

[0277] Merrifield, 1986

[0278] O'Shannessy et al., 1987

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Claims

1. Use of a dendritic cell-targeting complex in the preparation of a medicament for treating or preventing human papillomavirus (HPV)-related diseases, said dendritic cell-targeting complex comprising: An anti-CD40 antibody or a fragment thereof, said anti-CD40 antibody or fragment thereof comprising at least six complementarity-determining regions derived from the anti-CD40 antibody; at least one peptide linker; and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein said E6 or E7 antigen or multiple E6 or E7 antigens are HPV type 16 or HPV type 18 antigens.

2. The use according to claim 1, wherein the anti-CD40 antibody or a fragment thereof comprises an anti-CD40 antibody light chain variable region and an anti-CD40 antibody heavy chain variable region.

3. The use according to claim 1, wherein the anti-CD40 antibody or a fragment thereof is humanized.

4. The use according to any one of claims 1 to 3, wherein at least one HPV E6 antigen is an HPV 16 antigen and at least one HPV E7 antigen is an HPV 16 antigen.

5. The use according to any one of claims 1 to 3, wherein at least one HPV E6 antigen is an HPV 18 antigen and at least one HPV E7 antigen is an HPV 18 antigen.

6. The use according to any one of claims 1 to 3, wherein at least one HPV E6 antigen is an HPV 16 antigen, at least one HPV E7 antigen is an HPV 16 antigen, at least one HPV E6 antigen is an HPV 18 antigen, and at least one HPV E7 antigen is an HPV 18 antigen.

7. The use according to claim 4, wherein the dendritic cell targeting complex comprises SEQ ID NO:

19.

8. The use according to claim 5, wherein the dendritic cell targeting complex comprises SEQ ID NO:

21.

9. The use according to any one of claims 1 to 8, wherein the composition further comprises an adjuvant.

10. The use according to any one of claims 1 to 8, wherein the HPV-related disease is a developmental disorder, a benign tumor, a precancerous tumor, or cancer.

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