Peptide-based vaccines for inducing immune responses, methods for their production and use - Patents.com

Abstract

Peptide-based vaccines for inducing an immune response, methods for their production and use are provided. [Solution] The present disclosure relates to novel peptide-based vaccines, methods for producing novel peptide-based vaccines, and their use for delivering peptide antigens to subjects to induce immune responses, particularly T cell responses. The inventors have developed novel compositions and methods for producing peptide-based vaccines that overcome at least one of the limitations of current peptide-based vaccine approaches. The novel peptide-based vaccine compositions and methods for production disclosed herein take into account the variability in the physical and chemical properties of peptide antigens and are therefore generalizable to any peptide antigen.

Description

[Technical Field]

[0001] Priority document This application is a continuation of U.S. Provisional Patent Application No. 62 / 481,432, filed April 4, 2017, and U.S. Provisional Patent Application No. 62 / 617,519, filed January 15, 2018, both entitled "PEPTIDE-BASED VACCINES, METHODS OF This application claims priority from "Patent #1111116" and "Patent #1111116" ("Patent #1111116").

[0002] This invention was made in fulfillment of a Cooperative Research and Development Agreement with the U.S. Department of Health and Human Services, National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] The present disclosure relates to novel peptide-based vaccines, methods for producing novel peptide-based vaccines, and their use for inducing immune responses, particularly T cell responses, in subjects. Embodiments of the peptide-based vaccines of the present disclosure can be used to prevent or treat infectious diseases and cancer, as well as to induce tolerance or modulate immunity to prevent or treat autoimmunity and allergies. [Background technology]

[0004] Vaccines containing immunogenic compositions of antigens can be used to induce immune responses in subjects, including inducing immune responses for the treatment or prevention of cancer or infectious diseases, or can even be used to induce tolerance and / or immunosuppression for the treatment or prevention of autoimmunity or allergy. Vaccine compositions for inducing immune responses for the treatment or prevention of cancer and infectious diseases contain an antigen that mediates pathogen elimination or killing of virus-infected cells or cancerous cells, and a specific type of adjuvant that induces cytotoxic T cell responses and / or antibodies against the antigen. In contrast, vaccine compositions that induce tolerogenic or suppressive responses may contain an antigen, a vehicle (e.g., a delivery system such as a particle carrier), and / or an immunosuppressive compound, such as an mTOR inhibitor, but will lack a specific adjuvant that induces cytotoxic T cells. Instead of inducing cytotoxic T cells, they may induce T cell tolerance or the activation of regulatory cells, such as regulatory T cells, that downregulate or modulate the qualitative characteristics of the response.

[0005] It is becoming increasingly recognized that a patient's genetic background, particularly the composition of major histocompatibility complex (MHC) alleles, and environment can influence their susceptibility to cancer, infectious diseases, autoimmunity, and allergies, as well as their response to vaccines used to treat such conditions.With the continued development of new diagnostic and informatics methods that lead to an understanding of the molecular basis of disease, patient-specific information is becoming increasingly accessible.Therefore, there is growing interest in using information about an individual's genes, proteins, and environment to direct the medical care that an individual receives, including the selection of specific immunotherapies, including vaccines, for treating or preventing cancer, infectious diseases, autoimmunity, and allergies.

[0006] In cancer therapy, specific information about an individual's tumor can be used to aid in diagnosis, plan treatment, assess the effectiveness of treatment, or predict prognosis.For example, specific information about an individual's genes, proteins, and environment can be used to develop vaccines based on this information, thereby tailoring preventive approaches to cancer treatment.Vaccines used for immunological treatment or prevention of a particular cancer must induce an immune response against tumor-associated antigens.One preferred immune response is a CD8 T cell response and / or a CD4 T cell response that recognizes tumor-associated antigens.

[0007] Similarly, personalized approaches to treating autoimmunity or allergies are possible by identifying the specific autoantigens or foreign antigens responsible for the immune-mediated pathology, respectively. While some autoantigens and allergens are known and common to all patients, others may be patient-specific, and therefore treatment for these patients must be tailored to each individual. Antigens identified as the cause of the pathology can be administered in the form of peptides as vaccines that can induce tolerance to autoantigens (i.e., self-antigens). Alternatively, the immune response to foreign antigens generated in allergies may be of a specific immune response type that leads to the pathology. Therefore, vaccines against such foreign antigens can be provided as peptide-based vaccines to shift the immune response to a qualitatively distinct immune response type that does not lead to the pathology.

[0008] The role of T cells in cancer treatment or prevention It is known that T cells mediate tumor regression and improve patient survival, as demonstrated by adoptive T cell therapy and immune checkpoint inhibitor-based immunotherapy.Several human studies have shown that intravenous infusion of large numbers of expanded CD8 and / or CD4 T cells that recognize a single tumor-associated antigen can mediate tumor regression and improve survival (see E. Tran et al., N Engl J Med 375:2255-2262, 2016; and E. Tran et al., Science 344:641-645, 2014). In addition, treatment of certain cancers with drugs that block or reverse T-cell suppression, such as monoclonal antibodies that block CTLA-4, PD-1, and / or PD-L1 (so-called "checkpoint inhibitors"), results in tumor regression and improved overall survival (see D. M. Pardoll, Nat Rev Cancer, 12:252-264, 2012; F. S. Hodi et al., N Engl J Med, 363:711-723, 2010; and M. Reck et al., N Engl J Med, 375:1823-1833, 2016; J. E. Rosenberg et al., Lancet, 387:1909-1920, 2016). PD-1 / PD-L1 is a receptor-ligand pair in which PD-1 is expressed on T cells and PD-L1 is expressed on cells in tumors. PD-1 / PD-L1 interaction prevents T cells from performing their effector functions that would otherwise result in the slowing of growth or elimination of tumor cells that express the tumor-associated antigen recognized by the T cell. Blockade of PD-1 / PD-L1 interaction with monoclonal antibodies abolishes the inhibitory signal, thereby freeing T cells to perform effector functions that result in the slowing of tumor cell growth or tumor cell death (DM Pardoll, Nat Rev Cancer 12:252-264, 2012).

[0009] The role of T cells in promoting tumor elimination in patients is supported by human studies showing that checkpoint inhibitors are more effective (and have lower mortality) in patients with higher levels of T cell infiltration in tumor biopsies at the start of treatment (see JE Rosenberg et al., Lancet 387:1909-1920, 2016). Furthermore, checkpoint inhibitors have also been shown to be more effective in patients with higher levels of tumor mutations (see A. Snyder et al., N Engl J Med 371:2189-2199, 2014; and NA Rizvi et al., Science 348:124-128, 2015), likely because increasing mutation counts result in more mutant proteins, thereby providing a greater number of potential targets for T cells to recognize and target tumor cells for elimination.

[0010] However, a major challenge is the low mutational burden of many prevalent tumors (e.g., prostate, breast, and pancreatic cancers) (see: T.N. Schumacher, RD Schreiber, Science 348:69-74, 2015), and patients with these cancers derive little or no benefit from checkpoint inhibitors. Furthermore, even among tumors with high mutational burden, only a small subset of patients benefit from checkpoint inhibitor treatment because the majority of subjects lack the requisite pre-existing tumor-specific T cell responses required for immunotherapy to mediate efficacy.

[0011] Vaccines targeting tumor-associated antigens for the treatment or prevention of cancer - Patents.com Generating an immune response, particularly a T cell response, for the treatment or prevention of cancer requires the identification of suitable tumor-associated antigens. Tumor-associated antigens include autoantigens, which are present on certain healthy cells but are preferentially expressed by tumor cells; pathogen-derived antigens from oncogenic microbial pathogens; and / or neoantigens, which are abnormal proteins specific to tumor cells and are often, but not always, unique to individual patients.

[0012] Suitable autoantigens can be antigens derived from proteins that are no longer expressed in postnatal subjects.For example, suitable autoantigens can include antigens that are preferentially expressed by tumor cells, such as NY-ESO-1 and MAGE-A3 (see NN Hunder et al., N Engl J Med, 358:2698-2703, 2008).Alternatively, suitable autoantigens can include antigens that are expressed in healthy tissues, and the T cell response against these healthy tissues is not excessively harmful to patients, such as prostatic acid phosphatase (PAP) (see PW Kantoff et al., N Engl J Med, 363:411-422, 2010).

[0013] Tumor-associated antigens can be antigens derived from pathogens, such as viruses or other pathogens that are the underlying drivers of the neoplastic process, e.g., proteins from human papillomavirus (HPV) (see GG Kenter et al., N Engl J Med, 361:1838-1847, 2009).

[0014] Neoantigens are antigens that are the result of mutations present only in tumor cells and not in normal cells (see TN Schumacher, RD Schreiber, Science 348:69-74, 2015). Neoantigens can be created by nucleotide polymorphisms that result in non-conservative amino acid changes. Neoantigens can also be created by insertions and / or deletions, which can result in peptide antigens containing insertions, deletions, or frameshift mutations. Neoantigens can also be created by the introduction of a stop codon, which is not recognized by the stop codon machinery in its new context, resulting in the ribosome skipping the codon and generating a peptide containing a single amino acid deletion. Neoantigens can also be created by mutations at splice sites that result in incorrectly spliced ​​mRNA transcripts. Neoantigens can also be created by inversions and / or chromosomal translocations that result in fusion proteins.

[0015] Finally, tumor-associated antigens may contain novel post-translational modifications of the unmutated or mutant peptide that are not present in normal tissue.

[0016] In summary, a tumor-associated antigen is any antigen produced by neoplastic cells that, when targeted by a T cell response, ideally results in significant regression of tumor growth or prevents tumor formation and has limited effects on healthy, non-cancerous cells.

[0017] Challenges facing the vaccine field The preferred immune response for the treatment of certain cancers is a CD8 T cell response (see E. Tran et al., N Engl J Med 375:2255-2262, 2016) and / or a CD4 T cell response (see also E. Tran et al., Science 344:641-645, 2014; and NN Hunder et al., N Engl J Med 358:2698-2703, 2008) and are preferentially sustained, large, and highly functional (high quality) CD8 and / or CD4 T cell responses (L. Gattinoni et al., Nature Medicine 17:1290-1297, 2011).

[0018] One way to induce T cell responses against antigens, such as tumor-associated antigens, in subjects is through vaccination. Numerous approaches to induce CD8 and / or CD4 T cell responses against tumor-associated antigens, including DNA / RNA-based, viral vector-based, and peptide-based approaches, are currently being investigated (see, for example, LM Kranz et al., Nature 534, 396-401, 2016; and CJ Melief, SH van der Burg, Nat Rev Cancer 8: 351-360, 2008). However, a major challenge to generating effective T cell immunity against cancer is that most current vaccine approaches are limited by their weak immunogenicity in eliciting CD8 T cell immunity and the limited antigenic breadth of responses against most predicted neoantigens (see, for example, S. Kreiter et al., Nature 520, 692-696, 2015).

[0019] Many efforts to induce antitumor T cell immunity have focused on the use of synthetic peptide antigens simply mixed with different immunostimulatory agents and / or vehicles (adjuvants) (see: CJ Melief, SH van der Burg, Nat Rev Cancer 8:351-360, 2008; and MS Bijker et al., Eur J Immunol 38:1033-1042, 2008) or RNA (see Kranz LM et al., Nature 534(7607):396-401, 2016 and Kreiter S et al., Nature 520(7549):692-696, 2015).

[0020] However, a major challenge to generating effective T cell immunity against cancer is that most current vaccine approaches based on peptide antigens or RNA have been hampered by the small magnitude and limited antigenic breadth of responses to tumor-associated antigens, including neoantigens. For example, in terms of antigenic breadth, current standard peptide-based vaccine approaches based on peptide antigens (e.g., 25-amino acid synthetic long peptides mixed with the adjuvant polyIC:LC) or RNA induce T cell responses against less than 10% of predicted neoantigens (see S. Kreiter et al., Nature 520, 692-696, 2015). Therefore, improved vaccine approaches, especially peptide-based vaccine approaches, are needed.

[0021] A satisfactory explanation for the small magnitude and poor antigenic breadth of T cell responses with modern vaccine technologies remains uncertain, and therefore, a continuing challenge is the current lack of consensus on optimal parameters for delivery of peptide antigens to ensure reliable priming of T cell immunity for cancer treatment and prevention, as well as for other applications, including the treatment and prevention of infectious diseases. Similarly, optimal parameters for peptide-based vaccines to induce suppression and tolerance remain unknown.

[0022] Current challenges surrounding peptide-based T cell vaccines A major challenge in developing vaccines for the treatment or prevention of infectious diseases and cancer is the lack of consensus on the best way to construct peptide-based vaccines that reliably elicit large-scale T cell immunity against most antigens. Similarly, there is no consensus on the best way to construct peptide-based vaccines to induce immune tolerance or shift the immune response from allergy-inducing to a harmless type of response. For example, there is still considerable debate about the optimal peptide antigen length, the physical form of peptide antigen delivery (e.g., soluble vs. particulate), and the type of innate immune stimulation (e.g., adjuvant selection) required to induce optimal T cell immunity for the treatment or prevention of cancer and infectious diseases, as well as for the treatment or prevention of autoimmunity and allergy. Indeed, the effects of many peptide-based vaccine parameters, including the amino acids flanking CD4 and CD8 T cell epitopes, the linker chemistry used, charge, and so on, on immune responses remain largely unknown. This problem is particularly pronounced given that personalized vaccine approaches must be uniquely tailored to each patient; therefore, there is a need for universal peptide-based vaccine approaches to reliably elicit immunity, particularly T cell immunity, against patient-specific antigens.

[0023] Another major challenge facing current peptide-based vaccine approaches is that they do not take into account the wide range of possible physical and chemical properties of peptide antigens. For example, personalized cancer vaccine approaches require generating a unique set of peptide antigens for each patient, which may have a wide range of possible physical and chemical properties. In the case of autoimmunity, multiple different antigens may be identified as the cause of the pathology. Therefore, tolerance-inducing vaccines must contain a set of peptide antigens that is unique to each patient. The problem is that variability in peptide antigen composition can result in some peptide-based antigens being difficult or impossible to produce as native peptide antigens, estimated to be approximately 10–30% of peptide-based antigens 25 amino acids or longer. Therefore, because native peptide antigens cannot be efficiently produced or isolated after synthesis, many antigens cannot be targeted by current vaccine approaches. Additionally, variability in peptide antigen composition affecting charge and solubility, which is not controlled by current peptide-based vaccine approaches, can affect various attributes of the vaccine formulation, including the peptide's mass loading, and / or can result in adverse interactions between the peptide antigen and either other peptide antigens or other components of the vaccine. Therefore, to overcome the limitations of current peptide-based vaccine approaches, there is a need for new compositions and methods for producing peptide-based vaccines that (i) take into account the variability of the physical and chemical properties of peptide antigens not only in vaccine formulations but also during production, and (ii) ensure optimal delivery of peptide antigens to reliably induce immune responses to most antigens, particularly T cell responses in subjects. Such vaccines and methods that take into account peptide antigen variability and are therefore generalizable to any peptide antigen would be particularly useful in the field of personalized cancer treatment, where the characteristics of peptide antigens used in personalized cancer vaccines may vary from patient to patient. However, the applicability of peptide-based vaccine compositions to any peptide antigen means that these compositions can also be used in other personalized immunological-based treatments, such as inducing tolerance for the treatment of autoimmunity and allergies or modulating immune responses to allergens, respectively. [Prior art documents] [Non-patent literature]

[0024] [Non-Patent Document 1] CJMelief, SHvan der Burg, Nat Rev Cancer (2008) 8:351-360 [Non-patent document 2] MS Bijker et al., Eur J Immunol (2008) 38:1033-1042 [Non-patent document 3] Kranz LM et al., Nature (2016) 534(7607):396-401 [Non-patent document 4] Kreiter S et al., Nature (2015) 520(7549):692-696 [Non-patent document 5] S. Kreiter et al., Nature (2015) 520, 692-696 Summary of the Invention [Means for solving the problem]

[0025] The present inventors have developed novel compositions and methods for producing peptide-based vaccines that overcome at least one of the limitations of current peptide-based vaccine approaches. The novel peptide-based vaccine compositions and production methods disclosed herein take into account the variability in the physical and chemical properties of peptide antigens and are therefore generalizable to any peptide antigen. Furthermore, the novel peptide-based vaccine compositions disclosed herein ensure optimal delivery of a variety of different peptide antigens to induce an immune response in a subject.

[0026] The novel compositions disclosed herein relate to immunogenic compositions comprising peptide-antigen conjugates, which comprise a peptide antigen (A) linked either directly to a particle-forming hydrophobic molecule (H) or to a preformed particle (P), or indirectly via an optional linker (L) and / or optional N- or C-terminal extensions (B1 or B2) linked to either the N- or C-terminus of the peptide antigen, respectively.

[0027] In some embodiments, the particles formed by the peptide antigen conjugate further comprise an optional charged molecule (C). In some embodiments, the charged molecule is linked to the peptide antigen conjugate to stabilize the particle in aqueous conditions. In other embodiments, the charged molecule (C) is provided on a separate molecule and is incorporated into the particle formed by the peptide antigen conjugate.

[0028] The addition of certain N- and / or C-terminal extensions (B1 and / or B2) and / or charged molecules (C) provides unexpected improvements in the production of peptide-based vaccines by solid-phase synthesis, as well as improved ease of purification through improved organic solvent solubility and unexpectedly improved control over the size and stability of particles formed by peptide-antigen conjugates. These compositions demonstrate unexpected improvements in T cell immunity and tumor elimination, particularly in the magnitude and breadth of T cells generated against tumor-associated antigens.

[0029] Embodiments of the present disclosure include a peptide antigen conjugate comprising a peptide antigen (A) and either a hydrophobic molecule (H) or a particle (P), where the peptide antigen (A) is linked to either the hydrophobic molecule (H) or the particle (P) directly or indirectly via an N-terminal extension (B1) linked to the N-terminus of the peptide antigen (A) or a C-terminal extension (B2) linked to the C-terminus of the peptide antigen (A). Further embodiments of the present disclosure include immunogenic compositions comprising the peptide antigen conjugate. Yet further embodiments of the present disclosure include methods of treating a patient suffering from a disease, comprising administering the peptide antigen conjugate or immunogenic composition to a patient suffering from a disease.

[0030] In some embodiments, vaccines used in cancer treatment can be used alone or in combination with other immunotherapy and treatment modalities, including but not limited to chemotherapy, checkpoint inhibitors, radiation, and any other techniques used to fight cancer.

[0031] Accordingly, also disclosed herein are methods of treating a patient suffering from a disease, comprising administering to the patient a peptide-antigen conjugate or immunogenic composition of the present disclosure.

[0032] Further disclosed herein is the use of a peptide-antigen conjugate or immunogenic composition of the present disclosure in the manufacture of a medicament for the treatment of disease.

[0033] The unexpected discoveries disclosed herein relate to: (i) The optimal length of peptide antigens (A) used in cancer vaccines to ensure reliable priming of T cell immunity; (ii) the use of peptide antigen extension sequences, i.e., N-terminal or C-terminal extensions (B1 and / or B2), and / or optionally charged molecules (C), to facilitate the production of peptide-based vaccines; (iii) How the properties of the hydrophobic molecules (H) that make up the peptide-antigen conjugate affect manufacturability and particle size and stability, and how these parameters affect biological activity; (iv) the composition of charged molecules (C) and net charge of peptide-antigen conjugates required to induce and stabilize particles of optimal size for promoting T-cell immunity; (v) the composition of an enzyme-degradable peptide sequence that facilitates efficient processing of the minimal epitope delivered within the context of the peptide-antigen conjugate; and / or (vi) How do the potency and qualitative characteristics of adjuvant ligands (e.g., PRR agonists) delivered in peptide-antigen conjugates affect the breadth, magnitude, and quality of T cell responses? In an embodiment of the present invention, for example, the following items are provided: (Item 1) a. Peptide antigen (A), and b. Either hydrophobic molecules (H) or particles (P) 1. A peptide-antigen conjugate comprising: 1. A peptide antigen conjugate, wherein the peptide antigen (A) is either linked to the hydrophobic molecule (H) or to the particle (P), either directly or indirectly via an optional N-terminal extension (B1) linked to the N-terminus of the peptide antigen (A) or an optional C-terminal extension (B2) linked to the C-terminus of the peptide antigen (A), and optionally the hydrophobic molecule (H) or the particle (P) is indirectly linked to the extension (B1 or B2) via a linker (L). (Item 2) 2. The peptide-antigen conjugate according to item 1, further comprising a charged molecule (C). (Item 3) 3. The peptide-antigen conjugate of item 2, having a net electrostatic charge of greater than or equal to about +3, or less than or equal to about -3 in an aqueous buffer solution at a pH of about 7.4. (Item 4) 4. The peptide antigen conjugate according to any one of items 1 to 3, wherein the charged molecule (C) and either the hydrophobic molecule (H) or the particle (P) are both bound to either the N-terminus or the C-terminus of the peptide antigen (A) directly or via the optional N-terminal extension (B1) or the optional C-terminal extension (B2), respectively. (Item 5) 4. The peptide antigen conjugate according to any one of items 2 and 3, wherein the charged molecule (C) and either the hydrophobic molecule (H) or the particle (P) are bound to both ends of the peptide antigen (A). (Item 6) 6. The peptide antigen conjugate according to item 5, wherein (i) the charged molecule (C) is attached to the N-terminus of the peptide antigen (A) via the N-terminal extension (B1), and (ii) either the hydrophobic molecule (H) or the particle (P) is attached to the C-terminus of the peptide antigen (A) via the C-terminal extension (B2). (Item 7) 7. The peptide-antigen conjugate according to any one of items 1 to 6, wherein the N-terminal extension (B1) comprises an enzymatically degradable peptide sequence comprising the amino acid PN1, wherein PN1 is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine. (Item 8) The enzyme-degradable peptide sequence comprising the amino acid PN1 is: a. an amino acid PN2 selected from glycine, valine, leucine, or isoleucine; b. an amino acid PN3 selected from glycine, serine, proline, or leucine; c. an amino acid PN4 selected from arginine, lysine, glutamic acid, aspartic acid, glycine, or serine; or Any combination of da~c 8. The peptide-antigen conjugate according to item 7, further comprising at least one of: (Item 9) The enzyme-degradable peptide sequence is: a. Ser-Leu-Val-Cit; b. Ser-Leu-Val-Leu (SEQ ID NO: 4); c. Ser-Pro-Val-Cit; d. Gly-Pro-Val-Cit; e. Gly-Pro-Leu-Cit; f. Glu-Leu-Val-Arg (SEQ ID NO: 5); g. Ser-Pro-Val-Arg (SEQ ID NO: 6); h. Ser-Pro-Leu-Arg (SEQ ID NO: 21); i. Ser-Leu-Val-Arg (SEQ ID NO: 7); j. Lys-Pro-Leu-Arg (SEQ ID NO: 8); k. Glu-Leu-Val-Cit; l. Glu-Leu-Val-Leu (SEQ ID NO: 10); m. Glu-Pro-Val-Cit; or n. Glu-Gly-Val-Cit wherein Glu is optionally replaced by Asp and / or Arg is optionally replaced by Lys. (Item 10) The C-terminal extension (B2) is: i. a single amino acid PC1' selected from glycine, serine, arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine; ii. a dipeptide wherein PC1' is selected from glycine or serine and PC2' is selected from glycine, serine, proline, arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine; iii. A tripeptide wherein PC1' is selected from glycine or serine, PC2' is selected from glycine, serine or proline, and PC3' is selected from glycine, serine, arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine; iv. A tetrapeptide wherein PC1' is selected from glycine or serine, PC2' is selected from glycine, serine, proline or leucine, PC3' is selected from glycine, valine, leucine or isoleucine, and PC4' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine; v. a pentapeptide in which PC1' is selected from glycine or serine, PC2' is selected from glycine, serine, proline, arginine, lysine, glutamic acid or aspartic acid, PC3' is selected from glycine, serine, proline or leucine, PC4' is selected from glycine, valine, leucine or isoleucine, and PC5' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine; or vi. A hexapeptide wherein PC1' is selected from glycine or serine, PC2' is selected from glycine, serine or proline, PC3' is selected from glycine, serine, proline, arginine, lysine, glutamic acid or aspartic acid, PC4' is selected from proline or leucine, PC5' is selected from glycine, valine, leucine or isoleucine, and PC6' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine. 10. The peptide-antigen conjugate according to any one of items 1 to 9, comprising an enzymatically degradable peptide sequence comprising: (Item 11) The C-terminal extension (B2) is a. i. Gly-Gly-Lys-Leu-Val-Arg (SEQ ID NO: 11); ii. Gly-Gly-Ser-Leu-Val-Arg (SEQ ID NO: 13); iii. Gly-Gly-Ser-Pro-Val-Arg (SEQ ID NO: 61); iv. Gly-Gly-Ser-Leu-Val-Cit; v. Gly-Gly-Ser-Pro-Val-Cit; vi. Gly-Gly-Ser-Pro-Leu-Cit; vii. Gly-Gly-Ser-Leu-Val-Leu (SEQ ID NO: 14); viii. Gly-Gly-Glu-Leu-Val-Arg (SEQ ID NO: 15); ix. Gly-Gly-Glu-Leu-Val-Leu (SEQ ID NO: 16); x. Gly-Gly-Glu-Leu-Val-Cit wherein Glu may be replaced by Asp and / or Arg may be replaced by Lys; or b. i. Gly-Ser-Leu-Val-Arg (SEQ ID NO: 17); ii. Gly-Ser-Pro-Val-Cit; iii. Gly-Ser-Pro-Leu-Cit; iv. Gly-Ser-Leu-Val-Cit; v. Gly-Lys-Pro-Val-Cit; vi. Gly-Lys-Pro-Val-Arg (SEQ ID NO: 18); vii. Gly-Ser-Leu-Val-Leu (SEQ ID NO: 19); viii. Gly-Glu-Leu-Val-Leu (SEQ ID NO: 20) wherein Glu may be replaced by Asp and / or Arg may be replaced by Lys; or c. i. Ser-Leu-Val-Cit; ii. Ser-Leu-Val-Leu (SEQ ID NO: 4); iii. Ser-Pro-Val-Cit; iv. Ser-Pro-Leu-Cit; v. Glu-Leu-Val-Arg (SEQ ID NO: 5); vi. Ser-Pro-Val-Arg (SEQ ID NO: 6); vii. Ser-Leu-Val-Arg (SEQ ID NO: 7); viii. Lys-Pro-Leu-Arg (SEQ ID NO: 8); ix. Glu-Leu-Val-Cit; x. Glu-Leu-Val-Leu (SEQ ID NO: 10); xi. Glu-Pro-Val-Cit; or xii. Glu-Gly-Val-Cit wherein Glu may be replaced by Asp and / or Arg may be replaced by Lys; or d. i. Gly-Ser-Gly; ii. Gly-Ser-Arg; iii. Gly-Ser-Leu; iv. Gly-Ser-Cit; v. Gly-Pro-Gly; vi. Gly-Pro-Arg; vii. Gly-Pro-Leu; viii. Gly-Pro-Cit a tripeptide selected from: e. i. Gly-Ser; ii. Gly-Pro; iii. Gly-Arg; iv. Gly-Cit a dipeptide selected from: f. A single amino acid selected from the group consisting of Gly, Ser, Ala, Arg, Lys, Cit, Val, Leu, Met, Thr, Gln, or Nle (Item 12) The peptide-antigen conjugate according to Item 10, comprising an enzyme-degradable peptide sequence comprising: 2. The peptide-antigen conjugate of any one of the preceding items, wherein the particles (P) comprise polystyrene, poly(lactic-co-glycolic acid) (PLGA), liposomes, inorganic salts of aluminum, or emulsions. (Item 13) 12. The peptide-antigen conjugate of any one of items 1 to 11, wherein the hydrophobic molecule (H) is a branched long-chain or cyclic aliphatic molecule, or a fatty acid, lipid or sterol derivative that is insoluble in aqueous buffer at a pH of about 7.4 down to about 0.1 mg / mL. (Item 14) 12. The peptide-antigen conjugate according to any one of items 1 to 11, wherein the hydrophobic molecule comprises a polymer based on monomer units selected from acrylate, acrylamide, meth(acrylate), meth(acrylamide), styryl monomer, vinyl monomer, dioxophosphorane, cyclic ester, natural or unnatural amino acid, and a polymer resulting from the reaction of a diol or diamine with a diisocyanate or polyisocyanate, and optionally an adjuvant is linked to one or more of the monomers comprising the polymer. (Item 15) The hydrophobic molecule (H) has the formula I: [ka] wherein R 2 is selected from one of hydrogen, hydroxyl or amine, x is between 3 and 300, y3 is 1 to 8, and the ligand is attached via a linker X by any suitable means; Item 15. The peptide-antigen conjugate according to item 14. (Item 16) The hydrophobic molecule (H) has the formula II: [ka] wherein l is between 3 and 300, o is between 0 and 300, and R 3 is selected from one of hydrogen, hydroxyl, or amine; R 4 is hydrogen, lower alkyl, [ka] wherein y11 is typically selected from 1 to 8, FG is a functional group selected from a carboxylic acid, an aldehyde, a ketone, an amine, a thiol, an azide or an alkyne, which is linked to a ligand having adjuvant properties, and the N-terminus is linked to said peptide antigen either directly or via said extension (B1 or B2) and / or a linker (L) or a charged molecule (C), either directly via an amide bond or indirectly via a linker, Item 15. The peptide-antigen conjugate according to item 14. (Item 17) 15. The peptide-antigen conjugate of item 14, wherein the hydrophobic molecule (H) comprises a homopolymer or copolymer of poly(leucine), poly(tryptophan), poly(γ-glutamyldiethylene glycol monomethyl ether), poly(γ-benzyl glutamate), poly(N-isopropylacrylamide) (NIPAM), poly[2(2-methoxyethoxy)ethyl methacrylate] (DEGMA) or poly[N-(2-hydroxypropyl)methacrylamide] (HPMA). (Item 18) 16. The peptide-antigen conjugate of item 15, wherein the hydrophobic molecule (H) comprising the poly(amino acid) of formula I is grafted or terminally linked to a second polymer based on poly(leucine), poly(tryptophan), poly(γ-glutamyldiethylene glycol monomethyl ether), poly(γ-benzyl glutamate), poly(N-isopropylacrylamide) (NIPAM), poly[2(2-methoxyethoxy)ethyl methacrylate] (DEGMA) or poly[N-(2-hydroxypropyl)methacrylamide] (HPMA). (Item 19) 17. The peptide-antigen conjugate of claim 16, wherein the hydrophobic molecule (H) comprising the poly(amino acid) of formula II is grafted or terminally linked to a second polymer based on poly(leucine), poly(tryptophan), poly(γ-glutamyldiethylene glycol monomethyl ether), poly(γ-benzyl glutamate), poly(N-isopropylacrylamide) (NIPAM), poly[2(2-methoxyethoxy)ethyl methacrylate] (DEGMA) or poly[N-(2-hydroxypropyl)methacrylamide] (HPMA). (Item 20) 20. The peptide-antigen conjugate of either item 18 or item 19, wherein the second polymer comprises a plurality of monomer units of about 30 to 500 monomer units. (Item 21) 21. The peptide-antigen conjugate according to any one of items 14 to 20, wherein one or more adjuvants are linked as pendant side chains to the polymer constituting the hydrophobic molecule (H). (Item 22) 21. The peptide-antigen conjugate according to any one of items 14 to 20, wherein one or more adjuvants are linked to one end of the polymer (semi-telechelic). (Item 23) Item 24. The peptide-antigen conjugate according to either Item 21 or Item 22, wherein the adjuvant is either a Toll-like receptor agonist or a STING agonist. 24. The peptide-antigen conjugate according to item 23, wherein the Toll-like receptor agonist is either an agonist of Toll-like receptor-7 and / or -8. (Item 25) The adjuvant has formula III: [ka] wherein R 7is selected from one of hydrogen, optionally substituted lower alkyl, or optionally substituted lower ether; R 8 is selected from one of an optionally substituted aryl amine or an optionally substituted lower alkyl amine; 25. The peptide-antigen conjugate according to item 24. (Item 26) Three or more imidazoquinoline molecules of formula III are linked to the polymer constituting the hydrophobic molecule (H), and R 8 26. The peptide-antigen conjugate according to item 25, wherein is selected from one of optionally substituted lower alkyl amines. (Item 27) Three or fewer imidazoquinoline molecules of formula III are linked to the polymer constituting the hydrophobic molecule (H), and R 8 27. The peptide antigen conjugate according to item 26, wherein is selected from one of the optionally substituted arylamines. (Item 28) The peptide-antigen conjugate according to any one of the preceding items, wherein the particle (P) or the hydrophobic molecule (H) is insoluble in aqueous buffer at a pH of about 7.4 and a temperature between about 20°C and 40°C, down to a concentration of 0.1 mg / mL. (Item 29) The peptide-antigen conjugate of any one of the preceding items, which assembles in an aqueous buffer solution at a pH of about 7.4 to form particles with a diameter greater than 5 nm, composed of micelles or other types of nano- or micro-sized supramolecular assemblies comprising two or more peptide-antigen conjugates. (Item 30) 30. The peptide-antigen conjugate of item 29, which assembles in an aqueous buffer solution at a pH of about 7.4 to form micelles with diameters between about 5 and 200 nm. (Item 31) 30. The peptide-antigen conjugate of item 29, which aggregates in aqueous buffer at a pH of about 7.4. (Item 32) 2. The peptide antigen conjugate of claim 1, wherein a linker precursor X1 is linked to the peptide antigen (A) either directly or via a C-terminal extension (B2) and forms a link with a linker precursor X2 either on the hydrophobic molecule (H) or on the particle (P). (Item 33) The linker precursor X1 has the formula: [ka] wherein the N-terminal amine of the tag is linked to the peptide antigen (A) either directly or via said C-terminal extension B2; 1 is selected from one of hydrogen, hydroxyl, or amine; y1 is an integer from 1 to 8; FG is selected from one of thiol, amine, azide, or alkyne (or DBCO). 33. The peptide-antigen conjugate according to item 32. (Item 34) 34. The peptide antigen conjugate of claim 33, wherein FG is azide, y1 is 4, and the azide is linked to a DBCO molecule that is linked to a particle (P) or a hydrophobic molecule (H). (Item 35) A linker precursor X1 is linked to said N-terminal extension (B1) and has the formula: [ka] wherein the carbonyl of the tag is linked to the N-terminus of the peptide antigen (A) either directly or via a B1 extension; y2 is an integer from 1 to 8; and FG is selected from one of a thiol, an amine, an azide or an alkyne (or DBCO). (Item 36) 36. The peptide antigen conjugate according to any one of items 1 to 35, wherein the peptide antigen (P), optional N-terminal extension (B1), optional C-terminal extension (B2) and optional charged molecule (C) and optional linker precursor X1 are synthesized as a single peptide by solid phase peptide synthesis, and wherein the C-terminal extension (B2) contains proline (or pseudoproline during solid phase peptide synthesis while the sequence is attached to the resin used to synthesize the peptide). (Item 37) 37. The peptide-antigen conjugate according to any one of items 2 to 36, wherein the charged molecule (C) comprises one or more functional groups selected from a primary amine, a secondary amine, a tertiary amine, a quaternary amine, guanidinium, imidazolium, ammonium, sulfonium, phosphonium, carboxylate, sulfate, phosphate, phosphoramidate or phosphonate. (Item 38) 38. The peptide-antigen conjugate according to item 37, wherein the charged molecule (C) contains both negatively charged and positively charged functional groups. (Item 39) The number of charged functional groups constituting the charged molecule (C) is as follows: a. when the peptide antigen (A) has a total average hydropathy value greater than or equal to about 0.75, the net electrostatic charge of the peptide antigen conjugate comprises a positive value greater than or equal to about +6; b. when the peptide antigen (A) has a total average hydropathy value that is greater than or equal to about 0.25 and less than about 0.75, the net electrostatic charge of the peptide antigen conjugate comprises a positive value that is greater than or equal to about +5; c. when the peptide antigen (A) has a total average hydropathy value that is less than about 0.25, the net electrostatic charge of the peptide antigen conjugate comprises a positive value greater than or equal to about +4; d. when the peptide antigen (A) has a total average hydropathy value greater than or equal to about 0.75, the net electrostatic charge of the peptide antigen conjugate comprises a negative value less than or equal to about -6; e. when the peptide antigen (A) has a total average hydropathy value that is greater than or equal to about 0.25 and less than about 0.75, the net electrostatic charge of the peptide antigen conjugate comprises a negative value that is less than or equal to about -5; or f. if the peptide antigen (A) has a total average hydropathy value that is less than about 0.25, then the net electrostatic charge of the peptide antigen conjugate comprises a positive value greater than or equal to about -4; 39. The peptide-antigen conjugate according to any one of items 2 to 38, which is prepared. (Item 40) 40. The peptide antigen conjugate of item 39, wherein the peptide antigen (A), the N-terminal extension (B1), the C-terminal extension (B2), the charged molecule (C) and the linker precursor X1 are produced as a single peptide that is soluble in an aqueous buffer at a pH of about 7.4 up to about 1.0 mg / mL. (Item 41) 41. A particle comprising the peptide-antigen conjugate according to any one of items 1 to 40. (Item 42) 42. The particle according to item 41, comprising a plurality of different peptide antigen conjugates. (Item 43) An immunogenic composition comprising the peptide-antigen conjugate according to any one of items 1 to 40 or the particle according to items 41 to 42. (Item 44) 44. The immunogenic composition of item 43, further comprising a vaccine adjuvant. (Item 45) 45. The immunogenic composition of either item 43 or item 44, comprising a plurality of different peptide-antigen conjugates. (Item 46) 46. ​​The peptide-antigen conjugate according to any one of items 1 to 40, the particle according to any one of items 41 and 42, or the immunogenic composition according to any one of items 43 to 45, wherein the peptide antigen (A) comprises a tumor-associated antigen. (Item 47) 47. The peptide-antigen conjugate of claim 46, the particle of claim 46, or the immunogenic composition of claim 46, wherein the tumor-associated antigen is a neoantigen. (Item 48) 48. The peptide-antigen conjugate according to any one of items 1 to 40 and 46 to 47, the particle according to any one of items 41 to 42 and 46 to 47, or the immunogenic composition according to any one of items 43 to 47, wherein the peptide antigen (A) is a minimal CD4 or CD8 T-cell epitope. (Item 49) 48. The immunogenic composition of any one of items 43 to 47, comprising 10 to 50 different peptide-antigen conjugates composed of peptide antigens (A) selected from minimal CD4 T-cell epitopes, CD8 T-cell epitopes, or both minimal CD4 and CD8 T-cell epitopes. (Item 50) Minimal CD4 T-cell epitopes, minimal CD8 T-cell epitopes, or minimal CD4 and CD8 T-cell epitopes, plus CD8 T-cell epitopes and / or CD4 48. The immunogenic composition according to any one of Items 43 to 47, comprising 10 to 50 different peptide-antigen conjugates composed of a peptide antigen (A) selected from a 15 to 35 amino acid synthetic long-chain peptide containing a T-cell epitope and a peptide antigen (B) selected from a 15 to 35 amino acid synthetic long-chain peptide containing a T-cell epitope. (Item 51) 51. The peptide-antigen conjugate of Item 48, the particle of Item 48, or the immunogenic composition of any one of Items 48 to 50, wherein the minimal CD8 T cell or CD4 T cell epitope has a predicted binding affinity that is less than or equal to the 0.5 percentile by the IEDB consensus algorithm for an MHC molecule that matches the subject. (Item 52) 52. The immunogenic composition according to any one of items 43 to 51, further comprising a peptide-antigen conjugate, wherein the peptide antigen (A) is a universal CD4 T-cell epitope. (Item 53) A method of treating a patient suffering from a disease, comprising administering to said patient suffering from said disease a peptide-antigen conjugate or immunogenic composition described in any one of the preceding items. (Item 54) 54. The method of claim 53, further comprising administering an additional immunotherapeutic modality, including an immune checkpoint inhibitor, such as an anti-PD1, anti-PD-L1, or anti-CTLA-4 antibody; tumor-infiltrating lymphocyte and chimeric antigen receptor T cell-based therapy; a bispecific antibody; an anti-tumor antibody; or a drug designed to overcome immunosuppression. (Item 55) 55. The method of either item 53 or item 54, comprising a heterologous immunization scheme in which the immunogenic composition or peptide-antigen conjugate of any preceding item is used as either a prime or a boost in conjunction with a heterologous vaccine. (Item 56) 56. The method according to any one of items 53 to 55, wherein the treatment is used in combination with radiation therapy, chemotherapy or surgery. (Item 57) 53. Use of the peptide-antigen conjugate, particle or immunogenic composition according to any one of items 1 to 52 in the manufacture of a medicament for the treatment of a disease. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 is a schematic diagram of one particular embodiment of a peptide-antigen conjugate.

[0035] [Figure 2]FIG. 2 is a diagram of a general scheme for the synthesis of peptide antigen conjugates by reacting a peptide antigen fragment containing an azide-bearing linker precursor X1 with a DBCO-bearing linker precursor X2 to form a triazole linker (L) that connects the peptide antigen (A) to a hydrophobic molecule (H).

[0036] [Figure 3] FIG. 3 is a schematic diagram of the synthesis of compound 1.

[0037] [Figure 4] Figure 4 shows the molecular weight and hydrodynamic behavior of peptide antigens (A) composed of either synthetic long peptides (SLP or "LP") or minimal ("Min") CD8 T cell epitopes derived from the MC38 tumor cell line, and of peptide antigen conjugates that deliver the peptide antigens (A). Peptide antigens (A) composed of LP were linked to the linker precursor X1, azido-lysine (K'), which was either left unlinked or linked to a hydrophobic molecule (H) composed of either DBCO-W3 or DBCO-2BXy3. Peptide antigens (A) composed of LP were linked to an extension (B1), which was linked to the linker precursor X1, azido-lysine (K'), which was either left unlinked or linked to a hydrophobic molecule (H) composed of either DBCO-W3 or DBCO-2BXy3.

[0038] [Figure 5]Figure 5 shows the effect of peptide antigen (A) length, hydrodynamic behavior, and co-delivery of a TLR-7 / 8 agonist (TLR-7 / 8a) on the immunogenicity of peptide-based neoantigens (Irgq). Mice (N = 5 per group) were immunized with different immunogenic compositions containing peptide antigens (A) based on either a synthetic long peptide (LP, sometimes referred to as "SLP") or the minimal CD8 T cell epitope (Min, sometimes referred to as ME) of the antigen Irgq derived from the MC38 tumor cell line, either as the sole peptide antigen (A) mixed with a TLR-7 / 8a adjuvant, as peptide antigen (A) linked to the hydrophobic molecule (H) W3 and mixed with a TLR-7 / 8a adjuvant, or as peptide antigen (A) linked to the hydrophobic molecule (H) co-delivering the TLR-7 / 8a adjuvant 2BXy3. Mice were immunized on days 0 and 14, and antigen-specific CD8 T cell responses (% IFNg+ relative to total CD8 T cells) were assessed from whole blood on days 0 and 25. Open circles indicate that the peptide antigen (A) was soluble (i.e., non-particulate), whereas closed circles indicate that mice received a particulate immunogenic composition of peptide antigen (A).

[0039] [Figure 6]Figure 6 shows the effect of peptide antigen (A) length, hydrodynamic behavior, and co-delivery of TLR-7 / 8a adjuvant on the immunogenicity of a peptide-based neoantigen (Cpne1). Mice (N=5 per group) were immunized with different immunogenic compositions containing peptide antigens (A) based on either a synthetic long peptide (LP) or the minimal CD8 T cell epitope (Min) of the MC38 tumor cell line-derived antigen Cpne1, either as the sole peptide antigen (A) mixed with TLR-7 / 8a adjuvant, as peptide antigen (A) linked to the hydrophobic molecule (H) W3 and mixed with TLR-7 / 8a adjuvant, or as peptide antigen (A) linked to the hydrophobic molecule (H) co-delivered with the TLR-7 / 8a adjuvant 2BXy3. Mice were immunized on days 0 and 14, and antigen-specific CD8 T cell responses (% IFNg+ relative to total CD8 T cells) were assessed from whole blood on days 0 and 25. Open circles indicate that the peptide antigen (A) was soluble (i.e., non-particulate), whereas closed circles indicate that mice received a particulate immunogenic composition of peptide antigen (A).

[0040] [Figure 7] Figure 7 shows the molecular weight and hydrodynamic behavior of peptide antigens (A) composed of either synthetic long peptides (SLP or "LP") or minimal ("Min") CD8 T cell epitopes derived from the B16 tumor cell line, and of peptide antigen conjugates that deliver the peptide antigens (A). Peptide antigens (A) composed of LP were linked to the linker precursor X1, azido-lysine (K'), which was either left unlinked or linked to a hydrophobic molecule (H) composed of either DBCO-W3 or DBCO-BXy3. Peptide antigens (A) composed of LP were linked to an extension (B1), which was linked to the linker precursor X1, azido-lysine (K'), which was either left unlinked or linked to a hydrophobic molecule (H) composed of either DBCO-W3 or DBCO-2BXy3.

[0041] [Figure 8] Figure 8 shows the effect of peptide antigen (A) length and potency of TLR-7 / 8a linked to a hydrophobic molecule (H) on the immunogenicity of peptide antigen conjugates. Mice (N=5 per group) were immunized with different immunogenic compositions containing peptide antigen conjugates delivering either a synthetic long peptide (LP) of B16 tumor-derived peptide antigen (A) linked to either the hydrophobic molecule (H), DBCO-2BXy5 or DBCO-2B5, a minimal CD8 T cell epitope (Min), or both the SLP and Min. Mice were immunized on days 0 and 14, and antigen-specific CD4 and CD8 T cell responses (% IFNg+ relative to total) were assessed from whole blood on day 24. Bar graphs show the sum of the mean responses to each peptide antigen (M47, M44, M30, M25, M33, M27, and M08).

[0042] [Figure 9] Figure 9 shows the effect of the potency of TLR-7 / 8a linked to a hydrophobic molecule (H) on the immunogenicity of peptide neo-antigens and the breadth of T cell responses generated against a library of peptide neo-antigens. Mice (N=5 per group) were immunized with immunogenic compositions containing four unique peptide antigen conjugates delivering distinct MC38 tumor-derived minimal CD8 T cell epitopes (40 epitopes total) linked to either 2BXy5 or 2B5 hydrophobic molecules (H). Mice were immunized on days 0 and 14, and antigen-specific CD8 T cell responses (% IFNg+ relative to total CD8 T cells) were assessed from whole blood on day 24. CD8 T cell responses to each of the 40 epitopes are shown. The percentage of peptide antigens (A) containing minimal CD8 T cell epitopes that resulted in a CD8 T cell response is shown in a pie chart.

[0043] [Figure 10]Figure 10 shows the effect of the number and dose of peptide antigens (A) and the number of vaccination sites on the immunogenicity of peptide-based neoantigens. Mice (N=5 per group) were immunized with either a peptide antigen conjugate containing a single minimal CD8 T cell epitope (Adpgk) or peptide antigen conjugates containing four different minimal CD8 T cell epitopes (Dpagt, Cpne1, Adpgk, and Aatf). The dose of peptide antigen (A) and the number of vaccination sites were varied for the various groups. Mice were immunized on days 0 and 14, and antigen-specific CD8 T cell responses (%IFNg+ relative to total CD8 T cells) were assessed from whole blood on day 24.

[0044] [Figure 11] FIG. 11 is a schematic diagram of a peptide-antigen conjugate of Formula V.

[0045] [Figure 12] Figure 12: Effect of N- and C-terminal extensions (B1 and B2) on in vitro potency for CD8 T cell activation. Splenocyte cultures with APCs and neoantigen (Cpne1)-specific CD8 T cells were stimulated in vitro with minimal CD8 T cell epitope (Cpne1) peptide antigens (A) linked to different extensions (B1 and B2) and assessed for their ability to stimulate IFNg production from Cpne1-specific CD8 T cells.

[0046] [Figure 13]Figure 13 is a diagram of the peptide antigen conjugate evaluated in Figure 12. A peptide antigen (A) consisting of the minimal epitope Cpne1 (Ser-Ser-Pro-Tyr-Leu-His-Tyr-Leu SEQ ID NO: 1) was linked to either or both of the B1 and / or B2 extensions containing cathepsin and / or immunoproteasome cleavage sites, and in this case, the peptide antigen (A) was linked, either directly or through the B2 extension, to the linker precursor X1, azido-lysine (K'), which in turn was linked to a DBCO molecule linked to the hydrophobic molecule (H), 2BXy3.

[0047] [Figure 14] Figure 14 shows the effect of charged molecules (C) and extensions (B1 and / or B2) on the in vivo immunogenicity of peptide-antigen conjugates delivering peptide antigens (A) composed of minimal CD8 T cell epitopes. Mice (N=5 per group) were immunized with peptide-antigen conjugates containing different charged molecules (C) and extensions (B1 and / or B2) on days 0 and 14, and antigen-specific CD8 T cell responses (% IFNg+ relative to total CD8 T cells) were assessed from whole blood on days 10 (upper panel) and 24 (lower panel).

[0048] [Figure 15] Figure 15 shows the effect of N- and C-terminal extensions (B1 and B2) on in vitro potency for CD8 T cell activation. The minimal CD8 T cell epitope (Adpgk) peptide antigen (A) was linked to different extensions (B1 and B2) and evaluated for their ability to stimulate IFNg production from Adpgk-specific CD8 T cells.

[0049] [Figure 16] Figure 16 shows the effective concentration at half-maximal activity (EC50) for the different peptide-antigen conjugates evaluated in Figure 15. The lower the EC50, the higher the potency.

[0050] [Figure 17] Figure 17 shows the effect of N- and C-terminal extensions (B1 and B2) on in vitro potency for CD8 T cell activation and in vivo potency for eliciting de novo CD8 T cell responses. (A) The minimal CD8 T cell epitope (Adpgk) peptide antigen (A) was linked to different extensions (B1 and B2) and evaluated for its ability to stimulate IFNg production from Adpgk-specific CD8 T cells (B). The ability of peptide antigen conjugates with different N- and C-terminal extensions (B1 and B2) linked to the hydrophobic molecule (H), 2B3W2, was evaluated on day 13 after a single immunization.

[0051] [Figure 18-1] Figure 18 is a diagram of the effect of the net charge and hydrophobic molecular composition of peptide-antigen conjugates on hydrodynamic behavior. The hydrodynamic behavior of various different peptide-antigen conjugates of Formula V at 0.1 mg / mL or 0.5 mg / mL in PBS at a pH of 7.4 was assessed by dynamic light scattering. The net charge and number- or mass-average particle size of the peptide-antigen conjugates are reported. Turbidity was determined by measuring the absorbance of the solution at 490 nm. A turbidity >0.04 indicates aggregation. [Figure 18-2] Same as above [Figure 18-3] Same as above [Figure 18-4] Same as above [Figure 18-5] Same as above [Figure 18-6] Same as above [Figure 18-7] Same as above [Figure 18-8] Same as above [Figure 18-9] Same as above [Figure 18-10] Same as above

[0052] [Figure 19-1]Figure 19 is a diagram of the effect of net charge of peptide antigen conjugates on hydrodynamic behavior. (A) The effect of net charge on the hydrodynamic behavior of various different peptide antigen conjugates of Formula V suspended at 0.1 mg / mL or 0.5 mg / mL in PBS at a pH of 7.4 was assessed by dynamic light scattering. (B) Figure panel B shows a curated list of the data shown in figure panel (A). (C) GRAVY frequency distribution and (D) charge frequency distribution of antigens delivered as peptide antigen conjugates. (E) Particle size of matched LP neoantigens (n=39) prepared as peptide antigen conjugates of Formula V with a net charge of +6 or ≥ +8 that self-assemble into nanoparticles that codelidate TLR-7 / 8a ("SNP-7 / 8a"). (F) Effect of net charge and grand average hydropathy (GRAVY) of peptide antigen conjugates on hydrodynamic behavior. The hydrodynamic behavior of a variety of different peptide-antigen conjugates suspended at 0.1 mg / mL in PBS at a pH of 7.4 was assessed by dynamic light scattering. Number-average particle size is reported as a function of net charge and GRAVY value. [Figure 19-2] Same as above [Figure 19-3] Same as above [Figure 19-4] Same as above

[0053] [Figure 20]Figure 20 is a diagram of the effect of the net charge of the peptide-antigen conjugate and the length and composition of the hydrophobic molecule (H) on the hydrodynamic behavior of the peptide-antigen conjugate of Formula V. The hydrodynamic behavior of different peptide-antigen conjugates with varying net charge and hydrophobic molecule composition delivering a model neoantigen-based minimal epitope (Min), Ala-Ser-Met-Thr-Asn-Met-Glu-Leu-Met-Ser-Ser (SEQ ID NO: 2) ("Adpgk"), suspended at 0.1 mg / mL in PBS at a pH of 7.4, was assessed by dynamic light scattering. (A) Number-average particle size is reported as a function of the net charge of the peptide-antigen conjugate for four different hydrophobic molecules (2B2W8, 2BXy5, 2B5, W5). (B) Size distribution data are shown for a peptide antigen conjugate with a net charge of +6 that self-assembles into nanoparticles ("Min-SNP-7 / 8a"), and for a peptide antigen conjugate delivered with the same peptide antigen (A) but with a net charge of 0 that assembles into microparticles / aggregates ("Min-MP-7 / 8a").

[0054] [Figure 21] Figure 21 illustrates the effect of net charge on the hydrodynamic behavior of peptide-antigen conjugates of Formula V delivering hydrophobic peptide antigens. (A) Grand average hydropathy (GRAVY) distribution plot of 1,377 LP neoantigens from four mouse tumor models (B16.F10, MC38, 3123, and Panc02). (B) The most hydrophobic LP neoantigens identified in panel (A) (red circles) were prepared as LP and Min delivered as peptide-antigen conjugates of Formula V that self-assemble into nanoparticles co-delivering TLR-7 / 8a ("SNP-7 / 8a"), and the effect of net charge (absolute value) on particle size was evaluated. Particle size was assessed by dynamic light scattering using peptide-antigen conjugates suspended at 0.5 mg / mL in PBS at a pH of 7.4. Mass-average particle size is reported as a function of the net charge of the peptide-antigen conjugate.

[0055] [Figure 22] Figure 22 is a diagram of the effect of charged molecule (C or "C block") and hydrophobic molecule (H or "H block") composition on the immunogenicity of generating CD8 T cell responses to the neoantigen minimal epitope, Adpgk, delivered as a peptide-antigen conjugate of Formula V. (A-E) Peptide-antigen conjugates with various charged molecule compositions (either poly(K), poly(R), poly(D), or none) and hydrophobic molecule compositions (either W5, 2BXy3W2, or 2B3W2) delivering the minimal epitope, Adpgk, were administered to mice (N=3 per group per dose) at different doses on days 0, 14, and 28, after which neoantigen (Adpgk)-specific CD8 T cell responses were assessed from whole blood at a series of time points.

[0056] [Figure 23] Figure 23 is a diagram of the effect of charged molecule (C or "C-block") composition on the immunogenicity of generating CD8 T cell responses against the neoantigen minimal epitope, Adpgk, delivered as a peptide-antigen conjugate of Formula V. (A) Peptide-antigen conjugates with either a poly(K)-based C-block, a poly(D)-based C-block, or a PEG-based C-block delivering the minimal epitope, Adpgk, were administered to mice (N=3 per group per dose) at different doses on days 0, 14, and 28, and neoantigen (Adpgk)-specific CD8 T cell responses were assessed from whole blood on day 36. (B)

[0057] [Figure 24] Figure 24 is a diagram of the effect of administration route on CD8 T cell responses generated by a peptide antigen conjugate ("SNP-7 / 8a"). A peptide antigen conjugate with a poly(K) charged molecule (C) and a 2B3W2 hydrophobic molecule (H) delivering the neoantigen minimal epitope (Adpgk = Ala-Ser-Met-Thr-Asn-Met-Glu-Leu-Met (SEQ ID NO: 77)) was administered to mice on days 0, 14, and 28, and neoantigen (Adpgk)-specific CD8 T cell responses were assessed from whole blood on day 36.

[0058] [Figure 25] Figure 25 shows the effect of hydrophobic molecule (H) composition and hydrodynamic behavior of peptide-antigen conjugates on CD4 and CD8 T cell responses. Immunological compositions containing minimal CD8 and CD4 T cell epitopes (PADRE epitopes) were used in the form of either microparticles (MP) (diameter >500 nm) or nanoparticle (NP) micelles (diameter <200 nm) bearing various amounts and potencies of TLR-7 / 8 agonists. Mice were immunized on days 0 and 14, and antigen (Adpgk)-specific (A) CD8 T cell responses (% IFNg+ of total CD8 T cells) and (B) PADRE-specific CD4 T cell responses were assessed from whole blood on day 24.

[0059] [Figure 26] FIG. 26 is a schematic and chemical overview of peptide antigen conjugates with poly(K) charged molecules (C) and 2B3W2-based hydrophobic molecules (H) that deliver either the LP or Min epitope forms of the peptide antigen.

[0060] [Figure 27-1]Figure 27 shows that particulate delivery of peptide neoantigens enhances CD8 T cell responses to peptide antigen-based neoantigens (A). (A) Turbidity of different neoantigens as native LP, LP linked to a hydrophobic molecule (2B3W2) that assembles into microparticles ("MP-7 / 8a"), or LP synthesized as a peptide antigen conjugate of Formula V that self-assembles into nanoparticles ("SNP-7 / 8a"). A turbidity >0.05 OD indicates aggregation. (B-E) Mice were immunized on days 0 and 14 with native LP mixed with poly-ICLC or LP delivered as MP-7 / 8a or SNP-7 / 8a, and CD8 T cell responses were assessed from whole blood on day 28. Data on a logarithmic scale are reported as geometric means with 95% CI. Comparisons of multiple groups for statistical significance were determined using one- or two-way ANOVA. ns=not significant; *, p=0.05; **, p=0.01. [Figure 27-2] Same as above [Figure 27-3] Same as above

[0061] [Figure 28] Figure 28 shows a mechanistic illustration demonstrating the enhanced immunogenicity of peptide-antigen conjugates that self-assemble into nanoparticles that co-deliver TLR-7 / 8a (SNP-7 / 8a). (A-C) The minimal epitope, Adpgk, was fluorescently labeled and then administered subcutaneously into the hind footpad of mice on day 0 as either a soluble peptide mixed with the small molecule TLR-7 / 8a (7 / 8a), a soluble peptide mixed with MP-7 / 8a, or a soluble peptide covalently linked to MP-7 / 8a or SNP-7 / 8a. Lymph nodes draining the immunization site (N = 5 per time point per group) were subsequently harvested at sequential time points and assessed for peptide amount (E), peptide uptake by lymph node APCs per cell (F), and IL-12p40 cytokine production (G). Data are reported as mean ± SEM. Comparisons of multiple groups for statistical significance were determined using one-way or two-way ANOVA. ns = not significant; *, p = 0.05; **, p = 0.01.

[0062] [Figure 29-1] Figure 29 shows the immunogenicity of peptide-based neoantigens delivered as either LP or Min peptide-antigen conjugates of Formula V, which self-assemble to form nanoparticles that co-deliver TLR-7 / 8a (SNP-7 / 8a). Different peptide-based neoantigens were prepared as either LP or Min peptide-antigen conjugates, where the charged molecule (C) is poly(lysine) and the hydrophobic molecule (H) is 2B3W2, which self-assemble to form nanoparticles that co-deliver TLR-7 / 8a (referred to as "SNP-7 / 8a"). Mice (N=7 / group) were immunized on days 0 and 14 with either the LP or Min form of the peptide-based neoantigen linked to SNP-7 / 8a. (A) CD8 T cell responses and (B) CD4 T cell responses were assessed from whole blood on day 28. Data on a logarithmic scale are reported as geometric means with 95% CI. Comparisons of multiple groups for statistical significance were determined using one-way or two-way ANOVA. ns = not significant; *, p = 0.05; **, p = 0.01. [Figure 29-2]Figure 29 shows the immunogenicity of peptide-based neoantigens delivered as either LP or Min peptide-antigen conjugates of Formula V, which self-assemble to form nanoparticles that co-deliver TLR-7 / 8a (SNP-7 / 8a). Different peptide-based neoantigens were prepared as either LP or Min peptide-antigen conjugates, where the charged molecule (C) is poly(lysine) and the hydrophobic molecule (H) is 2B3W2, which self-assemble to form nanoparticles that co-deliver TLR-7 / 8a (referred to as "SNP-7 / 8a"). Mice (N=7 / group) were immunized on days 0 and 14 with either the LP or Min form of the peptide-based neoantigen linked to SNP-7 / 8a. (A) CD8 T cell responses and (B) CD4 T cell responses were assessed from whole blood on day 28. Data on a logarithmic scale are reported as geometric means with 95% CI. Comparisons of multiple groups for statistical significance were determined using one-way or two-way ANOVA. ns = not significant; *, p = 0.05; **, p = 0.01.

[0063] [Figure 30] Figure 30 shows the immunogenicity of peptide-based neoantigens delivered as LP-based peptide-antigen conjugates of Formula V, where the charged molecule (C) is poly(lysine) and the hydrophobic molecule (H) is 2B3W2, which self-assemble into nanoparticles that codelidate TLR-7 / 8a (referred to as "SNP-7 / 8a"), compared to native LPs mixed with poly-ICLC. Mice were immunized on days 0 and 14, and CD8 T cell responses were assessed from whole blood on day 28.

[0064] [Figure 31]Figure 31 is a diagram of the relationship between immunogenicity and predicted binding affinity. (A) Immunogenicity of minimal epitopes (N=179) that generate a CD8 T cell response when delivered as a peptide-antigen conjugate where the hydrophobic molecule (H) is polymer-TLR-7 / 8a, plotted against predicted binding affinity using the Immune Epitope Database (IEDB) consensus algorithm. (B) Receiver operating characteristic (ROC) curves for the sensitivity and specificity of different MHC-I binding prediction algorithms based on a cutoff of a consensus score of 0.5 or 500 nM as a binder.

[0065] [Figure 32] Figure 32 shows that autoantigens and neoantigens delivered as peptide antigens (A) on peptide-antigen conjugates of Formula V, which self-assemble into nanoparticles that codelidate TLR-7 / 8a (SNP-7 / 8a), elicit CD8 and CD4 T cell-mediated elimination of established melanoma. Mice with established B16.F10 tumors were treated on days 2, 9, and 16 with PD1 and either SNP-7 / 8a-delivering Adpgk LP (a CD8 T cell epitope absent from B16.F10), autoantigen (Trp1), a minimal CD8 T cell epitope, or M30 LP, a neoantigen with a CD4 T cell epitope.

[0066] [Figure 33] Figure 33 shows that neoantigens delivered as peptide-antigen conjugates of Formula V, which self-assemble into nanoparticles that codelidate TLR-7 / 8a (SNP-7 / 8a) administered by subcutaneous and intravenous routes, elicit robust CD8 T cell-mediated elimination of established tumors. Mice with established MC38 tumors were vaccinated with Adpgk LP-SNP-7 / 8a by either subcutaneous or intravenous routes on days 9 and 16, and tumor volume was assessed at subsequent time points.

[0067] [Figure 34]Figure 34 shows that neoantigens delivered as peptide-antigen conjugates of Formula V, which self-assemble into nanoparticles that co-deliver TLR-7 / 8a (SNP-7 / 8a), elicit CD8 T cell-mediated elimination of established melanoma. Mice with established B16.F10 tumors were treated with PDL1 and either SNP-7 / 8a-delivered Med12 (MC38 neoantigen with a CD8 T cell epitope), Trp1 (B16 autoantigen with a CD8 T cell epitope), or M39 (B16 neoantigen with a CD8 T cell epitope) on days 1, 8, and 15. (A) Tumor growth was monitored, and (B) CD8 T cell responses were assessed on day 17.

[0068] [Figure 35] Figure 35 shows that viral (HPV) antigens delivered as peptide-antigen conjugates of Formula V, which self-assemble into nanoparticles that co-deliver TLR-7 / 8a (SNP-7 / 8a), elicit CD8 T cell-mediated rejection of HPV+ tumors. (A and B) Mice were immunized on days 0 and 14 with different E6 and E7 peptide minimal epitopes (min) derived from HPV delivered as peptide-antigen conjugates of Formula V (where the charged molecule (C) is poly(lysine) and the hydrophobic molecule (H) is 2B3W2), which self-assemble into nanoparticles that co-deliver TLR-7 / 8a (referred to as "SNP-7 / 8a"). (A) CD8 T cell responses were assessed on day 28, and mice were challenged with an HPV+ cancer cell line (TC1). (B) Tumor volume was assessed 14 days post-challenge.

[0069] [Figure 36-1]Figure 36 shows the particle size and stability of multi-antigen particles. (A) Neoantigen LP as peptide antigens (A1-A9) with various charges (-6 to +6) and hydropathy (GRAVY from -2 to +2) were synthesized as (B) peptide antigen conjugates of formula V (where the charged molecule (C) is poly(K), B1 is VR, B2 is SPVZ, the X1 linker precursor is azido-lysine ("X" is also referred to as K'), and the hydrophobic molecule (H) is 2B3W2) that self-assemble to construct nanoparticles that codelidate TLR-7 / 8a (SNP-7 / 8a). The peptide antigen conjugates were suspended at 0.5 mg / mL in PBS pH 7.4 and evaluated for turbidity (OD490nm) and particle size (diameter, nm). (C) Multi-antigen particles containing several different peptide-antigen conjugates (A1-A9) were mixed in different ratios in DMSO solution (Scenarios 1-7), then suspended in PBS pH 7.4 at 0.5 mg / mL and evaluated for turbidity (OD490nm) and particle size (diameter, nm). The percentage of each peptide-antigen conjugate containing multi-antigen particles for each scenario is provided in the table. [Figure 36-2]Figure 36 shows the particle size and stability of multi-antigen particles. (A) Neoantigen LP as peptide antigens (A1-A9) with various charges (-6 to +6) and hydropathy (GRAVY from -2 to +2) were synthesized as (B) peptide antigen conjugates of formula V (where the charged molecule (C) is poly(K), B1 is VR, B2 is SPVZ, the X1 linker precursor is azido-lysine ("X" is also referred to as K'), and the hydrophobic molecule (H) is 2B3W2) that self-assemble to construct nanoparticles that codelidate TLR-7 / 8a (SNP-7 / 8a). The peptide antigen conjugates were suspended at 0.5 mg / mL in PBS pH 7.4 and evaluated for turbidity (OD490nm) and particle size (diameter, nm). (C) Multi-antigen particles containing several different peptide-antigen conjugates (A1-A9) were mixed in different ratios in DMSO solution (Scenarios 1-7), then suspended in PBS pH 7.4 at 0.5 mg / mL and evaluated for turbidity (OD490nm) and particle size (diameter, nm). The percentage of each peptide-antigen conjugate containing multi-antigen particles for each scenario is provided in the table.

[0070] [Figure 37] FIG. 37 shows T cell responses induced by peptide-antigen conjugates of Formula V delivering tumor-associated self-antigens, infectious disease (HIV) antigens, and foreign antigens.

[0071] [Figure 38-1] FIG. 38 shows the particle size and bioactivity of peptide-antigen conjugates of formula V, composed of different charged molecules (C) or different hydrophobic molecules (H) based on poly(amino acid)s of formula II, linked to adjuvants of formula III. [Figure 38-2] FIG. 38 shows the particle size and bioactivity of peptide-antigen conjugates of formula V, composed of different charged molecules (C) or different hydrophobic molecules (H) based on poly(amino acid)s of formula II, linked to adjuvants of formula III. [Figure 38-3]FIG. 38 shows the particle size and bioactivity of peptide-antigen conjugates of formula V, composed of different charged molecules (C) or different hydrophobic molecules (H) based on poly(amino acid)s of formula II, linked to adjuvants of formula III.

[0072] [Figure 39-1] FIG. 39 shows the particle size and bioactivity of peptide antigen conjugates of formula V composed of different hydrophobic molecules (H) based on poly(amino acid) of formula II linked to different PRR agonists. [Figure 39-2] FIG. 39 shows the particle size and bioactivity of peptide antigen conjugates of formula V composed of different hydrophobic molecules (H) based on poly(amino acid) of formula II linked to different PRR agonists.

[0073] [Figure 40] FIG. 40 shows the particle size and bioactivity of peptide antigen conjugates of formula V, in which the peptide antigen (A) is linked to a hydrophobic molecule (H) using different linker chemistries (amide and thio-ether).

[0074] [Figure 41-1] FIG. 41 shows the particle size and bioactivity of peptide-antigen conjugates of formula V composed of different hydrophobic molecules (H) based on fatty acids, lipids and cholesterol. [Figure 41-2] FIG. 41 shows the particle size and bioactivity of peptide-antigen conjugates of formula V composed of different hydrophobic molecules (H) based on fatty acids, lipids and cholesterol.

[0075] [Figure 42] FIG. 42 shows the particle size and bioactivity of peptide antigen conjugates of formulas IV and V composed of different hydrophobic molecules (H) based on AB-type diblock copolymers.

[0076] [Figure 43]FIG. 43 shows the particle size and stability of peptide antigen conjugates of formula V, which are based on peptide antigens (A) linked to preformed particles (P).

[0077] [Figure 44] FIG. 44 shows particle size and bioactivity of peptide antigen conjugates delivering autoantigens used to induce tolerance.

[0078] [Figure 45] FIG. 45 shows the particle size and bioactivity of particles composed of A-H+CH or AH(C) (Formula VI). DETAILED DESCRIPTION OF THE INVENTION

[0079] In order to guide those skilled in the art in the practice of the present disclosure, details of terms and methods are provided below to provide greater clarity regarding the compounds, compositions, methods, and their use to induce an immune response in a subject. The terminology in this disclosure is understood to be useful for the purpose of providing a better description of certain embodiments, and should not be considered limiting.

[0080] About: In the context of this disclosure, "about" means plus or minus 5% from the set amount. For example, "about 10" refers to 9.5 to 10.5. A ratio of "about 5:1" refers to a ratio of 4.75:1 to 5.25:1.

[0081] Adjuvant: Any material added to a vaccine to enhance or modify the immunogenicity of an antigen. The adjuvant can be a delivery system, such as particles based on inorganic salts (e.g., aluminum hydroxide, or a phosphate salt called alum), water-in-oil or oil-in-water emulsions, or polymer particles (e.g., PLGA), with which the antigen is simply mixed or adsorbed, incorporated therein, or indirectly or directly linked by covalent interactions. Alternatively, adjuvants can be pattern recognition receptor (PRR) agonists, e.g., synthetic or naturally occurring agonists of Toll-like receptors (TLRs), stimulator of interferon genes (STING), nucleotide-binding oligomerization domain-like receptors (NLRs), retinoic acid-inducible gene I-like receptors (RLRs), or C-type lectin receptors (CLRs), chemically defined molecules that bind to specific receptors and induce downstream signaling pathways, as well as biomolecules ("biological adjuvants"), e.g., IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL. Small molecule analogs of nucleotide bases, such as hydroxyadenine and imidazoquinoline, which bind to Toll-like receptor-7 (TLR-7) and TLR-7 / 8a, respectively, and agonists of TLR-2 / 6, TLR-4, STING, and NOD, are used as exemplary PRR agonists in the present disclosure. Those skilled in the art are familiar with adjuvants (see Perrie et al., Int J Pharm 364:272-280, 2008 and Brito et al., Journal of controlled release, 190C:563-579, 2014). Generally, any PRR agonist or biological adjuvant listed herein can be attached to the peptide-antigen conjugate of the present disclosure by any suitable means.

[0082] Administration: Providing or giving to a subject an agent, for example, an immunogenic composition comprising a peptide-antigen conjugate described herein, by any effective route.

[0083] Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0084] "Administration of" a compound and "administering" should be understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition described herein. The compound or composition may be administered to the subject by another person or may be self-administered by the subject.

[0085] Antigen-presenting cell (APC): Any cell that presents antigen bound to an MHC class I or class II molecule to a T cell, including but not limited to monocytes, macrophages, dendritic cells, B cells, T cells, and Langerhans cells.

[0086] Antigen: Any molecule that contains an epitope that binds to a T cell or B cell receptor and can stimulate an immune response in a subject, particularly a B cell response and / or a T cell response. An epitope can be composed of a peptide, glycopeptide, lipid, or any suitable molecule that contains an epitope that can interact with a component of a specific B cell or T cell protein. Such an interaction can generate a response by an immune cell. "Epitope" refers to the region of a peptide antigen that interacts with a B and / or T cell protein, i.e., a B cell receptor and a T cell receptor.

[0087] Antigens used in embodiments of the present disclosure can be selected from pathogens, cancerous cells, autoantigens, or allergens. In some embodiments, antigens can be peptide-based antigens, which can include regions of polypeptides or proteins from pathogens (e.g., viruses, bacteria, or fungi) or tissues of interest (e.g., cancerous cells). In other embodiments, antigens can be whole proteins or glycoproteins derived from pathogens, or peptide or glycopeptide fragments of said proteins or glycoproteins. In other embodiments, antigens can be proteins or peptide fragments of proteins that are primarily expressed by tumor tissues (but not healthy tissues) and are tumor-associated antigens. In other embodiments, antigens are proteins or peptides associated with autoimmunity. In still other embodiments, antigens are proteins or glycoproteins associated with allergies.

[0088] Many such antigens may be used in accordance with embodiments of the present invention, and many such antigens are discussed in more detail herein.

[0089] Aromatic: Aromatic compounds are unsaturated cyclic rings with an odd number of delocalized pi-orbital electron pairs between the carbon or nitrogen atoms that form the ring. Aromatic amino acids include those with side chains containing aromatic groups, such as phenylalanine, tyrosine, or tryptophan. Benzene, a six-carbon ring containing three double bonds, is the prototypical aromatic compound. Phenylalanine (Phe) and tryptophan (Trp) are prototypical aromatic amino acids. Aryl can also refer to an aromatic substituent, and aryl-amine can also refer to an aromatic group containing an amine. An exemplary aromatic amine is aniline. Aromatic heterocycles refer to aromatic rings containing carbon and another atom, such as nitrogen, oxygen, or sulfur, in a cyclic ring structure. Nucleotide bases, such as adenine and cytosine, are exemplary aromatic heterocycles.

[0090] Biocompatible: A material is considered biocompatible if it exerts little destructive or host response effect when in contact with body fluids, cells, or tissues.

[0091] Biocompatible groups can contain chemical moieties, including those from the aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, or heteroaryl classes, although, depending on the molecular composition, such moieties are not necessarily biocompatible.

[0092] Alternatively, the term "biocompatible" may be taken to mean substances and functional groups that are specifically intended to interact with components of biological systems (e.g., drugs and prodrugs) such that the consequences of the interaction are not substantially negative or destructive, or that interact little with recognition proteins and / or other components of biological systems (e.g., naturally occurring antibodies, cellular proteins including glycoproteins, or cells).

[0093] CD4: Cluster of differentiation 4, a surface glycoprotein that interacts with MHC class II molecules present on the surface of other cells. A subset of T cells expresses CD4; these cells are commonly called helper T cells.

[0094] CD8: Cluster of differentiation 8, a surface glycoprotein that interacts with MHC class I molecules present on the surface of other cells. A subset of T cells expresses CD8; these cells are commonly called cytotoxic or killer T cells.

[0095] Charge: A physical property of a substance that affects its interactions with other atoms and molecules (including solutes and solvents). Charged substances are subject to electrostatic forces from other types of charged substances and from molecules that do not carry a full integer value of charge, such as polar molecules. Two charged molecules of the same charge repel each other, but two charged molecules of different charges attract each other. Charge is often described in whole numbers, either positive or negative.

[0096] Charged molecule (C): A charged molecule (C) refers to any molecule that has one or more functional groups that are positively or negatively charged. The functional groups that make up a charged molecule can have partial or full charge integer values. A charged molecule can be a molecule with a single charged functional group or multiple charged functional groups. The functional groups can be permanently charged, or the functional groups that make up a charged molecule can have a charge that is pH-dependent. A charged molecule can be composed of positively charged functional groups, negatively charged functional groups, or both positively and negatively charged functional groups. The net charge of a charged molecule can be positive, negative, or neutral. The charge of a molecule can be easily estimated based on the Lewis structure of the molecule and accepted methods known to those skilled in the art. Charge can also arise as a result of inductive effects; for example, atoms bonded to each other with different electron affinities can result in a polar covalent bond that results in some atoms being negatively charged and some atoms being positively charged. For example, nitrogen bonding to a hydrogen results in a negative partial charge on the nitrogen and a positive partial charge on the hydrogen atom. Alternatively, an atom can be considered to have a full charge integer value if the number of electrons assigned to that atom is less than or equal to the atom's atomic number. The charge of a functional group is determined by adding up the charges of each atom that makes up that functional group. The net charge of a charged molecule (C) is determined by adding up the charges of each atom that makes up that molecule. Those skilled in the art are familiar with how to estimate the charge of a molecule or individual functional group by adding up the formal charges of each atom in the molecule or functional group, respectively.

[0097] Charged molecules (C) may contain negatively charged functional groups, such as those that exist as the conjugate base of an acid at physiological pH (e.g., functional groups with a pKa of less than about 6.5), e.g., those that exist as the conjugate base of an acid at a pH of about 7.4. These include, but are not limited to, molecules with carboxylate, sulfate, phosphate, phosphoramidate, and phosphonate. Charged molecules may contain positively charged functional groups, such as those that exist as the conjugate acid of a base at physiological pH (e.g., functional groups with a pKa of the conjugate acid of a base greater than about 8.5). These include, but are not limited to, molecules with primary, secondary, and tertiary amines, as well as molecules with ammonium and guanidinium. Charged molecules may also contain functional groups with pH-independent charges, including quaternary ammonium, phosphonium, and sulfonium functional groups. In some embodiments, the charged molecule is a poly(amino acid) composed of negatively charged amino acids, positively charged amino acids, or both negatively and positively charged amino acids. In some embodiments, the negatively charged amino acid is glutamic acid or aspartic acid. In other embodiments, the positively charged amino acid is lysine or arginine. Those skilled in the art will recognize that many such embodiments are possible.

[0098] Click chemistry reaction: This may refer to a bioorthogonal reaction that couples two compounds together under mild conditions in a high-yield reaction that produces minimal biocompatible and / or harmless by-products. An exemplary click chemistry reaction used in this disclosure is the reaction of an azide group present on a linker precursor, X1, with an alkyne present on a linker precursor, X2, to form a triazole linker (L) via a strain-promoted [3+2] azide-alkyne cycloaddition.

[0099] Effective amount: The amount necessary to induce a desired response, e.g., the amount of an agent, either alone or with one or more additional agents, necessary to induce an immune response, e.g., an immune response to a peptide-antigen conjugate.

[0100] Extension: The term extension is used herein to describe a molecule linked to the N- or C-terminus of a peptidic antigen (A), which is composed of amino acids, unnatural amino acids, hydrophilic ethylene oxide monomers (i.e., PEG), hydrophobic alkane chains, or combinations thereof, and which functions to regulate the degradation rate of the peptidic antigen (A). An extension linked to the N-terminus of a peptidic antigen is referred to as B1, and an extension linked to the C-terminus of a peptidic antigen is referred to as B2. The extensions (B1 and B2) primarily function to control the degradation rate of the peptidic antigen, but may also perform any one or more additional functions. In some embodiments, the extensions (B1 and / or B2) may be linked to another molecule, e.g., a charged molecule (C) or a hydrophobic molecule (H), may function as a linker, and may control the release rate of the peptidic antigen (A) from other molecules. In further embodiments, the extensions (B1 and / or B2) function to provide distance, i.e., spacing, between any two heterologous molecules. In other embodiments, the extensions (B1 and / or B2) function to confer hydrophobicity or hydrophilicity to the peptide antigen conjugate. In still other embodiments, the composition of the extensions (B1 and / or B2) used as linkers can be selected to confer rigidity or flexibility between the peptide antigen (A) and the heterologous molecule. In preferred embodiments, the extensions (B1 and / or B2) are peptide sequences selected for recognition and hydrolysis by enzymes such as proteases. Note that the B1 and B2 extensions are sometimes referred to as B1 and B2 linkers, or B1 and B2, respectively. Specific compositions of extensions that may be suitable for practicing the present disclosure are described throughout.

[0101] Graft polymer: can be described as a polymer resulting from the linking of a polymer of one composition to the side chain of a second polymer of a different composition. A first polymer linked to a second polymer by a comonomer is a graft copolymer. A first polymer linked to a second polymer by an end group may also be described as a block polymer (e.g., AB diblock) or end-graft polymer.

[0102] Hydropathic Index / GRAVY Value: A number that represents the hydrophobic or hydrophilic properties of an amino acid. There are various scales that can be used to represent the relative hydrophobic and hydrophilic properties of the amino acids that make up a peptide. In this disclosure, the Kyte and Doolittle hydropathy scale (Kyte J, Doolittle RF, J. Mol. Biol. 157:105-32, 1983) is used to calculate the grand average hydropathy (GRAVY) value, sometimes referred to as the GRAVY score, of a sequence of amino acids that constitutes a peptide-antigen conjugate, including a peptide antigen (A), optional N- and C-terminal extensions (B1 and B2) of the peptide base, and an optional charged molecule (C). The GRAVY value of a peptide is the sum of the hydropathy values ​​of all amino acids that make up the peptide, divided by the length of the peptide (i.e., the number of amino acids). GRAVY values ​​are relative values. A peptide sequence with a higher GRAVY value is considered more hydrophobic, while a peptide sequence with a lower GRAVY value is considered more hydrophilic.

[0103] Hydrophilicity: Refers to the tendency of a material to disperse freely in an aqueous medium. A material is considered hydrophilic if it prefers to interact with other hydrophilic materials and avoids interacting with hydrophobic materials. In some cases, hydrophilicity is used as a relative term; for example, the same molecule may or may not be described as hydrophilic, depending on what it is being compared to. Hydrophilic molecules are often polar and / or charged and have good aqueous solubility, for example, soluble up to 0.1 mg / mL or higher.

[0104] Hydrophobicity: Refers to a material's tendency to avoid contact with water. A material is considered hydrophobic if it prefers to interact with other hydrophobic materials and avoids interacting with hydrophilic materials. Hydrophobicity is a relative term; the same molecule may or may not be described as hydrophobic, depending on what it is being compared to. Hydrophobic molecules are often non-polar and uncharged and have poor aqueous solubility, e.g., insoluble down to 0.1 mg / mL or less.

[0105] Hydrophobic Ligand: A molecule that binds to a biological receptor and has hydrophobic properties. In some embodiments, the hydrophobic ligand is arranged along the backbone of a polymer, thereby imparting hydrophobicity to the polymer to which it is attached. In some embodiments, the hydrophobic ligand is a pattern recognition receptor agonist that has limited aqueous solubility and therefore can be described as hydrophobic. In further embodiments, the hydrophobic ligand is a TLR-7 or TLR-7 / 8 agonist, e.g., an imidazoquinoline.

[0106] Hydrophobic molecule (H): In this disclosure, the term "hydrophobic molecule" (H) is used as a general term to describe a molecule with limited aqueous solubility or amphipathic properties that can be linked to a peptide antigen to result in a peptide-antigen conjugate that forms particles in aqueous conditions. Hydrophobic molecules (H) in this context promote particle assembly or a tendency to assemble into particles due to their poor solubility in aqueous conditions over a certain temperature and pH range.

[0107] Hydrophobic molecules (H) described herein include amphiphilic molecules that can form supramolecular structures, such as micelles or bilayer-forming lamellar or multilamellar structures (e.g., liposomes or polymersomes), as well as compounds that are completely insoluble and form aggregates by themselves. The hydrophobic properties of the molecules can also be temperature and / or pH responsive. In some embodiments, the hydrophobic molecule (H) is a polymer that is water-soluble at low temperatures but is insoluble or micelle-forming at temperatures above 20°C, such as, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C. In other embodiments, the hydrophobic molecule (H) is a polymer that is water-soluble at low pH, e.g., below 6.5, but insoluble at pH above 6.5. Examples of hydrophobic molecules (H) include, but are not limited to, fatty acids, cholesterol and its derivatives, long-chain aliphatics, lipids, and various polymers, such as polystyrene, poly(lactic-co-glycolic acid) (PLGA), and poly(amino acids) composed primarily of hydrophobic amino acids. In some embodiments, the hydrophobic molecule (H) is a hydrophilic polymer to which multiple hydrophobic ligands are attached. Various hydrophobic molecules useful for implementing the present disclosure are disclosed herein.

[0108] Immune response: A change in the activity of a cell of the immune system, such as a B cell, T cell, or monocyte, as a result of stimulation, either directly or indirectly (e.g., via cell or cytokine mediation). In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, e.g., a CD4 T cell response or a CD8 T cell response. In one embodiment, the immune response results in the production of additional T cell progeny. In one embodiment, the immune response results in the migration of T cells. In another embodiment, the response is a B cell response, resulting in the production of specific antibodies or the production of additional B cell progeny. In other embodiments, the response is an antigen-presenting cell response. "Enhancing an immune response" refers to the co-administration of an adjuvant with an immunogenic agent, such as a peptide antigen, as part of a peptide-antigen conjugate, where the adjuvant increases the desired immune response to the immunogenic agent compared to administration of the immunogenic agent to a subject in the absence of the adjuvant. In some embodiments, antigens are used to stimulate immune responses, resulting in the activation of cytotoxic T cells that kill virus-infected or cancerous cells. In some embodiments, antigens are used to induce tolerance or immunosuppression. A tolerogenic response can result from the unresponsiveness of T cells or B cells to an antigen. An inhibitory immune response can also result from the activation of regulatory cells, such as regulatory T cells, that downregulate the immune response, thereby attenuating the immune response. Antigens administered to patients without an adjuvant are generally tolerogenic or suppressive, while antigens administered with an adjuvant are generally stimulatory, resulting in the recruitment, proliferation, and activation of immune cells.

[0109] Immunogenic composition: A formulation of material containing an antigen and, optionally, an adjuvant that induces a measurable immune response to the antigen.

[0110] Ligand: A general term used to describe any molecule that binds to a biological receptor. Pattern recognition receptor agonists are a specific type of ligand that binds to pattern recognition receptors and are sometimes referred to as adjuvants or ligands with adjuvant properties. For example, PRR agonists are ligands that bind to PRRs, such as TLRs. Ligands that bind to PRRs (or PRRs) are sometimes referred to as adjuvants, molecular adjuvants, adjuvant molecules, or ligands with adjuvant properties. Ligands that have limited solubility in water are sometimes referred to as hydrophobic ligands, while ligands that are water-soluble are sometimes referred to as hydrophilic ligands. Hydrophobic or hydrophilic ligands with adjuvant properties are sometimes referred to as hydrophobic adjuvants or hydrophilic adjuvants, respectively.

[0111] Linked or Coupled: The terms "linked" or "coupled" mean attached to one another, either directly or indirectly. A first moiety may be covalently or non-covalently linked to a second moiety. In some embodiments, a first molecule is linked to another molecule by a covalent bond. In some embodiments, a first molecule is linked to another molecule by electrostatic attraction. In some embodiments, a first molecule is linked to another molecule by dipole-dipole forces (e.g., hydrogen bonding). In some embodiments, a first molecule is linked to another molecule by van der Waals forces (also known as London forces). A first molecule may also be linked to another molecule by any and all combinations of such couplings.

[0112] Molecules may be indirectly linked, for example, through the use of a linker. Molecules may also be indirectly linked through an intervening moiety that is independently non-covalently bound to both molecules.

[0113] As used herein, "linked" and variations thereof refer to maintaining molecules in chemical or physical association, including at least until they contact cells, particularly immune cells, after immunization.

[0114] In some embodiments, the linked components are associated such that they cannot disperse freely from one another, at least until they come into contact with cells, such as immune cells. For example, two components may be covalently linked to one another such that they cannot disperse or diffuse separately. In a preferred embodiment, the peptide antigen conjugate is composed of a peptide antigen (A) covalently linked to a hydrophobic molecule (H) or a particle (P), either directly or indirectly via an extension (B1 or B2). The peptide antigen conjugates comprising the hydrophobic molecule (H) assemble into particles in aqueous conditions, where two or more peptide antigen conjugates associate to form a stable entity in which the individual peptide antigen conjugates and the components that make up the peptide antigen conjugate cannot disperse or diffuse before encountering cells, such as immune cells.

[0115] Conjugation is particularly distinguished from a simple mixture of antigen and adjuvant, as may be found, for example, in conventional vaccines, e.g., vaccines containing a water-soluble peptide antigen mixed with an adjuvant, in which the components are free to disperse separately within and beyond the vaccinated tissue.

[0116] Linkers, linker precursors, and linkers (L): A linker is a molecule or group of atoms that connects, couples, or bonds two or more moieties to each other. The peptide antigen conjugates disclosed herein are composite molecules containing multiple different functional components (such as a peptide antigen (A), a hydrophobic molecule (H) or particle (P), an optional extension (B1 and / or B2), an optional charged molecule (C), an optional linker (L), or an optional adjuvant) that may be connected or bonded to each other by any suitable means. For example, a peptide antigen (A) linked to a hydrophobic molecule (H) may use a linker between the peptide antigen (A) and the hydrophobic molecule (H). The peptide antigen (A) may be connected to the hydrophobic molecule (H) or particle (P) either directly or indirectly via the linker (L), extension (B1 or B2), or charged molecule (C) by any suitable means, including any suitable linker. In some embodiments, the linker is covalently bonded to both of the moieties to be coupled. In some embodiments, the linker is bifunctional, meaning that it has functional groups at two sites, and these functional groups are used to couple the linker to the two moieties. The two functional groups can be the same (considered a homobifunctional linker) or different (considered a heterobifunctional linker). For example, in some embodiments, a linker precursor X2 further comprising an alkyne and an acid is used to link an amine-bearing hydrophobic molecule (H) to a peptide antigen (A) linked to an azide-bearing linker precursor X1, where the acid and alkyne of the linker precursor X2 react with the amine and azide, respectively, to form amide and triazole bonds, thus linking the two heterogeneous molecules. In some embodiments, the linker precursor X2 of the heterobifunctional linker is a dibenzocyclooctyne (DBCO) molecule linked to an acid. In other embodiments, the linker precursor is an acid linked to a maleimide that couples an amine and a thiol, or to a bis(carboxylic acid) that couples two amines.In still other embodiments, trifunctional or multifunctional linkers, with the linkages being the same or different, may be used. In other embodiments, a cleavable N- or C-terminal peptide extension (B1 or B2) is used to link the peptidic antigen (A) to the hydrophobic molecule (H). In some embodiments, the cleavable peptide extension (B1 or B2) is heterobifunctional, e.g., the N-terminal amine of the B2 extension is linked to the C-terminus of the peptidic antigen (A) and the C-terminal carboxyl group of the B2 extension is linked directly to the hydrophobic molecule (H). While the extension (B1 or B2) can function as a linker, not all linkers are extensions.

[0117] Linker (L) is a specific subset of linkers resulting from the reaction of linker precursor X1 with linker precursor X2, which specifically functions to bind a peptide antigen (A) to a hydrophobic molecule (H) or particle (P) either directly or indirectly, via an extension (B1 or B2) or a charged molecule (C). The linker performs the specific function of site-selectively coupling, i.e., binding or linking, the peptide antigen (A) and the hydrophobic molecule (H) or particle (P) to each other. Linker precursor X1 can be linked to the peptide antigen directly or indirectly via an extension (B1 or B2), usually during solid-phase peptide synthesis. Note that linker precursor X1 directly linked to the N- or C-terminus of the peptide antigen (A) is not considered an extension because it does not specifically function to regulate the degradation rate of the peptide antigen. Although the linker precursor X1 may have some effect on the degradation rate of the peptide antigen (A), the linker precursor X1 is not selected to regulate the degradation rate of the peptide antigen (A) or its release from other molecules, but instead functions specifically to bind the peptide antigen (A) to the hydrophobic molecule (H) or particle (P).

[0118] In some embodiments, linker precursor X1 can be linked to the peptide antigen (A) during solid-phase peptide synthesis, either directly or indirectly via an extension (B1 or B2) comprising a degradable peptide linker. Typically, linker precursor X1, which is directly or indirectly linked to the peptide antigen (A), is selected to facilitate bioorthogonal reaction with linker precursor X2 provided on the hydrophobic molecule (H) or particle (P). The bioorthogonal reaction allows for site-selective linkage of the peptide antigen (A) to the hydrophobic molecule (H) or particle (P) without modifying any of the amino acids that make up the peptide antigen (A). Preferred linker precursors X1 that allow for bioorthogonal reaction include those with azides or alkynes. Depending on the composition of the antigen, additional linker precursors X1 that allow site-selective reactivity include thiols, hydrazines, ketones, and aldehydes. In some embodiments, the linker precursor has an azide functional group. In some embodiments, the linker precursor X1 is an azide-bearing unnatural amino acid, e.g., azido-lysine, Lys(N3). In such embodiments, a peptide antigen (A) linked to the azide-functionalized linker precursor X1 can react with an alkyne-containing linker precursor X2 provided on a hydrophobic molecule (H), resulting in the formation of a triazole linker linking the peptide antigen (A) and the hydrophobic molecule (H). Various linker precursors (X1 and X2) and linkers are described throughout.

[0119] Herein, both the linker and the linker precursor X1 are sometimes referred to as Tag (T), but the context of Tag (T) is used to distinguish whether the Tag (T) is a linker or a linker precursor (X1). A Tag (T) that is linked to a peptide antigen (A) directly or indirectly, either through an optional extension (B1 or B2) or an optional charged molecule (C), but not to a hydrophobic molecule (H) or a particle (P) may also be referred to as a linker precursor X1. A Tag (T) that links a peptide antigen (A) to a hydrophobic molecule (H) or a particle (P) may also be referred to as a linker (L). A linker precursor X2 reacts with the linker precursor X1 to form a linker. The linker precursor X1 may also be referred to as a Tag, and the linker precursor X2 may also be referred to as a tag-reactive moiety or tag-reactive molecule that includes a functional group that is specific or reactive to the Tag.

[0120] Net Charge: The total electrostatic charge carried by a molecule, or, where specified, a section of a molecule.

[0121] Particle: A nano- or micro-sized supramolecular structure composed of an assembly of molecules. The peptide antigen conjugates of the present disclosure comprise either preformed particles (P) or peptide antigens (A) linked to hydrophobic molecules (H) that assemble into micelles or other supramolecular structures. Particles comprising the peptide antigen conjugates can be taken up by cells (e.g., immune cells such as antigen-presenting cells). In some embodiments, the peptide antigen conjugates form particles in aqueous solution. In some embodiments, particle formation by the peptide antigen conjugates is pH- or temperature-dependent. In some embodiments, nanoparticles comprised of the peptide antigen conjugates have an average diameter between 5 nanometers (nm) and 500 nm. In some embodiments, nanoparticles comprised of the peptide antigen conjugates can be larger than 100 nm. In some embodiments, nanoparticles comprised of the peptide antigen conjugates are contained within larger particle structures (e.g., particles larger than about 5000 nm) that are too large to be taken up by immune cells, and these larger particle structures slowly release smaller nanoparticles comprising the peptide antigen conjugates.

[0122] In some embodiments, a peptide-antigen conjugate comprising a hydrophobic molecule (H) forms a nanoparticle. The nanoparticle is formed by the association of the peptide-antigen conjugate through hydrophobic interactions and can therefore be considered a supramolecular assembly. In some embodiments, the nanoparticle is a micelle. In a preferred embodiment, the nanoparticle micelle has a diameter of about 5-50 nm. In some embodiments, the peptide-antigen conjugate forms a micelle, and the micelle formation is temperature-dependent, pH-dependent, or temperature- and pH-dependent. In some embodiments, the disclosed nanoparticles comprise a peptide-antigen conjugate composed of a peptide antigen (A) linked to a hydrophobic molecule (H), which is composed of a polymer linked to a ligand with adjuvant properties, such as a PRR agonist. The linkage between the PRR agonist and the peptide antigen in the nanoparticle prevents the PRR agonist from freely dispersing after administration to a subject, thereby preventing systemic toxicity.

[0123] The particles may be formed by assembly of the individual molecules that make up the peptide-antigen conjugate, or in the case of peptide-antigen conjugates that consist of a peptide antigen (A) linked to a preformed particle (P), the particles may be crosslinked by covalent or non-covalent interactions.

[0124] Preformed particle (P) / particle (P) according to the formula: A preformed particle (P) or simply "particle" (P) refers to a particle that has already formed prior to linkage to a peptide antigen (A). Thus, particle (P) is used to describe a particle according to the formula, and is distinct from a particle formed by assembly of two or more peptide antigen conjugates comprising a hydrophobic molecule (H). For clarity, a particle formed by assembly of a peptide antigen conjugate is distinct from a preformed particle (P) or particle (P) according to the formula. In some embodiments, a peptide antigen (A) can be directly or indirectly linked to a particle (P) to form a peptide antigen conjugate, which can become a particle in aqueous conditions.

[0125] To accurately describe the difference between particles formed by peptide antigen conjugates and preformed particles (P), when referring to preformed particles or particles (P) according to the formula, the letter p is always capitalized in "Particle" and is followed by a capital letter (P) in parentheses, i.e., "Particle (P)." In some embodiments, particles (P) can be PLGA particles (P) that are formed in aqueous conditions and then linked to peptide antigen (A) to form peptide antigen conjugates that remain particles in aqueous conditions. In some embodiments, particles (P) can be composed of lipids, such as liposomal particles (P), that are formed in aqueous conditions and then linked to peptide antigen (A) to form peptide antigen conjugates that remain particles in aqueous conditions.

[0126] Pattern recognition receptors (PRRs): Receptors expressed by various cell populations, particularly innate immune cells, that bind a diverse group of synthetic and naturally occurring molecules called pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs are conserved molecular motifs present on certain microorganisms and viruses. DAMPs are cellular components released or expressed during cell death or injury.

[0127] PAMP or DAMP activation of pattern recognition receptors induces intracellular signaling cascades that result in alterations in host cell physiology. Such physiological changes can include alterations in the cellular transcriptional profile, inducing the expression of various pro-inflammatory and pro-survival genes. The coordinated expression of these genes can enhance adaptive immunity.

[0128] There are several classes of PRR. Non-limiting examples of PRR include Toll-like receptor (TLR), RIG-I-like receptor (RLR), NOD-like receptor (NLR), stimulator of interferon genes (STING) receptor and C-type lectin receptor (CLR). Agonists of such PRR can be used to enhance the immune response to target antigens.

[0129] Agonists of PRRs are adjuvants and are sometimes referred to as ligands or ligands with adjuvant properties. In some embodiments of the present disclosure, PRR agonists are used as adjuvants to enhance immune responses to peptide antigens.

[0130] Toll-like receptors (TLRs) 1–13 are transmembrane PRRs that recognize a variety of PAMPs. There are two broad categories of TLRs: cell surface-localized TLRs and endosomal lumen-localized TLRs. Cell surface TLRs are typically important for bacterial recognition. Endosomal lumen-localized TLRs, such as TLRs 3, 7, 8, and 9, serve to recognize nucleic acids and are therefore typically important for viral recognition and, therefore, for promoting antiviral immune responses. Polyinosinic-polycytidylic acid is a ligand for TLR-3. TLR-7 and TLR-8 recognize single-stranded RNA as well as nucleotide base analogs and imidazoquinolines. TLR-9 recognizes unmethylated deoxycytidylate-phosphate-deoxyguanylate (CpG) DNA, primarily found in bacteria.

[0131] NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) are located in the cytoplasm. Non-limiting examples of RLRs include RIG-I, MDA5 and LGP2. There are 22 human NLRs, which can be subdivided into five structurally related NLR families A, B, C, P and X. All NLRs have three domains: an N-terminal domain involved in signal transduction, a nucleotide-binding NOD domain, and a C-terminal leucine-rich region (LRR) that is important for ligand recognition. Non-limiting examples of NLRs include NALP3 and NOD2.

[0132] For more information on pattern recognition receptors, see Wales et al., Biochem Soc Trans. 35:1501-1503, 2007.

[0133] Peptide or Polypeptide: Two or more natural or non-natural amino acid residues linked together by an amide bond. The amino acid residues may have post-translational modifications (e.g., glycosylation and / or phosphorylation). Such modifications may mimic post-translational modifications that occur naturally in vivo or may be non-natural. Any one or more of the components of a peptide-antigen conjugate may be composed of a peptide.

[0134] There is no conceptual upper limit to the length of the peptide. The length of the peptide is typically selected depending on the application. In some embodiments, the hydrophobic molecule (H) is comprised of a peptide that can be between 3 and 1,000 amino acids in length, typically 300 amino acids or less in length. In some embodiments, the N-terminal and / or C-terminal extensions (B1 and / or B2) are peptides about 1 to 8 amino acids in length. In some embodiments, the charged molecule (C) is a peptide comprised of positively charged amino acids, negatively charged amino acids, or both positively and negatively charged amino acids, typically 16 amino acids or less in length.

[0135] In preferred embodiments, the peptide antigen (A) is a peptide of between 5 and about 50 amino acids, typically about 7 to 35 amino acids, e.g., 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, or 35 amino acids. In other embodiments, the peptide antigen (A) is about 50 amino acids in length or longer. Thus, in some embodiments, the peptide antigen (A) can be considered a protein.

[0136] It should be noted that the peptide antigen (A) may be a minimal epitope (sometimes referred to as min or ME) or a long peptide comprising a minimal epitope (sometimes referred to as LP or SLP). Thus, when a minimal epitope or a long peptide is said to be delivered as a peptide antigen conjugate, it is understood that the minimal epitope or the long peptide is the peptide antigen (A) unless otherwise stated.

[0137] In some embodiments, the optional charged molecule (C), antigen (A), optional extensions (B1 and B2), and linker precursor X1 are amino acids and may be prepared by solid phase peptide synthesis as a contiguous peptide sequence, sometimes referred to as a "peptide antigen fragment." Note that the calculation of the net charge or GRAVY of the peptide antigen fragment does not include the linker precursor X1.

[0138] A peptide sequence that refers to a peptide antigen is designated "PA," a peptide sequence that refers to an N-terminal extension (B1) is designated "PN," and a peptide sequence that refers to a C-terminal extension (B2) is designated "PC." The sequence of amino acids that make up a peptide antigen (A) is represented by the formula PA1...PAn, where PA represents any amino acid residue that makes up the peptide antigen (A), and n is an integer value. For example, an 8-amino acid peptide antigen (A) can be represented as PA1-PA2-PA3-PA4-PA5-PA6-PA7-PA8. The sequence of amino acids that make up the N-terminal extension (B1) is represented by the formula PN...PNn, where PN represents any amino acid residue that makes up the N-terminal extension, and n is an integer value. The sequence of amino acids that make up the C-terminal extension (B2) is represented by the formula PC1...PCn, where PC represents any amino acid residue that makes up the C-terminal extension, and n is an integer value.

[0139] Peptide Modifications: Peptides may be altered or otherwise synthesized using one or more of several modifications, as shown below. In addition, analogs (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting from peptides), and variants (homologs) of these peptides can be utilized in the methods described herein. The peptides described herein are composed of sequences of amino acids, analogs, derivatives, and variants, which can be either L- and / or D-versions. Such peptides can contain naturally occurring and non-naturally occurring peptides, analogs, derivatives, and variants.

[0140] Peptides can be modified by a variety of chemical methods to produce derivatives that have essentially the same activity as the unmodified peptide, and optionally other desirable properties. For example, the carboxylic acid group of the peptide, whether at the carboxyl terminus or in a side chain, can be provided in the form of a salt of a pharmaceutically acceptable cation, or esterified to form CC1-CC2. 16It can be made into an ester (where CC refers to a carbon chain (thus CC1 refers to a single carbon and CC16 refers to 16 carbons)) or converted into an amide. The amino group of the peptide, whether at the amino terminus or in the side chain, can be in the form of a pharmaceutically acceptable acid addition salt, such as HCl, HBr, acetate, trifluoroacetate, formate, benzoate, toluenesulfonate, maleate, tartrate, and other organic salts, or can be modified or converted into an amide.

[0141] The amino acids can be modified such that they contain a PRR agonist, eg, a TLR agonist, eg, an imidazoquinoline-based TLR-7 or TLR-7 / 8 agonist, by a covalent linkage.

[0142] The peptides may be modified to contain substituents that contain positive or negative charges or both. The positive and / or negative charges may be affected by the pH at which the peptide is present.

[0143] Hydroxyl groups on the peptide side chains were cleaved from CC1 to CC2 using well-recognized techniques. 16 Alkoxy or CC1~CC 16 They may be converted to esters, or the hydroxyl groups may be converted (e.g., sulfated or phosphorylated) to introduce a negative charge. The phenyl and phenol rings of the peptide side chains may be substituted with one or more halogen atoms, e.g., fluorine, chlorine, bromine, or iodine, or may be substituted with CC1-CC 16 Alkyl, CC1 to CC 16Substitution with alkoxy, carboxylic acids and their esters, or amides of such carboxylic acids may be used. Methylene groups on peptide side chains can be extended to homologous CC2-CC4 alkylenes. Thiols can be used to form disulfide bonds or thioethers, for example, by reaction with maleimide. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of the present invention to select and equip the structure with conformational constraints that result in enhanced stability. For further details on modifications that can be made to functional groups, see Greene et al., "Greene's Protective Groups in Organic Synthesis," 4th Edition, John Wiley & Sons, Inc., 2006.

[0144] Peptidomimetic and organomimetic embodiments of peptide antigens (A) are contemplated in which the three-dimensional arrangement of the chemical moieties of such peptidomimetics and organomimetics mimics the three-dimensional arrangement of the peptide backbone and constituent amino acid side chains, resulting in such peptidomimetics and organomimetics of immunogenic peptides having a measurable ability to induce tolerance or immunosuppression, or an enhanced ability to generate a stimulatory immune response, e.g., a cytotoxic T cell or antibody response.

[0145] Pharmaceutically acceptable vehicles: Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes compositions and formulations suitable for the pharmaceutical delivery of one or more therapeutic compositions, such as one or more therapeutic cancer vaccines and additional pharmaceutical agents.

[0146] Generally, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually contain injectable fluids that contain pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like, as a vehicle. Conventional non-toxic solid carriers for solid compositions (e.g., powder, pill, tablet, or capsule forms) can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, and the like, for example, sodium acetate or sorbitan monolaurate.

[0147] Polarity: A description of the properties of a substance. Polarity is a relative term and can describe a molecule or a portion of a molecule that has a partial charge resulting from the difference in electronegativity between atoms bonded to each other in the molecule, such as the bond between nitrogen and hydrogen. Polar molecules prefer to interact with other polar molecules and do not usually associate with non-polar molecules. In certain non-limiting cases, polar groups may contain hydroxyl groups, or amino groups, or carboxyl groups, or charged groups. In certain non-limiting cases, polar groups may also prefer to interact with polar solvents such as water. In certain non-limiting cases, the introduction of additional polar groups may increase the solubility of a portion of the molecule.

[0148] Polymer: A molecule containing repeating structural units (monomers). As described in more detail throughout this disclosure, polymers can be used for any number of components of a peptide-antigen conjugate and can be natural or synthetic. In preferred embodiments, a hydrophobic or amphiphilic polymer is used as the hydrophobic molecule (H) to drive particle assembly of the peptide-antigen conjugate. In some embodiments, the peptide antigen (A) is a polymer containing amino acids. In some embodiments, the extensions (B1 and B2) comprise a polymer, such as, for example, PEG, poly(amino acids), or combinations thereof. The polymers included in the disclosed embodiments can form polymeric nanoparticles that can be administered to a subject without causing adverse side effects. The polymers included in the disclosed embodiments can form polymeric nanoparticles that can be administered to a subject to generate an immune response or to treat and / or ameliorate a disease. The polymers included in the disclosed embodiments can include side chains with functional groups that can be utilized to facilitate linkage to, for example, adjuvants or molecules used to induce immunosuppression or tolerance, such as macrolides, e.g., rapamycin. In some embodiments, the polymer can contain two or more polymer blocks linked by a linker to create a block copolymer, such as an amphiphilic diblock copolymer. In some embodiments, the polymer blocks can be primarily hydrophobic in nature. In some embodiments, the polymer is comprised of peptides, their analogs, derivatives, and variants. Various compositions of polymers useful in the practice of the present invention are discussed in more detail elsewhere.

[0149] Polymerization: A chemical reaction, usually performed with a catalyst, heat, or light, in which monomers combine to form chains or cross-linked macromolecules (polymers). Further chains can be joined by further chemical synthesis using appropriate substituents and chemical reactions. Monomers may contain reactive substances. Polymerization generally occurs by addition or condensation. Addition polymerization occurs when an initiator, usually a free radical, reacts with a double bond of a monomer. The free radical adds to one side of the double bond, creating a free electron on the other side. This free electron then reacts with another monomer, and the chain self-propagates, thus adding one monomer unit at a time to the end of the growing chain. Condensation polymerization involves the reaction of two monomers, resulting in cleavage from a water molecule. In other forms of polymerization, monomers are added to a growing chain one at a time by the stepwise introduction of activated monomers, for example, during solid-phase peptide synthesis.

[0150] Purified: Having a composition that is relatively free from impurities or substances that degrade or contaminate a substance. The term purified is a relative term and does not require absolute purity. Thus, for example, a purified peptide preparation is one in which the peptide or protein is enriched relative to the peptide or protein in its natural environment, e.g., within a cell. In one embodiment, the preparation is purified such that the peptide-antigen conjugate represents at least 50% of the total contents of the preparation. Substantial purification refers to purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free from other proteins or cellular components or contaminating peptides.

[0151] Soluble: Able to be molecularly or ionically dispersed in a solvent to form a homogeneous solution. When referring to peptides, a soluble peptide is understood to be a single molecule in solution that does not assemble into multimers or other supramolecular structures through hydrophobic or other non-covalent interactions. A soluble molecule is understood to be freely dispersed in a solution as a single molecule. In some embodiments, the peptide antigen may be a soluble peptide antigen that is soluble in phosphate-buffered saline, pH 7.4, at room temperature up to at least 0.1 mg / ml. In other embodiments, the peptide-antigen conjugate may be soluble in dimethyl sulfoxide and / or other organic solvents at room temperature, but not soluble in aqueous solvents such as phosphate-buffered saline, pH 7.4, at room temperature. The hydrophobic molecules described herein are insoluble down to about 0.1 mg / ml. Solubility can be determined by visual inspection, turbidity measurement, or dynamic light scattering.

[0152] Subject: Refers to both humans and non-human animals, including birds and non-human mammals, such as rodents (e.g., mice and rats), non-human primates (e.g., rhesus monkeys), companion animals (e.g., domestic dogs and cats), livestock (e.g., pigs, sheep, cattle, llamas, and camels), and non-domesticated animals (e.g., big cats).

[0153] Supramolecular: Refers to two or more molecules associated by non-covalent interactions. In some embodiments, the molecules associate due to hydrophobic interactions. In some embodiments, the molecules associate due to electrostatic interactions. The association confers new properties to the supramolecular complex that were not shared by either of the component molecules, such as increased size and a different immune response that affect the material's interaction with the immune system. For example, peptide-antigen conjugates can aggregate to form supramolecular complexes.

[0154] T cell: a type of white blood cell that is part of the immune system and can participate in an immune response. T cells are composed of CD4 These include, but are not limited to, CD4 T cells and CD8 T cells. CD4 T cells present the CD4 glycoprotein on their surface and are often referred to as helper T cells. These cells often coordinate immune responses, including antibody responses and cytotoxic T cell responses, although CD4 T cells can also suppress immune responses, and CD4 T cells can also act as cytotoxic T cells. CD8 T cells present the CD8 glycoprotein on their surface and are often referred to as cytotoxic or killer T cells, although CD8 T cells can also suppress immune responses.

[0155] Telechelic: Used to describe polymers with one reactive end or two reactive ends that can be the same or different. The term is derived from the Greek words telos and chele, meaning end and claw, respectively. Semitelechelic polymers describe polymers with only one end group, such as a reactive functional group, that can undergo further reaction, such as polymerization. Heterotelechelic polymers describe polymers with two end groups, such as reactive functional groups, with different reactivity characteristics.

[0156] As used herein, the hydrophobic molecule (H) may be comprised of a polymer with reactive groups at one or both termini. In some embodiments, the adjuvant is placed at one terminus of the polymer, and the other terminus of the polymer can be reacted with a linker that is linked to the peptidic antigen directly or indirectly via an extension (B1 or B2) or linker (L). In this example, the polymer is semi-telechelic with respect to the adjuvant, meaning that the adjuvant is attached to only one terminus of the polymer chain comprising the hydrophobic molecule (H).

[0157] Treating, preventing, or ameliorating a disease: "Treating" refers to an intervention that reduces a sign or symptom or marker of a disease or pathological condition after it has begun to develop. For example, treating a disease may result in a reduction in tumor burden, which refers to a decrease in the number or size of tumors and / or metastases, or treating a disease may result in immune tolerance, which reduces systems related to autoimmunity. "Preventing" a disease means inhibiting the full development of the disease. A disease may also be prevented from occurring at all. A disease may also be prevented from progressing in severity or degree or type. "Ameliorating" refers to a reduction in the number or severity of signs or symptoms or markers of a disease, such as cancer.

[0158] Reducing the signs, symptoms, or markers of a disease or disease-related pathological condition refers to any observable beneficial effect of treatment, and / or any observable effect on a proximal surrogate endpoint, such as tumor volume, whether symptomatic or not. Reducing the signs or symptoms associated with tumors or viral infections can be demonstrated, for example, by delaying the onset of clinical symptoms of disease in susceptible subjects (e.g., subjects with tumors that have not yet metastasized, or subjects who may be exposed to viral infections), by reducing the severity of some or all clinical symptoms of disease, by slower disease progression (e.g., by extending the lifespan of subjects with tumors or infected with viruses), by reducing the number of disease relapses, by improving the subject's overall health or well-being, or by other parameters known in the art (e.g., parameters specific to a particular tumor or viral infection). A "prophylactic" treatment is a treatment administered to subjects who do not show signs of disease or who only show early signs, with the aim of reducing the risk or severity of the onset of a pathological condition.

[0159] In one example, the desired response is to induce an immune response that leads to a reduction in tumor size, volume, growth rate, or number (e.g., metastases) in the subject. For example, the agent(s) can induce an immune response that reduces tumor size, volume, or number by a desired amount, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95%, compared to the response in the absence of the agent.

[0160] Tumor or cancer or neoplastic: An abnormal growth of cells that may be benign or malignant, but often does not necessarily produce clinical symptoms. "Neoplastic" cell growth refers to cell growth that is not responsive to physiological cues, such as growth and inhibitory factors.

[0161] A "tumor" is a collection of neoplastic cells. In most cases, tumor refers to a collection of neoplastic cells that form a solid mass. Such tumors may be referred to as solid tumors. In some cases, such as in some leukemias, neoplastic cells may not form a solid mass. In such cases, the collection of neoplastic cells may be referred to as a liquid cancer.

[0162] Cancer refers to the malignant growth of neoplastic cells, which can be either solid or liquid. Characteristics of cancer that define it as malignant include metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products, and suppressed or exacerbated inflammatory or immunological responses, and invasion of surrounding or distant tissues or organs, such as lymph nodes.

[0163] Tumors that do not exhibit substantial adverse clinical symptoms and / or grow slowly are termed "benign."

[0164] "Malignant" means causing or likely to cause significant clinical symptoms in the future. A tumor is said to be "malignant" if it produces substantial clinical symptoms by infiltrating surrounding tissues and / or metastasizing and / or producing and secreting chemical mediators that affect nearby or distant body systems.

[0165] "Metastatic disease" refers to cancer cells that have left the original tumor site and traveled to other parts of the body, for example, via the bloodstream, via the lymphatic system, or via body cavities such as the abdominal or thoracic cavity.

[0166] The amount of tumor in an individual is the “tumor burden.” Tumor burden can be measured as the number, volume, or mass of tumors and is often assessed by physical examination, radiological imaging, or pathological examination.

[0167] An "established" or "pre-existing" tumor is one that is present at the time treatment is initiated. Often, an established tumor can be identified by a diagnostic test. In some embodiments, an established tumor can be palpated. In some embodiments, an established tumor is at least about 500 mm 3 , e.g., at least 600 mm 3 , at least 700mm 3 , or at least 800mm 3 In other embodiments, the tumor is at least 1 cm in size. With respect to solid tumors, established tumors are generally newly established, have a robust blood supply, and may induce regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs).

[0168] Those of ordinary skill in the art will recognize that the definitions provided above are not intended to include impermissible substitution patterns (e.g., methyl substituted with five different groups, and the like). Such impermissible substitution patterns are readily recognized by those of ordinary skill in the art. Any functional group disclosed herein and / or defined above may be substituted or unsubstituted, unless otherwise indicated herein. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referred to unless the context clearly dictates otherwise. The term "comprises" means "includes." Thus, comprising "A" or "B" refers to including A, including B, or including both A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weights or molecular mass values ​​given for nucleic acid or polypeptide are approximate and are provided for illustration purposes.Methods and materials similar or equivalent to those described herein can be used in carrying out or testing this disclosure, and suitable methods and materials are described herein.In case of discrepancies, the present specification shall prevail, including explanations of terms.In addition, materials, methods and examples are only illustrative and are not intended to be limiting.

[0169] immunogenic composition Described herein are novel immunogenic compositions comprising particles containing peptide antigen conjugates, which further comprise a peptide antigen (A) linked to a particle (P) or a hydrophobic molecule (H). A peptide antigen conjugate refers to a compound resulting from linking, e.g., covalently or otherwise, a peptide antigen (A) to a particle (P) or a hydrophobic molecule (H). The hydrophobic molecule (H) or particle (P) induces the peptide antigen conjugate to assemble into a particle, which results in an unexpected enhancement of the immune response to the peptide antigen (A). The peptide antigen conjugate may further comprise an optional N-terminal extension (B1) and / or a C-terminal extension (B2) linked to the N-terminus and C-terminus of the peptide antigen (A), respectively, which provides unexpected improvements in manufacturing and biological activity; an optional charged molecule (C) which provides unexpected improvements in the stability of particles formed by the peptide antigen conjugate, thereby providing improved manufacturing and biological activity; and an optional linker (L) which is generated as a result of the reaction of a linker precursor X1 linked to the peptide antigen (A) with a hydrophobic molecule (H) or a linker precursor X2 provided on the particle (P), thereby binding the peptide antigen (A), the hydrophobic molecule (H), and the particle (P) in an efficient process, which provides unexpected improvements in manufacturing efficiency of the peptide antigen conjugate. The components that make up the peptide-antigen conjugate can be linked by any suitable means, and are described in more detail elsewhere.

[0170] In some embodiments, the peptide antigen (A) is linked directly to the hydrophobic molecule (H) or particle (P) to form a peptide antigen conjugate of formula AH or AP. In other embodiments, the peptide antigen (A) is linked to the hydrophobic molecule (H) or particle (P) by a linker (L) to form a peptide antigen conjugate of formula ALH or ALP. In still other embodiments, the peptide antigen (A) is linked to an extension (B1 or B2) that is linked to the hydrophobic molecule (H) or particle (P) either directly or by a linker (L) to form a peptide antigen conjugate of any one of the formulas A-B2-H, A-B2-LH, H-B1-A, HL-B1-A, A-B2-P, A-B2-LP, P-B1-A, or PL-B1-P. In some embodiments, the peptide antigen (A) is linked to a linker precursor X1, either directly or via extensions (B1 and B2), to form a peptide antigen fragment of the formula A-X1, A-B2-X1, X1-A, or X1-B1-A, which reacts with a linker precursor X2, i.e., X2-H or X2-P, on a hydrophobic molecule (H) or particle (P) to form a linker (L) that connects the peptide antigen (A) to the hydrophobic molecule (H) or particle (P), resulting in a peptide antigen conjugate of any one of the formulas: ALH, ALP, A-B2-LH, A-B2-LP, HLA, PLA, H-B1-A, or P-B1-A. In the present disclosure, such embodiments are shown to form particles in aqueous conditions that are useful for inducing an immune response in a subject.

[0171] In some embodiments, the peptide antigen (A) is linked to both extensions (B1 and B2). Such embodiments include peptide antigen conjugates of the formula B1-A-B2-H, B1-A-B2-LH, B1-A-B2-P, B1-A-B2-LP, H-B1-A-B2, HL-B1-A-B2, P-B1-A-B2, or PL-B1-A-B2. In the present disclosure, such embodiments are shown to form particles in aqueous conditions that are proven useful for inducing an immune response in a subject.

[0172] In some embodiments, a molecule containing a functional group that confers an electrostatic charge, i.e., a charged molecule (C), is linked to the peptide antigen (A) directly or indirectly, via an optional extension (B1 and / or B2), an optional linker (L), or a hydrophobic molecule (H) or particle (P). The charge imparted to the peptide antigen conjugate by the charged molecule stabilizes the supramolecular structure that forms in aqueous conditions. Non-limiting examples of peptide-antigen conjugates comprising a charged molecule (C) include CAH, C-B1-AH, CA-B2-H, C-B1-A-B2-H, AH(C), A-B2-H(C), B1-AH(C), B1-A-B2-H(C), C1-AH(C2), C1-A-B2-H(C2), C1-B1-AH(C2), C1-B1-A-B2-H(C2), HAC, H-B1-AC, HA-B2-C, H-B1-A-B 2-C, H(C)-A, H(C)-B1-A, H(C)-A-B2, H(C)-B1-A-B2, H(C1)-A-C2, H(C1)-B1-A-C2, H(C1)-A-B2-C2, H(C1)-B1-A- B2-C2, CALH, C-B1-ALH, CA-B2-LH, C-B1-A-B2-LH, ALH(C), A-B2-LH(C), B1-ALH(C), B1-A-B2-LH(C), C1-ALH(C2) , C1-A-B2-LH(C2), C1-B1-ALH(C2), C1-B1-A-B2-LH(C2), HLAC, HL-B1-AC, HLA-B2-C, HL-B1-A-B2-C, H(C)-LA, H (C)-L-B1-A, H(C)-LA-B2, H(C)-L-B1-A-B2, H(C1)-LA-C2, H(C1)-L-B1-A-C2, H(C1)-LA-B2-C2, H(C1)-L-B1-A-B 2-C2, CAP, C-B1-AP, CA-B2-P, C-B1-A-B2-P, AP(C), A-B2-P(C), B1-AP(C), B1-A-B2-P(C), C1-AP(C2), C1-A-B2-P (C2), C1-B1-AP(C2), C1-B1-A-B2-P(C2), PAC, P-B1-AC, PA-B2-C, P-B1-A-B2-C, P(C)-A, P(C)-B1-A, P(C)-A-B2,P(C)-B1-A-B2, P(C1)-A-C2, P(C1)-B1-A-C2, P(C1)-A-B2-C2, P(C1)-B1-A-B2-C2, CALP, C-B1-ALP, CA-B2- LP, C-B1-A-B2-LP, ALP(C), A-B2-LP(C), B1-ALP(C), B1-A-B2-LP(C), C1-ALP(C2), C1-A-B2-LP(C2), C1-B1 -ALP(C2), C1-B1-A-B2-LP(C2), PLAC, PL-B1-AC, PLA-B2-C, PL-B1-A-B2-C, P(C)-LA, P(C)-L-B1-A, P(C)-L A-B2, P(C)-L-B1-A-B2, P(C1)-LA-C2, P(C1)-L-B1-A-C2, P(C1)-LA-B2-C2 or P(C1)-L-B1-A-B2-C2. ,

[0173] The charged molecule (C) stabilizes particles formed by the peptide antigen conjugate. The charged molecule (C) may be directly linked to the peptide antigen conjugate. Alternatively, the charged molecule (C) may be provided on a separate molecule that associates with the particles formed by the peptide antigen conjugate. In some embodiments, the charged molecule (C) is linked to a hydrophobic molecule (H) to form a charged molecule conjugate of the formula C-H or C-A'-H (where A' is a conserved antigen), and this conjugate is mixed with a peptide antigen conjugate of the formula [C]-[B1]-A-[B2]-[L]-H (where [ ] indicates that the group is optional) under aqueous conditions, and the resulting particles comprise C-H or C-A'-H and the peptide antigen conjugate.

[0174] The hydrophobic molecule (H) may include any suitable molecule that induces the peptide-antigen conjugate to assemble into particles in aqueous conditions. In some embodiments, the hydrophobic molecule (H) that constitutes the peptide-antigen conjugate is a polymer with limited aqueous solubility. In some embodiments, the hydrophobic molecule (H) is a temperature- or pH-responsive polymer with limited aqueous solubility at a specific temperature or pH value. In other embodiments, the hydrophobic molecule (H) is a lipid, a fatty acid, or cholesterol. Many hydrophobic molecules (H) are useful in the present disclosure and are described in more detail throughout.

[0175] Particles formed by the peptide antigen conjugates disclosed herein are useful for inducing an immune response in a subject. In some embodiments, particles comprising a peptide antigen conjugate containing a tumor-associated antigen are provided to a subject to induce a T cell response, such as a cytotoxic CD4 or CD8 T cell response, for the treatment or prevention of cancer. In some embodiments, particles comprising a peptide antigen conjugate containing an infectious disease antigen are provided to a subject to induce a T cell response, such as a cytotoxic CD4 or CD8 T cell response or an antibody response, for the treatment or prevention of infectious disease. In some embodiments, particles comprising a peptide antigen conjugate containing an autoantigen are provided to a subject to induce a tolerogenic or suppressive T cell response for the treatment of autoimmune disease.

[0176] Peptide antigen conjugates comprise a peptide antigen (A), optional N-terminal and / or C-terminal extensions (B1 and / or B2), an optional linker (L), a particle (P) or hydrophobic molecule (H), and an optional charged molecule (C). Each of these components is described in more detail below and elsewhere.

[0177] Peptide antigen (A) The peptide antigen (A) may be any antigen useful for inducing an immune response in a subject. The peptide antigen (A) can be used to induce either a proinflammatory or tolerogenic immune response, depending on the nature of the immune response desired for the application. In some embodiments, the peptide antigen (A) is a tumor-associated antigen, such as an autoantigen, a neoantigen, or a tumor-associated viral antigen (e.g., HPV E6 / E7). In other embodiments, the peptide antigen (A) is an infectious disease antigen, such as a peptide derived from a protein isolated from a viral, bacterial, fungal, or protozoan microbial pathogen. In yet other embodiments, the peptide antigen (A) is a peptide derived from an allergen or autoantigen known or suspected to cause allergy or autoimmunity.

[0178] The peptide antigen (A) is composed of a sequence of amino acids or peptidomimetics that can induce an immune response, such as a T cell or B cell response, in a subject. In some embodiments, the peptide antigen (A) comprises amino acids, or amino acids with post-translational modifications, unnatural amino acids, or peptidomimetics. The peptide antigen may be any sequence of natural, unnatural, or post-translationally modified amino acids, peptidomimetics, or any combination thereof, that comprises an antigen or predicted antigen, i.e., an antigen with a T cell or B cell epitope.

[0179] An immunogenic composition may comprise one or more different peptide antigen conjugates, each having a different peptide antigen (A) composition. In some embodiments, an immunogenic composition comprises particles having up to 50 different peptide antigen conjugates, each having a unique peptide antigen (A) composition. In some embodiments, an immunogenic composition comprises a mosaic particle comprising 20 different peptide antigen conjugates. In other embodiments, an immunogenic composition comprises a mosaic particle comprising five different peptide antigen conjugates. In some embodiments, an immunogenic composition comprises 20 different particle compositions, each assembled from a unique peptide antigen conjugate (i.e., each particle contains a single peptide antigen conjugate composition). In other embodiments, an immunogenic composition comprises five different particle compositions, each assembled from a unique peptide antigen conjugate (i.e., each particle contains a single peptide antigen conjugate composition). In yet other embodiments, an immunogenic composition comprises a single particle composition composed of a single peptide antigen conjugate composition.

[0180] The length of the peptide antigen (A) depends on the particular application and is typically between about 5 and about 50 amino acids. In a preferred embodiment, the peptide antigen (A) is between about 7 and 35 amino acids, e.g., 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, or 35 amino acids. In other embodiments, the peptide antigen is a fragment of a polypeptide. In still other cases, the peptide antigen is a full-length polypeptide, e.g., a recombinantly expressed protein antigen. Peptide antigens (A) based on tumor-associated antigens, infectious disease antigens, allergens, or autoantigens can be delivered as full-length sequences, preferably 50 amino acids or less in length. In a preferred embodiment, the peptide antigen (A) is between 7 and 35 amino acids, typically about 25. Thus, for tumor-associated antigens, infectious disease antigens, allergens, or autoantigens longer than 25 amino acids, e.g., 100-amino acid antigens, the antigen can be divided into 7-35 amino acid, e.g., 25 amino acid, peptide antigens (A), each with a unique amino acid composition; or the peptide antigens (A) can be an overlapping peptide pool, in which the antigen is divided into a set number of 7-35 amino acid, e.g., 25 amino acid, peptide antigens (A), each with overlapping sequences. For example, an overlapping peptide pool containing a 100-amino acid antigen can be divided into eight 25-amino acid peptide antigens (A), each offset by 12 amino acids (i.e., each resulting 25-amino acid peptide that makes up the 100-amino acid peptide sequence starts at the 13th amino acid position from the previous peptide). Those skilled in the art will appreciate that there are many permutations for generating peptide pools from an antigen.

[0181] In some embodiments, the peptide antigen (A) is a minimal CD8 or CD4 T cell epitope, including a portion of a tumor-associated antigen, infectious disease antigen, allergen, or autoantigen, predicted in silico (or experimentally measured) to bind to MHC-I or MHC-II molecules. For tumor-associated antigens, the peptide antigen (A) that is a minimal CD8 or CD4 T cell epitope predicted in silico (or experimentally measured) to bind to MHC-I or MHC-II molecules should also be a sequence of amino acids that is unique to tumor cells. Algorithms for predicting binding to MHC-I or MHC-II are widely available (see Lundegaard et al., Nucleic Acids Res., 36:W509-W512, 2008, and http: / / www.cbs.dtu.dk / services / NetMHC / ). In some embodiments of personalized therapy for a particular subject (e.g., patient), the peptide antigen (A) constituting the peptide-antigen conjugate may comprise a minimal CD8 T cell epitope from a tumor-associated antigen, infectious disease antigen, allergen, or autoantigen, typically a 7-13 amino acid peptide predicted to have a binding affinity of <1,000 nM for a particular MHC-I allele expressed by the subject. In some embodiments of personalized therapy for a particular subject (e.g., patient), the peptide antigen (A) may comprise a minimal CD4 T cell epitope from a tumor-associated antigen, infectious disease antigen, allergen, or autoantigen, typically a 10-16 amino acid peptide predicted to have a binding affinity of <1,000 nM for a particular MHC-II allele expressed by the subject.In a preferred embodiment, when a minimal CD8 or CD4 T cell epitope cannot be identified for a tumor-associated antigen, infectious disease antigen, allergen, or autoantigen, or when the tumor-associated antigen, infectious disease antigen, allergen, or autoantigen contains multiple CD8 and CD4 T cell epitopes, the peptide antigen (A) may be between 16 and 35 amino acids, or up to 50 amino acids, e.g., up to 35 amino acids, up to 25 amino acids, or up to 20 amino acids, or up to 16 amino acids, so as to contain all possible CD8 or CD4 T cell epitopes.

[0182] In some embodiments of the present disclosure, the peptide antigen (A) is derived from a tumor-associated antigen. Tumor-associated antigens can be either autoantigens present on healthy cells but preferentially expressed by tumor cells, or neoantigens, which are abnormal proteins specific to tumor cells and unique to individual patients. Suitable autoantigens include antigens preferentially expressed by tumor cells, such as CLPP, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAgE1b / GAGED2a, Melan-A / MART-1, TRP-1, tyrosinase, CD45, glypican-3, IGF2B3, kallikrein 4, KIF20A, Lengsin, Meloe, MUC5AC, survivin, prostatic acid phosphatase, NY-ESO-1, and MAGE-A3. Neoantigens arise from the inherent genetic instability of cancer, which can lead to mutations in DNA, RNA splice variants, and post-translational modifications, all of which can result in de novo protein products, collectively referred to as neoantigens or sometimes called predicted neoantigens. DNA mutations include alterations to DNA, including nonsynonymous missense mutations, nonsense mutations, insertions, deletions, chromosomal inversions, and chromosomal translocations, all of which can result in novel gene products and, therefore, neoantigens. Alterations in RNA splice sites can result in novel protein products, and missense mutations can introduce amino acids that allow post-translational modifications (e.g., phosphorylation) that can be antigenic. Furthermore, tumor cell instability can lead to epigenetic changes and activation of certain transcription factors, resulting in the selective expression of certain antigens by tumor cells that are not expressed by healthy, non-cancerous cells.

[0183] Peptide-antigen conjugates used in personalized cancer vaccines should contain a peptide antigen (A) comprising a portion of a tumor-associated antigen unique to tumor cells. Peptide antigens (A), including neoantigens resulting from missense mutations, should encompass the amino acid changes encoded by one or more nucleotide polymorphisms. Peptide antigens (A), including neoantigens resulting from frameshift mutations, splice site variants, insertions, inversions, and deletions, should encompass the novel peptide sequence and the junction of the novel peptide sequence. Peptide antigens (A), including neoantigens with novel post-translational modifications, should encompass amino acids with post-translational modifications, such as phosphates or glycans. In a preferred embodiment, peptide antigen (A) contains 0 to 25 amino acids flanked on both sides by amino acid changes or novel junctions resulting from mutations. In one embodiment, peptide antigen (A) is a neoantigen sequence containing 12 amino acids flanked on both sides by amino acid changes resulting from single nucleotide polymorphisms, e.g., a 25-amino acid peptide in which the 13th amino acid is the amino acid residue resulting from a single nucleotide polymorphism. In some embodiments, peptide antigen (A) is a neo-antigen sequence containing 12 amino acids flanked on either side by amino acids with a novel post-translational modification, e.g., a 25-amino acid peptide in which the 13th amino acid is the amino acid residue resulting from the novel post-translational modification site. In other embodiments, peptide antigen (A) is a neo-antigen sequence containing 0-12 amino acids flanked on either side by novel junctions created by insertion, deletion, or inversion. In some cases, peptide antigen (A) containing the neo-antigen resulting from the novel sequence can encompass the entire novel sequence, including 0-25 amino acids on either side of the novel junction, which may also occur.

[0184] Tumor-associated antigens suitable as peptide antigens (A) for the immunogenic compositions of the present disclosure can be identified by various techniques well known to those skilled in the art. Tumor-associated antigens can be identified by assessing protein expression in tumor cells compared with healthy cells, i.e., non-cancerous cells from the subject. Suitable methods for assessing protein expression include, but are not limited to, immunohistochemistry, immunofluorescence, Western blot, chromatography (i.e., size-exclusion chromatography), ELISA, flow cytometry, and mass spectrometry. Proteins that are preferentially expressed by tumor cells but not by healthy cells, or expressed only by a limited number of healthy cells (e.g., CD20), are suitable tumor-associated antigens. DNA and RNA sequencing of patient tumor biopsy samples, followed by bioinformatics, to identify mutations in protein-encoding DNA that produce peptides predicted to be expressed as RNA and bind to MHC-I or MHC-II alleles on patient antigen-presenting cells (APCs) can also be used to identify tumor-associated antigens suitable as peptide antigens (A) for the immunogenic compositions of the present disclosure.

[0185] In a preferred embodiment, tumor-associated antigens suitable as peptide antigens (A) for the immunogenic composition are identified using mass spectrometry. Suitable peptide antigens (A) are peptides identified by mass spectrometry after elution from MHC molecules from a patient tumor biopsy sample, but not from healthy tissue from the same subject (i.e., the peptide antigen is present only on tumor cells, not healthy cells, from the same subject). Mass spectrometry may be used alone or in combination with other techniques for identifying tumor-associated antigens. Those skilled in the art will recognize that there are many methods for identifying tumor-associated antigens, such as neoantigens, suitable as peptide antigens (A) for implementing the invention of the present disclosure (see Yadav et al., Nature, 515:572-576, 2014).

[0186] In a preferred embodiment, the tumor-associated antigen used as the peptide antigen (A) is clonal or near-clonal within a population of neoplastic cells, which may otherwise be considered heterogeneous.

[0187] Tumor-associated antigens selected for use as peptide antigens (A) in personalized cancer vaccination schemes can be selected based on peptide-MHC binding and / or in silico predicted MHC binding affinity and confirmation of RNA expression levels within tumors by mass spectrometry. These data provide information on whether the tumor-associated antigen is expressed and presented by tumor cells and therefore whether it is a suitable target for T cells. Such criteria can be used to select peptide antigens (A) for use in personalized cancer vaccines.

[0188] A personalized cancer vaccine based on an immunogenic composition can include one or more different peptide-antigen conjugates, each having a unique peptide antigen (A) composition. In some embodiments, a personalized cancer vaccine can contain 10 to 50 different peptide-antigen conjugate compositions, each having a unique peptide antigen (A). In a preferred embodiment, the immunogenic composition includes 20 different peptide-antigen conjugates, each comprising a peptide antigen (A) between 7 and 35 amino acids in length, e.g., 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, or 35 amino acids, typically 50 amino acids or less. In a non-limiting example, an immunogenic composition composed of particles formed from 20 different peptide antigen conjugates composed of a peptide antigen (A) 8 amino acids in length is used as a personalized cancer vaccine. In another non-limiting example, an immunogenic composition composed of particles formed from 20 different peptide antigen conjugates composed of a peptide antigen (A) 25 amino acids in length is used as a personalized cancer vaccine.

[0189] For patients with highly mutated tumors with more than 50 tumor-associated neoantigens, a down-selection process may be used to select peptide antigens (A) for use in personalized cancer vaccines composed of peptide-antigen conjugates. In some embodiments, the down-selection process is used to select peptide antigens (A) containing epitopes predicted to have the highest MHC binding affinity and RNA expression levels in tumor cells. Additional criteria may be applied to the selection of tumor-associated autoantigens or neoantigens. For example, the predicted immunogenicity or predicted ability of the peptide antigens (A) to elicit T cells that react with other autoantigens that may lead to autoimmunity is a possible additional criterion. For example, peptide antigens (A) including tumor-associated antigens that are predicted to be highly immunogenic but also predicted to be unlikely to lead to autoimmunity are criteria used to select potential peptide antigens (A) for use in personalized cancer vaccines. In some embodiments, neoantigens predicted to elicit T cell or antibody responses that react with autoantigens found on healthy cells are not selected for use as peptide antigens (A). For patients with fewer predicted neoantigens, e.g., 20-50, the down-selection process may not be significant, and therefore all 20-50 predicted neoantigens may be used as peptide antigens (A) in personalized cancer vaccines.

[0190] Cancer vaccines may contain peptide antigens (A) including patient-specific tumor-associated antigens and / or tumor-associated antigens shared among patients. For example, tumor-associated antigens may be conserved autoantigens, such as NY-ESO-1 (testicular cancer) or gp100 (melanoma), or the antigen may be a hidden epitope that is not normally expressed by healthy cells and is conserved among patients, such as Na17 (melanoma). The immunogenic compositions of the present disclosure may contain peptide antigens (A) resulting from so-called hotspot mutations, which are hypermutations of a particular gene or gene region that occur at a frequency higher than expected by chance. Non-limiting examples of hotspot mutations include the V600E mutation of the BRAF protein, which is commonly found in melanoma, papillary thyroid cancer, and colorectal cancer, or the KRAS G12 mutation, one of the most common mutations, such as KRAS G12C. Many suitable autoantigens, as well as neoantigens resulting from hotspot mutations, are known and are incorporated herein by reference: see Chang et al., Nature Biotechnology, 34:155-163, 2016; Vigneron, N. et al., Cancer Immunology, 13:15-20, 2013.

[0191] In some embodiments, the peptide antigen (A) may be from a blood tumor. Non-limiting examples of blood tumors include leukemia, including acute leukemia (e.g., 11q23-positive acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0192] In some embodiments, the peptide antigen (A) may be from a solid tumor. Non-limiting examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer (including basal, ductal, and lobular carcinoma), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, thyroid cancer, and thyroid cancer. These include papillary carcinoma, pheochromocytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, and CNS tumors (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In some examples, the tumor is melanoma, lung cancer, lymphoma, breast cancer, or colon cancer.

[0193] In some embodiments, the peptide antigen (A) is a tumor-associated antigen from breast cancer, such as ductal carcinoma or lobular carcinoma. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from prostate cancer. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from skin cancer, such as basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, or melanoma. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from lung cancer, such as adenocarcinoma, bronchiolaveolar carcinoma, large cell carcinoma, or small cell carcinoma. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from brain cancer, such as glioblastoma or meningioma. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from colon cancer. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from liver cancer, such as hepatocellular carcinoma. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from pancreatic cancer. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from kidney cancer, such as renal cell carcinoma. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from testicular cancer.

[0194] In some embodiments, the peptide antigen (A) is a tumor-associated antigen derived from a pre-malignant condition, such as intraepithelial carcinoma or a variant of vulvar intraepithelial neoplasia, cervical intraepithelial neoplasia, or vaginal intraepithelial neoplasia.

[0195] In some embodiments, the peptide antigen (A) is an antigen from an infectious agent, such as a virus, bacteria, or fungus. In further embodiments, the peptide antigen (A) is a peptide or glycopeptide derived from an infectious agent, for example, an HIV envelope fusion peptide, or a V3 or V1 / V2 glycopeptide from HIV.

[0196] In some embodiments, the peptide antigen (A) represents an autoantigen. The autoantigen can be identified and selected based on screening the subject's own T cells for autoreactivity to the autoantigen presented in the context of the patient's own MHC-I molecules. Alternatively, the peptide antigen can be selected using in silico methods to predict potential autoantigens that (i) are predicted to have high binding affinity to the subject's own MHC-I molecules and (ii) are known to be expressed and / or associated with the pathology underlying the subject's autoimmune syndrome. In other embodiments, the peptide antigen is selected based on a peptide antigen that represents a CD4 epitope derived from an allergen and has high binding affinity to the patient's own MHC-II molecules.

[0197] Those skilled in the art will recognize that any peptide, protein, or post-translationally modified protein (e.g., glycoprotein) that produces an immune response and is useful in the prevention or treatment of disease can be selected for use as a peptide antigen (A) for use in the immunogenic compositions of the invention.

[0198] Extensions (B1 and B2): The optional N- and C-terminal extensions (B1 and B2) refer to molecules linked to the N- and C-termini, respectively, of the peptidic antigen (A). The N- and C-terminal extensions B1 and B2 may be composed of any one or more of the following: amino acids, including unnatural amino acids; hydrophilic ethylene oxide monomers (e.g., PEG); hydrophobic alkane chains; or the like; or combinations thereof. The N- and C-terminal extensions B1 and B2 are linked to the peptidic antigen (A) by any suitable means, for example, by a stable amide bond.

[0199] In some embodiments, the extensions (B1 and B2) function to control the degradation rate of the peptidic antigen (A), but can also serve any one or more additional functions. In some embodiments, the N- or C-terminal extensions (B1 or B2) are free (in which case one end of the N- or C-terminal extension is linked to the peptidic antigen (A) and the other end is not linked to another molecule) and can function to slow degradation of the peptidic antigen. For example, a B1 peptide-based extension can be linked to the N-terminus of the peptidic antigen by an amide bond to slow degradation. In other embodiments, the N- and / or C-terminal extensions (B1 and / or B2) can be linked to heterologous molecules, and these extensions can also function as linkers to regulate peptidic antigen (A) degradation. The N- and / or C-terminal extensions providing a linker function allow the peptide antigen to be linked either directly or indirectly via a linker (L) to particles (P) or hydrophobic molecules (H) and / or charged molecules (C).

[0200] In some embodiments, the extensions (B1 and / or B2) function to provide distance, i.e., spacing, between any two heterologous molecules. In other embodiments, the extensions (B1 and / or B2) function to impart hydrophobic or hydrophilic properties to the peptide-antigen conjugate. In still other embodiments, the composition of the extensions (B1 and / or B2) can be selected to impart rigidity or flexibility. In other embodiments, the N-terminal and / or C-terminal extensions (B1 and / or B2) can serve to stabilize particles formed by the peptide-antigen conjugate.

[0201] In some embodiments, the extensions (B1 and / or B2) are composed of charged functional groups, e.g., charged amino acid residues (e.g., arginine, lysine), that impart an electrostatic charge at physiological pH. The number of charged residues present in the extensions can be used to adjust the net charge of the peptide-antigen conjugate. Disclosed herein is an algorithm that describes a systematic process for the selection of peptide-based extensions (B1 and / or B2) that are recognized by proteases and impart a specific electrostatic charge, and that function to stabilize particles formed by the peptide-antigen conjugate.

[0202] Additionally, in some embodiments, the C-terminal extension (B2) added to the peptide antigen (A) is selected to facilitate production of peptides comprising [C]-[B1]-A-B2-[X1] (where [ ] indicates that the group is optional) by incorporating an amino acid sequence in B2 that disrupts beta-sheet formation and prevents sequence shortening during solid-phase peptide synthesis. In a non-limiting example, the C-terminal dipeptide linker (B2), Gly-Ser, is incorporated as a pseudoproline dipeptide (e.g., Gly-Ser(Psi(Me,Me)pro)) during solid-phase peptide synthesis. In a further embodiment, proline is incorporated into a cathepsin-cleavable C-terminal extension (B2) sequence, e.g., Ser-Pro-Leu-Arg (SEQ ID NO: 21), thereby incorporating a proline to both facilitate production and promote processing of the extension by endosomal proteases.

[0203] In some embodiments, the peptide antigen (A) is linked at its C-terminus to a B2 extension, which is linked, either directly or indirectly via a linker (L), to a hydrophobic molecule (H) or a particle (P). In some embodiments, the B1 extension is linked to the N-terminus of the peptide antigen, and the B2 extension is linked to the C-terminus of the peptide antigen (A), in which case either B1 or B2 is linked, either directly or via a linker (L), to a particle (P) or a hydrophobic molecule (H). In other embodiments, the peptide antigen (A) is linked at its N-terminus to a B2 extension, which is linked, either directly or via a linker (L), to a particle or a hydrophobic molecule (H). In some embodiments, the charged molecule (C) is linked to an extension that is B1 or B2 linked to the N-terminus or C-terminus of the peptidic antigen (A), respectively, where the extension not linked to the charged molecule (C) is linked to a hydrophobic molecule (H) or a particle (P), either directly or via a linker (L). In further embodiments, the charged molecule (C) is linked to both extensions B1 and B2, which are linked to both the N-terminus and C-terminus of the peptidic antigen (A), respectively. In a further embodiment, the charged molecule (C) is linked to a B1 extension linked to the N-terminus of the peptidic antigen (A), but not to a B2 extension attached to the C-terminus of the peptidic antigen (A), which may be linked either directly or via a linker (L) to a hydrophobic molecule (H) or a particle (P). The linker precursor X1 or the linker (L) can be linked to either of the extensions (B1 or B2) by any suitable means, such as an amide bond. In a preferred embodiment, the extensions (B1 and B2) are peptide sequences selected for recognition and hydrolysis by an enzyme, such as a protease. The extensions (B1 and B2) are preferably cleavable peptides containing amino acids recognized by either or both of an endosomal protease or an immunoproteasome.

[0204] As described in more detail herein, the composition of the degradable peptide extensions (B1 and B2) depends on whether the extension is linked to the N-terminus (B1) or C-terminus (B2) of the peptide antigen (A).

[0205] In some embodiments, the N-terminal extension (B1) is a peptide sequence between about 1 and 8 amino acids in length, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 amino acids, typically 10 amino acids or less in length, that is linked to the peptide antigen (A) by, e.g., an amide bond formed between the carboxyl group of the extension (B1) and the alpha amine of the N-terminal residue of the peptide antigen (A). The amide bond between B1 and the peptide antigen (A) can be enzymatically cleaved. It is understood that, with respect to amino acid positions C-terminal to the cleavage site (e.g., Pn'), indicated by a dash, it is conventional to number the amino acid positions from proximal to distal to the cleavage site. For example, for a tetrapeptide extension (PN4-PN3-PN2-PN1), e.g., PN4-PN3-PN2-PN1-PA1'-PA2'-PA3'-PA4'-PA5'-PA6'-PA7'-PA8', linked to the N-terminus of a peptide antigen (A) that is an octapeptide (PA1'-PA2'-PA3'-PA4'-PA5'-PA6'-PA7'-PA8'), the amide bond between PN1 and PA1' is recognized and hydrolyzed by the enzyme.

[0206] In some embodiments, the N-terminal extension (B1) is an enzymatically degradable tetrapeptide recognized by endosomal proteases, wherein position PN1 of the tetrapeptide extension (e.g., PN4-PN3-PN2-PN1) is preferably selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine, e.g., PN4-PN3-PN2-Arg, wherein PN2 is selected from glycine, valine, leucine, or isoleucine, PN3 is selected from glycine, serine, alanine, proline, or leucine, and PN4 is selected from glycine, serine, arginine, lysine, aspartic acid, or glutamic acid. In some embodiments, the N-terminal extension (B1) is an enzymatically degradable tripeptide recognized by endosomal proteases, wherein position PN1 of the tripeptide extension (e.g., PN3-PN2-PN1) is preferably selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine, PN2 is selected from glycine, valine, leucine, or isoleucine, and PN3 is selected from glycine, serine, alanine, proline, or leucine. In some embodiments, the N-terminal extension (B1) is an enzymatically degradable dipeptide recognized by an endosomal protease, wherein the PN1 position of the dipeptide extension (e.g., PN2-PN1) is preferably selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine, and PN2 is selected from glycine, valine, leucine, or isoleucine. In yet further embodiments, the N-terminal extension (B1) is an amino acid recognized by an endosomal protease, wherein the PN1 position is preferably selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine.

[0207] In another embodiment, the N-terminal extension (B1) is an enzymatically degradable peptide recognized by the immunoproteasome, in which the P1 position of the tetrapeptide extension (PN4-PN3-PN2-PN1) is preferably selected from isoleucine, leucine, norleucine or valine, e.g., PN4-PN3-PN2-Leu.

[0208] In a further embodiment, the N-terminal extension (B1) is an enzymatically degradable peptide recognized by both endosomal proteases and immunoproteasomes, wherein the PN5 and PN1 positions of the octapeptide extension (PN8-PN7-PN6-PN5-PN4-PN3-PN2-PN1) are selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine for the PN5 position recognized by cathepsins, and isoleucine, leucine, norleucine, or valine for the PN1 position recognized by immunoproteasomes, e.g., PN8-PN7-PN6-Arg-PN4-PN3-PN2-Leu. A non-limiting example of an N-terminal extension (B1) recognized by cathepsins and immunoproteasomes is Lys-Pro-Leu-Arg-Tyr-Leu-Leu-Leu (SEQ ID NO: 3).

[0209] Non-limiting examples of tetrapeptide N-terminal extensions (B1) recognized by the immunoproteasome include Ser-Leu-Val-Cit, Ser-Leu-Val-Leu (SEQ ID NO: 4), Ser-Pro-Val-Cit, Glu-Leu-Val-Arg (SEQ ID NO: 5), Ser-Pro-Val-Arg (SEQ ID NO: 6), Ser-Leu-Val-Arg (SEQ ID NO: 7), Lys-Pro-Leu-Arg (SEQ ID NO: 8), Lys-Pro-Val-Arg (SEQ ID NO: 9), Glu-Leu-Val-Cit, Glu-Leu-Val-Leu (SEQ ID NO: 10), Glu-Pro-Val-Cit, and Lys-Pro-Val-Cit. Non-limiting examples of tripeptide N-terminal extensions (B1) include Leu-Val-Cit, Leu-Val-Leu, Pro-Val-Cit, Leu-Val-Arg, Pro-Val-Arg, Pro-Leu-Arg, and Gly-Val-Ser. Non-limiting examples of dipeptide N-terminal extensions (B1) include Val-Cit, Val-Leu, Val-Arg, and Leu-Arg. Non-limiting examples of single amino acid N-terminal extensions (B1) include Cit, Arg, Leu, or Lys. In the above examples, Arg can be replaced with Lys, Lys can be replaced with Arg, Glu can be replaced with Asp, and Asp can be replaced with Glu. Note that Cit = citrulline.

[0210] In some embodiments, extension (B2) is linked to the C-terminal residue of peptide antigen (A) and is a degradable peptide consisting of an amino acid sequence recognized and hydrolyzed by a specific protease. In some embodiments, C-terminal extension (B2) is a peptide sequence between about 1 and 8 amino acids in length, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 amino acids, typically 10 amino acids or less. In a preferred embodiment, C-terminal extension (B2) is linked to peptide antigen (A) by an amide bond formed between the C-terminal carboxyl group of peptide antigen (A) and the alpha amine of the N-terminal residue of extension (B2). The amide bond between B2 and peptide antigen (A) can be enzymatically cleaved. Note that for amino acid positions C-terminal to the cleavage site (e.g., Pn') indicated by a dash, it is conventional to number amino acid positions from proximal to distal to the cleavage site. For example, for a tetrapeptide extension (PC1'-PC2'-PC3'-PC4'), e.g., PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1-PC1'-PC2'-PC3'-PC4', linked to the C-terminus of an octapeptide antigen (PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1), the amide bond between PA1 and PC1' is recognized and hydrolyzed by the enzyme.

[0211] In a preferred embodiment, the C-terminal extension (B2) is an amino acid sequence selected to promote immunoproteasome recognition and cleavage, and optionally endosomal protease recognition. Since peptide antigens (A) typically contain a C-terminal residue, such as leucine, that promotes immunoproteasome hydrolysis of the amide bond proximal to the C-terminal residue of peptide antigen (A), the extension linked to the C-terminus of peptide antigen (A) should be selected to promote immunoproteasome recognition and cleavage of the amide bond proximal to the C-terminus of peptide antigen (A). Immunoproteasomes prefer small, uncharged amino acids at the PC1' position adjacent to the C-terminal amino acid PA1 of peptide antigen (A), for example, at the amide bond between PA1 and PC1'. However, endosomal proteases prefer bulky hydrophobic amino acids (e.g., leucine, norleucine, methionine, or glutamine) and basic amino acids (i.e., arginine and lysine). Thus, the C-terminal extension can be selected to promote recognition by either or both classes of proteases.

[0212] In some embodiments, a peptide antigen (A) having the sequence PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1 is linked to a C-terminal peptide extension (B2) having the sequence PC1'...PCn' (where n is an integer value between 1 and 8), e.g., PA8-PA7-PA6-PA4-PA3-PA2-PA1-PC1'...PCn'. The composition of the C-terminal extension (B2) depends on the length of the extension sequence used. In some embodiments, the C-terminal extension B2 is a single amino acid PC1' selected from Gly, Ala, Ser, Arg, Lys, Cit, Gln, Thr, Leu, Nle, or Met. In a further embodiment, C-terminal extension B2 is a dipeptide PC1'-PC2', where PC1' is selected from Gly, Ala or Ser and PC2' is selected from Gly, Ala, Ser, Pro, Arg, Lys, Cit, Gln, Thr, Leu, Nle or Met. In a further embodiment, C-terminal extension B2 is a tripeptide PC1'-PC2'-PC3', where P1' is selected from Gly, Ala or Ser, PC2' is selected from Gly, Ala, Ser or Pro and PC3' is selected from Gly, Ser, Arg, Lys, Cit, Gln, Thr, Leu, Nle or Met.

[0213] In a further embodiment, the C-terminal extension B2 is a tetrapeptide extension PC1'-PC2'-PC3'-PC4' (wherein PC1' is selected from glycine, alanine or serine, PC2' is selected from glycine, alanine, serine, proline or leucine, PC3' is selected from glycine, alanine, serine, valine, leucine or isoleucine, and PC4' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine). In a further embodiment, the C-terminal extension B2 is a pentapeptide PC1'-PC2'-PC3'-PC4'-PC5', where PC1' is selected from glycine, alanine or serine, PC2' is selected from glycine, alanine, serine, proline, arginine, lysine, glutamic acid or aspartic acid, PC3' is selected from glycine, alanine, serine, proline or leucine, PC4' is selected from glycine, alanine, valine, leucine or isoleucine, and PC5' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine. In a further embodiment, the C-terminal extension B2 is a hexapeptide PC1'-PC2'-PC3'-PC4'-PC5'-PC6', where PC1' is selected from glycine, alanine or serine, PC2' is selected from glycine, alanine, serine or proline, PC3' is selected from glycine, serine, proline, arginine, lysine, glutamic acid or aspartic acid, PC4' is selected from proline or leucine, PC5' is selected from glycine, alanine, valine, leucine or isoleucine, and PC6' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine.

[0214] Non-limiting examples of hexapeptide C-terminal extensions (B2) include Gly-Gly-Lys-Leu-Val-Arg (SEQ ID NO: 11), Gly-Gly-Lys-Pro-Leu-Arg (SEQ ID NO: 12), Gly-Gly-Ser-Leu-Val-Arg (SEQ ID NO: 13), Gly-Gly-Ser-Leu-Val-Cit, Gly-Gly-Ser-Pro-Val-Cit, Gly-Gly-Ser-Leu-Val-Leu (SEQ ID NO: 14), Gly-Gly-Glu-Leu-Val-Arg (SEQ ID NO: 15), Gly-Gly-Glu-Leu-Val-Leu (SEQ ID NO: 16). Non-limiting examples of pentapeptide C-terminal extensions (B2) include Gly-Ser-Leu-Val-Arg (SEQ ID NO: 17), Gly-Ser-Leu-Val-Cit, Gly-Lys-Pro-Val-Cit, Gly-Lys-Pro-Val-Arg (SEQ ID NO: 18), Gly-Ser-Leu-Val-Leu (SEQ ID NO: 19), Gly-Glu-Leu-Val-Leu (SEQ ID NO: 20). Non-limiting examples of tetrapeptide C-terminal extensions (B2) include Ser-Leu-Val-Cit, Ser-Leu-Val-Leu (SEQ ID NO: 4), Ser-Pro-Val-Cit, Glu-Leu-Val-Arg (SEQ ID NO: 5), Ser-Pro-Val-Arg (SEQ ID NO: 6), Ser-Leu-Val-Arg (SEQ ID NO: 7), Lys-Pro-Leu-Arg (SEQ ID NO: 8), Glu-Leu-Val-Cit, Glu-Leu-Val-Leu (SEQ ID NO: 10), Glu-Pro-Val-Cit, Glu-Gly-Val-Cit. Non-limiting examples of tripeptide C-terminal extensions (B2) include Gly-Ser-Gly, Gly-Ser-Arg, Gly-Ser-Leu, Gly-Ser-Cit, Gly-Pro-Gly, Gly-Pro-Arg, Gly-Pro-Leu, Gly-Pro-Cit. Non-limiting examples of dipeptide C-terminal extensions (B2) include Gly-Ser, Gly-Pro, Val-Cit, Gly-Arg, Gly-Cit.Non-limiting examples of single amino acid C-terminal extensions (B2) include Gly, Ser, Ala, Arg, Lys, Cit, Val, Leu, Met, Thr, Gln, or Nle. In the above examples, Arg can be replaced with Lys, Lys can be replaced with Arg, Glu can be replaced with Asp, and Asp can be replaced with Glu.

[0215] The C-terminal linker (B2) linked to the C-terminus of the peptide antigen (A) can be selected for recognition (i.e., hydrolysis) by both immunoproteasomes and endosomal proteases. In a non-limiting example, peptide antigen (A) having the sequence PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1 is linked to the C-terminus of a C-terminal tetrapeptide extension (B2) having the sequence PC1'-PC2'-PC3'-PC4' (where PC1' is selected from glycine, alanine, or serine, and PC4' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine), such as Ser-P3-P2-Arg. In some embodiments, an antigen having the sequence PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1 is linked to the C-terminal side of a C-terminal hexapeptide extension (B2) having the sequence PC1'-PC2'-PC3'-PC4'-PC5'-PC6' (wherein PC1' and PC2' are selected from glycine, alanine, proline, or serine, and PC6' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine), e.g., Gly-Gly-PC3'-PC4'-PC5'-Arg. A non-limiting example of a C-terminal extension (B2) linked to the C-terminus of peptide antigen (A) that promotes processing by both immunoproteasomes and cathepsins is Gly-Gly-Lys-Pro-Leu-Arg (SEQ ID NO: 12). Further non-limiting examples of C-terminal extensions (B2) linked to the C-terminus of peptide antigens (A) that favor processing by the immunoproteasome and cathepsins are Gly-Gly-Ser-Leu-Val-Cit or Gly-Gly-Ser-Pro-Val-Cit.

[0216] Linker (L) and Linker Precursors (X1 and X2) A subset of linkers that perform the specific function of site-selectively coupling, i.e., binding or linking, a peptide antigen (A) to a hydrophobic molecule (H) or particle (P) is referred to as a "linker (L)." The linker (L) is formed as a result of the reaction of a linker precursor X1 with a linker precursor X2. For example, a linker precursor X1 linked directly to a peptide antigen (A) or indirectly via an extension (B1 or B2) or a charged molecule (C) can react with a linker precursor X2 attached to a hydrophobic molecule (H) or particle (P) to form a linker (L) that links the peptide antigen (A) to the hydrophobic molecule (H) or particle (P). The linker precursor X1 enables site-selective linkage of the peptide antigen (A) to the particle (P) or hydrophobic molecule (H). In some embodiments, a peptide antigen (A) linked to a linker precursor X1, either directly or by an extension (B1 or B2), can be generated, isolated, and then separately attached to a particle (P) or hydrophobic molecule (H) containing a linker precursor X2 that selectively reacts to form a linker (L) that attaches the peptide antigen (A) to the particle (P) or hydrophobic molecule (H).

[0217] The linker (L) or linker precursor X1 can be linked to the peptide antigen (A) at either the N-terminus or C-terminus of the peptide antigen either directly or indirectly via an N-terminal extension (B1) or a C-terminal extension (B2), respectively. In a preferred embodiment, the linker (L) or linker precursor X1 is linked to the peptide antigen (A) or extension (B1 or B2) by an amide bond. Note that a linker (L) or linker precursor X1 directly linked to the N-terminus or C-terminus of the peptide antigen is not considered an extension.

[0218] A suitable linker precursor X1 selectively reacts with linker precursor X2 on the particle (P) or hydrophobic molecule (H), without forming a linkage at any other site on either the peptide antigen (A) or the optional extensions (B1 and / or B2), or the optional charged molecule (C). This selectivity is important to ensure that a linkage can be formed between the peptide antigen (A) and the particle (P) or hydrophobic molecule (H) without modifying the peptide antigen (A). The linker (L) can link the peptide antigen (A) to the particle (P) or hydrophobic molecule (H) directly through a covalent and / or high-affinity interaction, or indirectly through the extensions (B1 or B2). In a preferred embodiment, the linker precursor X1 forms a covalent bond with the linker precursor X2 on the particle or hydrophobic molecule (H).

[0219] In a preferred embodiment, the linker (L) is formed as a result of a bioorthogonal "click chemistry" reaction between linker precursors X1 and X2. In some embodiments, the click chemistry reaction is a catalyst-free click chemistry reaction, e.g., a strain-promoted azide-alkyne cycloaddition reaction, which does not require the use of copper or any catalyst. Non-limiting examples of linker precursors X1 that enable bioorthogonal reactions include molecules containing functional groups selected from azide, alkyne, tetrazine, and transcyclooctene. In some embodiments, the azide-containing linker precursor X1 reacts with the linker precursor X2 to form a triazole linker. In other embodiments, the tetrazine-containing linker precursor X1 reacts with the transcyclooctene (TCO)-containing linker precursor X2 to form a linker containing an inverse demand Diels-Alder ligation product. In a preferred embodiment, the linker precursor X1 is an unnatural amino acid bearing an azide functional group that reacts with an existing alkyne-containing linker precursor X2 by undergoing 1,3-dipolar cycloaddition to form a stable triazole ring. In a preferred embodiment, the X2 linker precursor linked to the particle (P) or hydrophobic molecule (H) comprises an alkyne, e.g., dibenzocyclooctyne (DBCO), that undergoes strain-promoted cycloaddition. In a further embodiment, the X1 linker precursor is an alkyne that reacts with an azide-containing linker precursor X2 present on the particle (P) or hydrophobic molecule (H).

[0220] In other embodiments, the linker precursor X1, which allows site-selective reactivity depending on the composition of the peptide antigen (A), can contain functional groups including thiol, hydrazine, ketone, and aldehyde. In some embodiments, a thiol-containing linker precursor X1 reacts with a pyridyl disulfide- or maleimide-containing linker precursor X2 to form a disulfide or thioether linker, respectively. In other embodiments, a hydrazine-containing linker precursor X1 reacts with a ketone- or aldehyde-containing linker precursor X2 to form a hydrazone linker. In some embodiments, the linker precursor X1 is a natural or unnatural amino acid residue bearing a thiol functional group, such as cysteine, that reacts with a linker precursor X2 containing a thiol-reactive functional group, such as maleimide or pyridyl disulfide.

[0221] In some embodiments, linker precursor X1 is a peptide sequence that is ligated to another peptide sequence that constitutes linker precursor X2 provided on a particle (P) or hydrophobic molecule (H). In other embodiments, linker precursor X1 binds to a complementary molecule that constitutes linker precursor X2 on a particle (P) or hydrophobic molecule (H) through a high-affinity, non-covalent interaction, such as a coiled-coil interaction or an electrostatic interaction. In other embodiments, linker precursor X1 binds to a protein, such as biotin, that forms a high-affinity interaction with a protein, such as streptavidin.

[0222] Those skilled in the art will recognize that suitable pairs of functional groups selected for linker precursors X1 and X2, or complementary molecules, can be interchangeable between X1 and X2. For example, a linker comprised of triazole can be formed from linker precursors X1 and X2 containing an azide and an alkyne, respectively, or from linker precursors X1 and X2 containing an alkyne and an azide, respectively. Thus, any suitable pair of functional groups that results in a linker can be placed at either X1 or X2.

[0223] The particular linker precursors (X1 and X2) and linkers (L) provided in this disclosure provide unexpected improvements in manufacturability and improved biological activity. Many such linker precursors (X1 and X2) and linkers (L) may be suitable for the practice of the present invention and are described in more detail elsewhere.

[0224] The linker precursor (X1) can be attached to either the N- or C-terminus of the peptide antigen (A) either directly or indirectly via an extension (B1 or B2) or a charged molecule (C).

[0225] In some embodiments, the linker precursor X1 is an amino acid that is linked to the C-terminus of the peptidic antigen (A), either directly or indirectly through a B2 extension or a charged molecule (C), and has the formula: [ka] where the functional group (FG) is selected to react specifically with FG on the linker precursor X2, and is typically selected from an amine, azide, hydrazine, or thiol; R 1 is typically selected from OH or NH; y is any integer, for example, 1, 2, 3, 4, 5, 6, 7, or 8; and the alpha amine of this amino acid is typically linked to the C-terminal amino acid of extension B, or to the C-terminal amino acid of the peptide antigen (A) if there is no B extension.

[0226] In other embodiments, the linker precursor X1 is linked to the N-terminus of the peptidic antigen (A), either directly or indirectly through an extension (B1 or B2) or a charged molecule (C), and has the formula: [ka] where the functional group (FG) is selected to react with the linker precursor X2 and is typically selected from an amine, azide, hydrazine, or thiol; y2 is any integer, typically 1, 2, 3, 4, 5, 6, 7, or 8; and the carbonyl is typically linked to the alpha amine of the N-terminal amino acid of the extension B1, or of the peptide antigen (A) if B1 is absent.

[0227] The linker precursor X2 provided on the hydrophobic molecule (H) or particle (P) contains a functional group selected to enable selective reaction with the linker precursor X1 to form the linker (L). In some embodiments, the functional group comprising X2 includes a carbonyl, e.g., an activated ester / carboxylic acid, or a ketone, which reacts with an amine or hydrazine provided on the linker precursor X1 to form an amide- or hydrazone-containing linker (L). In other embodiments, the functional group comprising X2 includes an azide, which reacts with an alkyne provided on the linker precursor X1 to form a triazole. In still other embodiments, the functional group comprising X2 is selected from a maleimide or disulfide, which reacts with a thiol provided on the linker precursor X1 to form a thioether- or disulfide-containing linker (L). The linker precursor X2 can be attached to the hydrophobic molecule (H) or particle (P) by any suitable means. In some embodiments, the hydrophobic molecule (H) includes a peptide, and X2 is linked to the N-terminus of the peptide-based hydrophobic molecule (H).

[0228] The linker (L) can comprise a triazole formed between azide- and alkyne-containing linker precursors X1 and X2, respectively. In some embodiments, the triazole-containing linker results from the reaction of an azide-containing linker precursor X1 with an alkyne (e.g., cyclooctyne)-containing linker precursor X2. In some embodiments, the azide-containing linker precursor X1 is an unnatural amino acid bearing an azide functionality that is linked to the N-terminus of the peptidic antigen (A) or extension (B1) or to the C-terminus of the peptidic antigen (A) or extension (B2). The azide functional group provides a reactive handle that is linked either directly to the peptidic antigen (A) or indirectly via the N-terminal extension (B1) or the C-terminal extension (B2) and selectively reacts with an alkyne (e.g., cyclooctyne)-containing linker precursor X2 that is linked to the hydrophobic molecule (H) or particle (P) to form a triazole linker (L) that connects the peptidic antigen (A) to the hydrophobic molecule (H) or particle (P).

[0229] In some embodiments, the linker precursor X1 is an azido amino acid linked to the C-terminus of the peptidic antigen (A), either directly or indirectly through a B2 extension or a charged molecule (C), and has the formula: [ka] where the alpha amine of the amino acid is linked to the C-terminal amino acid of B2, or of the peptide antigen (A) if there is no B2 extension; R 1 is selected from OH or NH2; n is any integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8). Non-limiting examples of azide-containing linker precursors X1 are 5-azido-2-aminopentanoic acid, 4-azido-2-aminobutanoic acid, and 3-azido-2-aminopropanoic acid.

[0230] In other embodiments, when the azide-containing linker precursor X1 is linked to the N-terminal extension (B1), or directly to the N-terminus of the peptidic antigen (A) if B1 is not present, the linker precursor X1 has the formula: [ka] wherein the carbonyl is typically linked to the alpha amine of the N-terminal amino acid of extension B1, or of peptide antigen (A) if B1 is absent; and y2 is any integer, typically 1, 2, 3, 4, 5, 6, 7, or 8. Non-limiting examples of azide-containing linker precursors include 6-azido-hexanoic acid, 5-azido-pentanoic acid, 4-azido-butanoic acid, and 3-azido-propanoic acid.

[0231] In some embodiments where the linker precursor X1 comprises an azide, the linker precursor X2 comprises an alkyne moiety. Non-limiting examples of alkynes include aliphatic alkynes, cyclooctynes, such as dibenzylcyclooctyne (DBCO or DIBO), difluorooctyne (DIFO), and biarylazacyclooctynone (BARAC). In certain embodiments, the alkyne-containing linker precursor X2 comprises a DBCO molecule.

[0232] In some embodiments, the C-terminal extension (B2) is linked to the peptide antigen (A) by an amide bond at the C-terminus of the peptide antigen (A), which in turn is linked to the hydrophobic block (H) via a linker (L). An example of such a C-terminally linked peptide antigen conjugate is (A) 7~35 -B2-LH (in the formula, (A) 7~35represents a peptide antigen containing 7 to 35 amino acids. In a non-limiting example, a peptide antigen (A) having the octapeptide sequence PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1 is linked at its C-terminus to a tetrapeptide extension (B2), e.g., Ser-Leu-Val-Arg (SEQ ID NO: 7), which is linked to an azide-containing linker precursor (X1) azido-lysine (6-azido-2-aminohexanoic acid, Lys(N3)), which in turn is reacted with the dibenzocyclooctyne (DBCO) moiety of a cyclooctyne-containing linker precursor (X2) linked to a hydrophobic molecule (H) to generate PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1-Ser-Leu-Val-Arg-Lys (N3-DBCO-H).

[0233] In some alternative embodiments, the peptidic antigen (A) is linked at the N-terminus of the peptidic antigen (A) to an N-terminal extension (B1), which is in turn linked to the hydrophobic block (H) via a linker (L). In a non-limiting example, a peptide antigen having the sequence PA1'-PA2'-PA3'-PA4'-PA5'-PA6'-PA7'-PA8' is linked at its N-terminus to a tetrapeptide extension (PN4-PN3-PN2-PN1), e.g., Ser-Leu-Val-Arg (SEQ ID NO: 7), which is linked to azidopentanoic acid (Azp), which in turn reacts with the dibenzocyclooctyne (DBCO) portion of a cyclooctyne-containing linker precursor (X2) linked to a hydrophobic molecule (H): H-DBCO-Azp-Ser-Leu-Val-Arg-PA1'-PA2'-PA3'-PA4'-PA5'-PA6'-PA7'-PA8'.

[0234] Other Linkers and Linkages A linker generally refers to any molecule that connects any two or more heterogeneous molecules of a peptide antigen conjugate to each other, and can further perform one or more of the following functions: I) increasing or decreasing aqueous solubility; II) increasing the distance between any two components, i.e., heterogeneous molecules, of the peptide antigen conjugate; III) imparting rigidity or flexibility; or IV) controlling / regulating the rate of degradation / hydrolysis of the linkage between any two or more heterogeneous molecules. Specific types of linkers used herein are designated. A linker (L) is a specific type of linker molecule used to site-specifically link a peptide antigen (A) to a particle (P) or a hydrophobic molecule (H) either directly or indirectly via an extension (B1 or B2). It should be noted that the N- and C-terminal extensions B1 and B2, respectively, located at the N- and C-termini of a peptide antigen (A), can be linked to a heterologous molecule, such as a charged molecule (C), Linkr (L), a particle (P), or a hydrophobic molecule (H), or any heterologous molecule, and thus B1 and B2 can function as linkers. For clarity, any molecule located at the N- or C-terminus of a peptide antigen (A) is an N- or C-terminal extension (B1 or B2), but an extension can also function as a linker when it is linked to another molecule, such as a charged molecule (C). Thus, as defined herein, a linker at the N- or C-terminus of an antigen is always an extension (B1 or B2), but an extension is not necessarily a linker.

[0235] The linker used to join any two components of the peptide antigen conjugate, e.g., a peptide antigen (A) indirectly linked to a hydrophobic molecule (H) by an extension (B1 or B2), can be by any suitable means. The linker can use covalent or non-covalent means to join any two or more components, i.e., heterologous molecules, e.g., a peptide antigen (A) and a hydrophobic molecule (H).

[0236] In a preferred embodiment, the linker can covalently bond, i.e., link, any two components of the peptide antigen conjugate. A covalent bond is the preferred linkage used to link any two components of the peptide antigen conjugate, i.e., heterologous molecules, ensuring that one component cannot disperse from the other component (e.g., the peptide antigen and the hydrophobic molecule (H)) immediately after administration to a subject. Furthermore, a covalent linkage typically provides greater stability than a non-covalent linkage and helps ensure that each component of the peptide antigen conjugate is co-delivered to antigen-presenting cells in the proportion or approximately the proportion of each administered component.

[0237] In a non-limiting example of covalent linkage, a click chemistry reaction can be used to obtain a triazole that links, i.e., bonds, any two components of a peptide antigen conjugate. In some embodiments, the click chemistry reaction is a strain-promoted [3 + 2] azide-alkyne cycloaddition reaction. Alkyne and azide groups can be provided on each molecule constituting the peptide antigen conjugate and linked via "click chemistry." In some embodiments, a ligand bearing an azide functional group, e.g., a TLR agonist, is coupled with a hydrophobic molecule (H) bearing a suitable reactive group, such as an alkyne, e.g., dibenzylcyclooctyne (DBCO). In further embodiments, an X1 linker precursor bearing a thiol functional group is linked to the peptide antigen (A) either directly or via an N- or C-terminal extension (B1 or B2). The thiol can be linked to a hydrophobic molecule (H) bearing a suitable reactive group, such as an alkyne, alkene, or maleimide, resulting in a thioether bond, or the thiol can be reacted with a pyridyl disulfide, for example, resulting in a disulfide linkage. In some embodiments, an amine can be provided on one molecule and linked to another by reacting the amine with any suitable electrophilic group, such as a carboxylic acid, acid chloride, or activated ester (e.g., NHS ester), resulting in the formation of an amide bond, or the amine can react with an alkene (via Michael addition), an aldehyde, or a ketone (via Schiff base). In a preferred embodiment, the linker precursor X1, optional N- and C-terminal extensions (B1 and B2), optional charged molecule (C), and peptide antigen (A) are linked to each other by amide bonds, such as a single peptide sequence generated by solid-phase peptide synthesis. Many suitable reactions that result in a covalent bond, ie, linkage, are available and will be well known to those skilled in the art.

[0238] The type of linker used to connect any two or more heterologous molecules, i.e., distinct components of a peptide antigen conjugate, can be selected to perform a specific function and meet the specific requirements of the application. Typically, the linker is capable of forming a covalent bond between two or more heterologous molecules of a peptide antigen conjugate, where the components are a peptide antigen (A), a particle (P) or a hydrophobic molecule (H), optional N-terminal and C-terminal extensions (B1 and B2), an optional linker (L), an optional charged molecule (C), an optional second polymer (linked to the hydrophobic molecule (H)), and an optional ligand, e.g., a PRR agonist.

[0239] Many suitable linkers are known to those skilled in the art, including, but not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, rigid aromatic linkers, flexible ethylene oxide linkers, peptide linkers, or combinations thereof. In some embodiments, the carbon linker can include a C1-C18 alkane linker, e.g., a lower alkyl C4. The alkane linker can serve to increase the spacing between two or more heterogeneous molecules, while longer-chain alkane linkers can be used to impart hydrophobic properties. Alternatively, a hydrophilic linker, such as an ethylene oxide linker, can be used in place of the alkane linker to increase the spacing between any two or more heterogeneous molecules and to increase aqueous solubility. In other embodiments, the linker can be an aromatic or poly(aromatic) compound that imparts rigidity. The linker molecule can include a hydrophilic or hydrophobic linker. In some embodiments, the linker comprises a degradable peptide sequence that is cleavable by an intracellular enzyme (eg, a cathepsin or an immunoproteasome).

[0240] In some embodiments, the linker can be composed of poly(ethylene oxide) (PEG). The length of the linker depends on the purpose of the linker. For example, the length of a linker, such as a PEG linker, can be increased to separate components of an immunogenic composition, e.g., to reduce steric hindrance, or, in the case of a hydrophilic PEG linker, to improve aqueous solubility. A linker, such as PEG, can be a short linker, at least two monomers in length. A linker, such as PEG, can be between about 4 and about 24 monomers in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 monomers in length, or longer. In some embodiments, the ligand is linked to a hydrophobic molecule (H) by a PEG linker.

[0241] In some embodiments, the linker comprises a carbon chain, the linker can comprise a chain between about 1 or 2 and about 18 carbons in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 carbons, or more. In some embodiments, the linker comprises a carbon chain, the linker can comprise a chain between about 12 and about 20 carbons. In some embodiments, the linker comprises a carbon chain, the linker can comprise a chain of 18 carbons or less. In some embodiments, a ligand, such as a PRR agonist, is linked to a hydrophobic molecule (H) by a lower alkyl.

[0242] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker results in release of any moiety, e.g., a peptide antigen, that is linked to the linker.

[0243] For example, the linker can be cleavable by an enzyme localized in intracellular vesicles (e.g., in lysosomes, endosomes, or caveolae) or by an enzyme in the cytosol, e.g., the proteasome or immunoproteasome. The linker can be, for example, a peptide linker that is cleaved by a protease enzyme localized in intracellular vesicles, e.g., including, but not limited to, a cathepsin in a lysosomal or endosomal compartment. Peptide linkers are typically 1 to 10 amino acids in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids in length, or longer (e.g., up to 20), e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length, or longer. It is known that certain dipeptides are hydrolyzed by proteases, including cathepsins, such as cathepsins B and D, and plasmin (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin B (e.g., a Phe-Leu or Gly-Phe-Leu-Gly (SEQ ID NO: 23) linker) can be used. Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In certain embodiments, the peptide linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker).

[0244] The specific sequence of the cleavable peptide in the linker can be used to promote processing by immune cells after intracellular uptake. For example, some embodiments of the immunogenic compositions disclosed herein form particles in aqueous conditions, and these particles are internalized by immune cells, such as antigen-presenting cells (e.g., dendritic cells). The cleavable peptide linker can be selected to promote processing (i.e., hydrolysis) of the peptide linker after intracellular uptake by immune cells. The sequence of the cleavable peptide linker can be selected to promote processing by intracellular proteases, such as cathepsins in intracellular vesicles or proteasomes or immunoproteasomes in the cytosolic space.

[0245] In some embodiments, linkers composed of peptide sequences of the formula Pn...P4-P3-P2-P1, where P1 is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine; P2 is selected from glycine, leucine, valine, or isoleucine; P3 is selected from glycine, serine, alanine, proline, or leucine; and P4 is selected from glycine, serine, arginine, lysine, aspartic acid, or glutamic acid, are used to promote recognition by cathepsins. In a non-limiting example, a tetrapeptide linker of the formula P4-P3-P2-P1, linked to a heterologous molecule by an amide bond, has the sequence Lys-Pro-Leu-Arg (SEQ ID NO: 8). For clarity, amino acid residues (Pn) are numbered from proximal to distal to the cleavage site C-terminal to the P1 residue, e.g., the P1-P1′ amide bond is hydrolyzed. Suitable peptide sequences that promote cleavage by endosomal and lysosomal proteases, such as cathepsins, are well described in the literature (see Choe et al., J. Biol. Chem., 281:12824-12832, 2006).

[0246] In some embodiments, linkers comprised of peptide sequences are selected to promote recognition by the proteasome or immunoproteasome. Peptide sequences of the formula Pn...P4-P3-P2-P1 are selected to promote recognition by the proteasome or immunoproteasome, where P1 is selected from basic residues and hydrophobic branched residues, such as arginine, lysine, leucine, isoleucine, and valine, and P2, P3, and P4 are optionally selected from leucine, isoleucine, valine, lysine, and tyrosine. In a non-limiting example, a cleavable linker of the formula P4-P3-P2-P1 recognized by the proteasome is linked to the heterologous molecule by an amide bond at P1 and has the sequence Tyr-Leu-Leu-Leu (SEQ ID NO: 24). Sequences that promote degradation by the proteasome or immunoproteasome may be used alone or in combination with a cathepsin-cleavable linker. In some embodiments, the amino acid that promotes immunoproteasomal processing is linked to a linker that promotes processing by endosomal proteases. Many suitable sequences for promoting cleavage by the immunoproteasome are well described in the literature (see Kloetzel et al., Nat. Rev. Mol. Cell Biol., 2:179-187, 2001; Huber et al., Cell, 148:727-738, 2012; and Harris et al., Chem. Biol., 8:1131-1141, 2001).

[0247] In a preferred embodiment, the N- and C-terminal extensions (B1 and B2) linked to the N- and C-termini of the peptide antigen (A), respectively, are composed of a cleavable peptide linked to additional molecules, e.g., a charged molecule (C) and a hydrophobic molecule (H), respectively. In some embodiments, the cleavable peptide extensions are located at either or both the N- and / or C-termini of the peptide antigen (A). The cleavable peptide extension located at the N-terminus of the peptide antigen (A) is an N-terminal extension (B1), but can further function as a linker when the extension is linked to another molecule, e.g., a charged molecule (C), in addition to the peptide antigen. The extension located at the C-terminus of the peptide antigen (A) is a C-terminal extension (B2), but can further function as a linker when the extension is linked to another molecule, e.g., a hydrophobic molecule (H). The preferred sequences of the cleavable peptides constituting the linkers (B1 and B2) at either the N- or C-terminus are distinct and are described in more detail elsewhere.

[0248] In other embodiments, any two or more components of the peptide-antigen conjugate can be linked to each other by a pH-sensitive linker that is susceptible to hydrolysis under acidic conditions. Numerous pH-sensitive linkages are well known to those skilled in the art, including, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, or the like (see, for example, U.S. Patent Nos. 5,122,368, 5,824,805, and 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). In a preferred embodiment, the linkage is stable at physiological pH, for example, at a pH of about 7.4, but is hydrolyzed at about pH 5-6.5, the pH of the lysosome. In some embodiments, a ligand such as a TLR-7 / 8 agonist is linked to a hydrophobic molecule (H) by FG, which forms a pH-sensitive bond, e.g., by reacting a ketone with a hydrazine to form a pH-labile hydrazone bond. In other embodiments, a peptide antigen (A) is linked, either directly or indirectly through an extension (B1 or B2), to a linker precursor X1 bearing a ketone group, which in turn is linked to a linker precursor X2 containing a hydrazine on the hydrophobic molecule (H). pH-sensitive linkages such as hydrazones offer the advantage that the bond is stable at physiological pH, about pH 7.4, but is hydrolyzed at lower pH values, such as the pH of intracellular vesicles.

[0249] In other embodiments, the linker comprises a linkage that can be cleaved under reducing conditions, such as a reducible disulfide bond.Many different linkers that can be used to introduce disulfide bonds are known in the art (see, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008).See also U.S. Patent No. 4,880,935. In some embodiments, the peptide antigen (A) is linked, either directly or indirectly through an extension (B1 or B2), to a linker precursor X1 bearing a thiol functional group, which forms a disulfide bond with an X2 linker precursor comprising a pyridyl disulfide linked to a hydrophobic molecule (H).

[0250] In still further embodiments, the linkage between any two components of the peptide-antigen conjugate is achieved by an enzymatic reaction, e.g., ligation of expressed proteins, or by sortase (see Fierer et al., Proc. Natl. Acad. Sci., 111:W1176-1181, 2014 and Theile et al., Nat. Protoc., 8:1800-1807, 2013), a chemoenzymatic reaction (Smith et al., Bioconjug. Chem., 25:788-795, 2014) or non-covalent high affinity interactions, such as biotin-avidin and coiled-coil interactions (Pechar et al., Biotechnol. Adv., 31:90-96, 2013) or any suitable means known to those skilled in the art (see Chalker et al., Acc. Chem. Res., 44:730-741, 2011; Dumas et al., Agnew Chem. Int. Ed. Engl. 52:3916-3921, 2013).

[0251] Particles (P) or hydrophobic molecules (H) The immunogenic compositions of the present disclosure include preformed particles (P) or peptide-antigen conjugates further comprising hydrophobic molecules (H). Peptide-antigen conjugates comprising particles (P) exist as particles in aqueous conditions. In some embodiments, peptide-antigen conjugates comprising hydrophobic molecules (H) may exist as single soluble molecules in organic solvents (e.g., DMSO) but may assemble into particles in aqueous conditions. In other embodiments, peptide-antigen conjugates comprising hydrophobic molecules (H) may exist as single soluble molecules in organic solvents (e.g., DMSO) and aqueous conditions over a certain temperature and pH range but may assemble into particles in aqueous conditions over a certain temperature and pH range, for example, at physiological temperature and pH.

[0252] The purpose of the particle (P) or hydrophobic molecule (H) is to force the peptide-antigen conjugate into a particulate form as a means of modulating pharmacokinetics and promoting uptake by antigen-presenting cells. Particles formed by the peptide-antigen conjugate should be between about 10 nm and 10,000 nm in diameter. In a preferred embodiment, the particles are nanoparticles, which are sized to be taken up by the endosomal system of cells (e.g., immune cells). Nanoparticles can range in average size from about 10 nm to about 500 nm in diameter. Thus, in some embodiments, nanoparticles can average about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 100 nm, 200 nm, 300 nm, 400 nm, or 500 nm in diameter. In other embodiments, nanoparticles can average about 10-50 nm, or about 10-100 nm, or about 10-200 nm, or about 10-500 nm in diameter. In a preferred embodiment, the particle size ranges from about 20 to 200 nm in diameter. The particles in the composition can vary in size, but generally will fall within the size ranges set forth herein. For example, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the particles in the composition will fall within the size ranges set forth herein. In some embodiments, the peptide antigen (A) can be linked to an extension (B1 or B2) that is linked directly or via a linker (L) to a particle (P) or a hydrophobic molecule (H), which assembles into particles that are too large to be taken up by immune cells (e.g., particles larger than about 5,000 nm) and forms a depot at the injection site.

[0253] In some embodiments, the peptide antigen (A) is linked to a preformed particle (P). The particle (P) may be composed of a hydrophobic material that retains its structure in aqueous conditions, such as a polymer or lipid, a cross-linked hydrophilic polymer, such as a hydrogel, or a cross-linked hydrophobic polymer, such as cross-linked polystyrene. Non-limiting examples of particles (P) include polymer particles, such as poly(lactic-co-glycolic acid) (PLGA), polymersomes, or poloxamers; lipid-based micelles, liposomes, or multilamellar vesicles; oil-in-water emulsions, such as mineral oil-in-water and water-in-mineral oil emulsions; and inorganic salt particles, such as aluminum phosphate or aluminum hydroxide salt particles (i.e., alum). In some embodiments, the particle (P) is a liposomal nanoparticle. In other embodiments, the particle (P) is an iron particle. In still other embodiments, the particle (P) is a polymer particle.

[0254] The efficiency of linking a peptide antigen (A) to a preformed particle (P) depends on the nature of the peptide antigen and the type of linkage used. For example, the peptide antigen (A) can be adsorbed or incorporated into the interior of the particle (P). Because the nature of the peptide antigen (A) can affect adsorption and incorporation, the efficiency of this process can be determined experimentally for each peptide antigen (A). The peptide antigen (A) can be linked to the particle (P) by high-affinity interactions (e.g., electrostatic) or covalent bonds. In this case, the particle (P) has a set number of reactive sites, which determines the number of peptide antigens (A) that can be linked to the particle (P). For an immunogenic composition containing multiple different peptide antigens (A), for example, 20 different peptide antigens (A), multiple copies of each type of peptide antigen (A) can be delivered on separate particles (P), or multiple copies of all 20 types of peptide antigens (A) can be delivered on the same particle (P).

[0255] A limitation of preformed particles (P) is that the ratio of antigen to particles (P) cannot be easily controlled. Alternatively, in a preferred embodiment, a peptide antigen (A) is linked, either directly or via a linker (L), to a hydrophobic molecule (H) that promotes particle assembly in aqueous conditions. A peptide antigen conjugate, consisting of a peptide antigen (A) optionally linked to a hydrophobic molecule (H) by an extension (B1 or B2) that is linked, either directly or via a linker (L), is a molecularly defined entity, allowing for precise control of the ratio of peptide antigen (A) to hydrophobic molecule (H). In a preferred embodiment, the ratio is 1:1 peptide antigen (A) to hydrophobic molecule (H). In a further non-limiting example, the ratio can be 1:3 to 3:1 peptide antigen (A) to hydrophobic molecule (H).

[0256] In contrast to preformed particles, peptide antigen conjugates can be formed by linking a peptide antigen (A) directly or indirectly via an extension (B1 or B2) and / or a linker (L) to a hydrophobic block (H) to generate a chemically defined single molecule. The hydrophobic molecule (H) is a molecule with substantially limited aqueous solubility or is amphiphilic in nature and can assemble into supramolecular structures, such as micellar particles, nanoparticles, or microparticles, under aqueous conditions. In a preferred embodiment, the hydrophobic molecule (H) is insoluble or forms micelles in aqueous conditions at concentrations below about 0.1 mg / mL or about 0.01 mg / mL.

[0257] The hydrophobic molecule (H) can be selected from any molecule, including higher alkanes, cyclic aromatics, fatty acids, terpene / isoprene-derived compounds, or polymers, that have limited aqueous solubility and / or amphiphilic properties, resulting in the molecules assembling into particles under aqueous conditions. Exemplary higher alkanes include, but are not limited to, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, and octadecane. Exemplary cyclic aromatics include, but are not limited to, benzene and fused benzene ring structures or heterocyclic aromatic molecules. Exemplary saturated and unsaturated fatty acids include, but are not limited to, myristic acid, palmitic acid, stearic acid, or oleic acid. In some embodiments, the hydrophobic molecule (H) is a fatty acid, such as myristic acid. In other embodiments, the hydrophobic molecule (H) comprises a diacyl lipid, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, or a lipopeptide, such as Pam2Cys. In some embodiments, the fatty acid or lipid-based hydrophobic molecule (H) may further comprise PEG. Exemplary compounds derived from terpenes / isoprenes include sterol derivatives, such as cholesterol and squalene. In some embodiments, the hydrophobic molecule (H) comprises cholesterol.

[0258] In preferred embodiments, the hydrophobic molecule (H) is a polymer with limited aqueous solubility or is amphiphilic and can assemble into particles, such as micelles, under aqueous conditions. Exemplary polymers include, but are not limited to, PLGA, hydrophobic poly(amino acids), poly(benzyl glutamate), polystyrene, poloxamers based on ethylene oxide propylene oxide monomers, and temperature-responsive polymers, such as poly(N,N'-diethylacrylamide), poly(Nn-propylacrylamide), poly(N-isopropylacrylamide), poly[di(ethylene glycol)methyl ether methacrylate], and certain PEGylated poly(amino acids), such as poly(γ-(2-methoxyethoxy)esteryl-L-glutamate). In some embodiments, the hydrophobic molecule (H) is a poly(amino acid) composed of hydrophobic amino acids. In preferred embodiments, the hydrophobic molecule (H) comprising a poly(amino acid) is composed of amino acids containing aromatic rings. In other embodiments, the hydrophobic molecule (H) is a poly(amino acid) linked to a ligand, such as a PRR agonist. In other embodiments, the hydrophobic molecule (H) is an AB-type diblock copolymer. In some embodiments, the diblock copolymer is temperature-responsive and can assemble into particles in response to a temperature shift. In some embodiments, the hydrophobic molecule (H) comprising an AB-type diblock copolymer further comprises a ligand, such as a PRR agonist, linked to one block of the diblock copolymer. Polymers useful as hydrophobic molecules (H) for peptide-antigen conjugates are described in more detail below.

[0259] polymer The hydrophobic molecule (H) or particle (P) may comprise a linear or branched polymer. The polymer may be a homopolymer, copolymer, or terpolymer. The polymer may be composed of one type or many different types of monomer units. The polymer may be a statistical copolymer or an alternating copolymer. The polymer may be a block copolymer, such as an AB type, or the polymer may consist of a graft type copolymer, in which two polymers are linked by a polymer-like reaction.

[0260] The hydrophobic molecule (H) or particle (P) can comprise a polymer containing naturally occurring and / or non-natural monomers and combinations thereof. Natural biopolymers can include peptides (sometimes called poly(amino acids)) composed of amino acids; a specific example is poly(tryptophan). Natural biopolymers can also be chemically modified. For example, biopolymers composed of glutamic acid or lysine residues can be modified at the gamma carboxyl or epsilon amino groups, respectively, for ligand attachment. The biopolymer can also be a polysaccharide, including, but not limited to, glycogen, cellulose, and dextran. Further examples include naturally occurring polysaccharides, including alginate and chitosan. The polymer can also be composed of naturally occurring small molecules, such as lactic acid or glycolic acid, or a copolymer of the two (i.e., PLGA).

[0261] In some embodiments, the hydrophobic molecule (H) or particle (P) is composed of an anionic (e.g., poly(acidic)) polymer, or a cationic (e.g., poly(basic)) polymer, or a combination of anionic and cationic polymers. Cationic polymers can bind negatively charged peptides through electrostatic interactions or may be useful for complexing with negatively charged nucleic acids such as DNA and RNA. In some embodiments, the polymer is a water-insoluble zwitterion at pH 7.4, but carries a net positive charge and is water-soluble at pHs below about 6. In some embodiments, the hydrophobic molecule (H) comprising a first polymer carries a positive or negative charge that is complementary to the negative or positive charge on the second polymer, respectively, and the first and second polymers form an electrostatic complex through charge neutralization, which renders the complex insoluble. In some embodiments, the cationic polymer can be a naturally occurring or synthetic poly(amine), such as poly(lysine) or poly(ethyleneimine) (PEI). In further embodiments, the cationic polymer can be a poly(amidoamine) (PAA) or poly(beta-aminoester) (PBAE) produced from the Michael addition reaction of an amine with a bis(acrylamide) or a bis(acrylic ester). Non-limiting examples of cationic polymers that can be used in the disclosed embodiments include poly(ethyleneimine), poly(allyl anion hydrochloride; PAH), putrescine, cadaverine, poly(lysine) (PL), poly(arginine), poly(trimethyleneimine), poly(tetramethyleneimine), poly(propyleneimine), aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, cadaverine, poly(2-dimethylamino)ethyl methacrylate, poly(histidine), cationized gelatin, dendrimers, chitosan, and any combination thereof. The cationic polymer can contain quaternary ammonium groups, such as those present on methylated chitosan. Alternatively, the polymer may be an anionic polymer. In some non-limiting examples, the polyanionic polymer is poly(glutamic acid). In alternative embodiments, the polyanionic polymer is poly(aspartic acid).The polymer may be a polyphosphoester polymer. The polymer may include natural anionic polysaccharides, such as alginic acid, composed of (1-4) linked β-D-mannuronate and guluronic acid. The polymer may also include nucleotides. Other polyanionic polymers may be suitable as well.

[0262] In some embodiments, the hydrophobic molecule (H) is a water-soluble cationic polymer over a certain pH range, but is uncharged and water-insoluble in a pH range around physiological pH 7.4. In some embodiments, the hydrophobic molecule (H) is a polymer containing an aromatic amine, where the pKa of the conjugate acid of the aromatic amine is less than 7.5. At a pH below the pKa of the aromatic amine, the aromatic amine becomes protonated, thus imparting a positive charge to the polymer. A non-limiting example of a hydrophobic molecule (H) composed of a polymer containing an aromatic amine is poly(phenylalanine amine). In some embodiments, the hydrophobic molecule (H) is a polymer containing a nitrogen heterocycle, where the pKa of the nitrogen atom constituting the heterocycle is less than 7.5. At a pH below the pKa of the nitrogen atom constituting the heterocycle, the nitrogen becomes protonated, imparting a positive charge to the polymer. A non-limiting example of a hydrophobic molecule (H) composed of a polymer containing a heterocycle with a protonatable (i.e., basic) nitrogen atom is poly(histidine). Herein, we report the unexpected discovery that hydrophobic molecules composed of polymers containing protonatable nitrogens (e.g., aromatic amines) provide unexpected improvements in manufacturing, particle stability, and biological activity.

[0263] In some embodiments, the hydrophobic molecule (H) can be a poly(diethylene glycol methyl ether methacrylate)-(DEGMA)-based polymer. In further embodiments, the hydrophobic molecule (H) is a polymer that can include monomers of (meth)acrylate, (meth)acrylamide, styryl, and vinyl moieties. Specific examples of (meth)acrylate, (meth)acrylamide, and styryl- and vinyl-based monomers include N-2-hydroxypropyl(methacrylamide) (HPMA), hydroxyethyl(methacrylate) (HEMA), styrene, and vinylpyrrolidone (PVP), respectively. The polymer can be a thermoresponsive polymer composed of monomers selected from N-isopropylacrylamide (NIPAAm), N-isopropylmethacrylamide (NIPMAm), N,N'-diethylacrylamide (DEAAm), N-(L)-(1-hydroxymethyl)propylmethacrylamide (HMPMAm), N,N'-dimethylethyl methacrylate (DMEMA), and 2-(2-methoxyethoxy)ethyl methacrylate (DEGMA). In some embodiments, the hydrophobic polymer is a polymer containing HPMA monomers or HPMA·DEGMA monomers. In some embodiments, the polymer containing HPMA and DEGMA monomers is an AB diblock polymer. The unexpected discovery reported herein is that peptide antigens (A) linked to AB diblock copolymers containing HPMA hydrophilic blocks assemble into nanoparticle micelles of uniform size, regardless of peptide antigen (A) composition.

[0264] The hydrophobic molecules (H) may also include polymers based on cyclic monomers including cyclic urethanes, cyclic ethers, cyclic amides, cyclic esters, cyclic anhydrides, cyclic sulfides, and cyclic amines.

[0265] Hydrophobic molecules (H) based on polymers containing cyclic monomers can be produced by ring-opening polymerization and include polyesters, polyethers, polyamines, polycarbonates, polyamides, polyurethanes, and polyphosphates, with specific examples including, but not limited to, polycaprolactone and poly(ethyleneimine) (PEI). Suitable polymers can also be produced by condensation reactions and include polyamides, polyacetals, and polyesters.

[0266] In some embodiments, the hydrophobic molecule (H) is a polymer that can contain 3 to 10,000 monomer units. In preferred embodiments, the polymer contains 3 to 300 monomer units, e.g., 3 to 10 monomer units, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 monomer units, or about 10 to 100 monomer units, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or about 100 to 200 monomer units, or about 200 to 300 monomer units, typically 1,000 or less. In some embodiments, the polymer can contain up to 1,000 to 10,000 monomer units. While typically at least five monomers are required to form a hydrophobic molecule (H) large enough to promote particle formation of peptide-antigen conjugates, surprisingly, a hydrophobic molecule (H) composed of a polymer with only three monomers containing aromatic rings was sufficient to drive particle assembly of peptide-antigen conjugates. Increasing the length of the polymer from 3 to 5 monomers, and then from 5 to 10 monomers, increases the strength of the forces promoting particle formation, resulting in more stable, larger particles formed by the peptide-antigen conjugate. In a preferred embodiment, the hydrophobic molecule (H) constituting the peptide-antigen conjugate is a polymer composed of between 5 and 100 monomers, resulting in the formation of particles with diameters of approximately 10 to 300 nm in aqueous conditions. In a further embodiment, the polymer constituting the hydrophobic molecule (H) is composed of approximately 300 monomers, resulting in the peptide-antigen conjugate assembling into particles between about 20 and 500 nm, or about 100 to 500 nm.

[0267] In some embodiments, the average molecular weight of the polymer constituting the hydrophobic molecule (H) can be between about 1,000 and 1,000,000 g / mol. In preferred embodiments, the average molecular weight of the polymer is between about 1,000 and 60,000 g / mol. In some embodiments, the polymer molecular weight is between about 1,000 and 5,000, or between about 5,000 and 10,000, or between about 10,000 and 20,000, or between about 20,000 and 30,000, or between about 25,000 and 60,000. In some embodiments, the hydrophobic molecule (H) is an AB diblock polymer having an average molecular weight of about 10,000 g / mol to about 60,000 g / mol, e.g., about 10,000 g / mol, 20,000 g / mol, 30,000 g / mol, 40,000 g / mol, 50,000 g / mol, or 60,000 g / mol. In some embodiments, the polymer is an AB diblock polymer having a ratio of the molecular weight of the A block to the B block of about 1:5 to about 5:1. In a non-limiting example, an AB diblock polymer having an average molecular weight of about 60,000 g / mol is composed of an A block having an average molecular weight of about 10,000 g / mol and a B block having an average molecular weight of about 50,000 g / mol; an A block having an average molecular weight of about 20,000 g / mol and a B block having an average molecular weight of about 40,000 g / mol; an A block having an average molecular weight of about 30,000 g / mol and a B block having an average molecular weight of about 30,000 g / mol; an A block having an average molecular weight of about 40,000 g / mol and a B block having an average molecular weight of about 20,000 g / mol; an A block having an average molecular weight of about 50,000 g / mol and a B block having an average molecular weight of about 10,000 g / mol.

[0268] The polydispersity, Mw / Mn, of the polymers can range from about 1.0 to about 5.0. Polymers can be formed by a variety of polymerization techniques. Peptide- and nucleotide-based polymers can be prepared by solid-phase synthesis, and molecularly defined polymers will have a polydispersity of 1.0. Polymers formed by chain-growth polymerization will have a polydispersity >1.0. Polymers can be synthesized by living polymerization techniques or solution free-radical polymerization. In a preferred embodiment, peptide-based biopolymers are synthesized by solid-phase peptide synthesis. Peptide (or "poly(amino acid)")-based polymers containing amino acids with aromatic rings, such as tryptophan, despite their hydrophobicity in aqueous conditions, have unexpectedly improved production by solid-phase synthesis compared to peptides (or "poly(amino acid)") without aromatic rings. Thus, in a preferred embodiment, peptide-based hydrophobic molecules (H) produced by solid-phase synthesis contain amino acids containing aromatic rings. In a further embodiment, acrylamide- and acrylate-based polymers are synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. In a further embodiment, the poly(amino acids) and poly(phosphoesters) are synthesized by ring-opening polymerization.

[0269] In some embodiments, the hydrophobic molecule (H) may comprise a polymer further comprising a ligand(s), e.g., a PRR agonist. In some embodiments, the polymer-based hydrophobic molecule (H) may comprise a monomer comprising at least one functional group capable of coupling to a ligand or at least one functional group capable of coupling to a linker capable of coupling to a ligand. In other embodiments, the polymer-based hydrophobic molecule (H) may comprise a ligand linked to the terminus and / or side chain of the polymer.

[0270] In a preferred embodiment of the immunogenic composition for the treatment or prevention of cancer and infectious diseases, the hydrophobic molecule (H) is a polymer linked to a ligand. In some embodiments, the ligand linked to the polymer-based hydrophobic molecule (H) comprises an aromatic ring structure. In some embodiments, the ligand linked to the polymer is hydrophobic and promotes increased stability of particles formed by the peptide-antigen conjugate in aqueous conditions. In other embodiments, the ligand linked to the polymer-based hydrophobic molecule (H) comprises a heterocyclic aromatic ring. In some embodiments, the ligand linked to the polymer-based hydrophobic molecule (H) comprises an aromatic ring further comprising an arylamine. In some embodiments, the ligand attached to the hydrophobic molecule (H) is a hydrophobic ligand comprising a heterocyclic aromatic ring, and optionally, the hydrophobic ligand further comprises an aromatic amine (i.e., Ar-NH). In embodiments where the hydrophobic molecule (H) comprises a ligand containing an aromatic group, optionally containing a heterocycle and / or an arylamine, the inventors report the unexpected discovery that such hydrophobic molecules (H) are highly soluble in pharmaceutically acceptable organic solvents such as DMSO and ethanol, but insoluble in aqueous buffers.

[0271] In some embodiments, the ligand linked to the polymer-based hydrophobic molecule (H) is a pattern recognition receptor agonist (PRRa), such as an agonist of STING, NOD receptor, or TLR, with adjuvant properties. The ligand with adjuvant properties linked to the polymer can be any suitable adjuvant compound, such as a PRR agonist, or can be derived from any suitable adjuvant compound. Suitable ligands with adjuvant properties include compounds containing small organic molecules, i.e., molecules with a molecular weight of less than about 3,000 daltons, although in some embodiments, adjuvants can have a molecular weight of less than about 700 daltons, and in some cases, adjuvants can have a molecular weight of about 200 daltons to about 700 daltons.

[0272] In a preferred embodiment of the immunogenic composition used for the treatment or prevention of cancer or infectious disease, the hydrophobic molecule (H) is a polymer linked to a ligand with adjuvant properties. The ligand with adjuvant properties, such as a PRR agonist, can be linked to the side chain or terminal group of the polymer via any suitable linker. In some embodiments, the monomers constituting the polymer-based hydrophobic molecule (H) contain a side chain containing at least one functional group that can be coupled to a ligand with adjuvant properties, or at least one functional group that can be coupled to a linker that can be coupled to a ligand with adjuvant properties. In some embodiments where the polymer-based hydrophobic molecule (H) contains a ligand with adjuvant properties, all of the monomers of the polymer are linked to a ligand with adjuvant properties. In other embodiments where the hydrophobic molecule (H) contains a ligand with adjuvant properties, not all of the monomers of the polymer are linked to an adjuvant.

[0273] In some embodiments, comprising polymers in which hydrophobic molecules (H) are linked by monomer units distributed along the backbone of the polymer to ligands with adjuvant properties, increasing the density of the ligands on the polymer leads to unexpected enhancement of the immune response to the peptide antigen (H).

[0274] In certain embodiments, the molar ratio of the ligand with adjuvant properties, such as a PRR agonist, to the monomer of the polymer can be selected from about 1:100 to 1:1 mol / mol (or about 1 mol% to about 100 mol%), e.g., 1:2.5 to 1:1 mol / mol.

[0275] The density of the ligand with adjuvant properties, such as a PRR agonist, linked to the polymer can be varied depending on the needs of a particular application. The ligand with adjuvant properties, such as a PRR agonist, can be linked to the polymer at 1 to 100 mol%, for example, 1 to 10 mol%, or 50 to 100 mol%. Mol% refers to the percentage of monomers constituting the polymer that are linked to the ligand with adjuvant properties, such as a PRR agonist. For example, 10 mol% ligand (e.g., a PRR agonist) is equivalent to 10 monomer units linked to the ligand out of a total of 100 monomer units. The remaining 90 can be polymer-forming monomer units that are not linked to the ligand.

[0276] The density of the ligands, such as ligands with adjuvant properties, linked to the polymer-based hydrophobic molecule (H) should be selected to ensure that the peptide-antigen conjugate is (i) soluble in a pharmaceutically acceptable organic solvent such as DMSO, (ii) capable of forming stable nanoparticles in aqueous conditions at physiological temperature and pH, and / or (iii) capable of inducing an immune response, particularly a T cell response, in a subject.

[0277] The optimal density of a ligand, such as a PRR agonist, linked to a polymer depends not only on the composition of the ligand but also on the polymer composition and polymer length. When the ligand is a hydrophobic / amphiphilic molecule with poor aqueous solubility, such as an imidazoquinoline-based Toll-like receptor-7 and -8 agonist (TLR-7 / 8a), and the polymer alone is water-soluble (i.e., the polymer unlinked to the ligand is water-soluble), the ligand is typically linked to the polymer at a density of about 20-100 mol% when the polymer is composed of between about 5-30 monomer units, 10-50 mol% when the polymer is composed of between 30-100 monomer units, or 5-20 mol% when the polymer is composed of between 100-300 monomer units. Generally, the mol% of the ligand with adjuvant properties is higher for shorter polymers and lower for longer polymers.

[0278] Hydrophilic or temperature-responsive polymers with a molecular weight greater than 10,000 g / mol, including N-2-hydroxypropyl(methacrylamide) (HPMA), hydroxyethyl(methacrylate) (HEMA), styrene, vinylpyrrolidone (PVP), N-isopropylacrylamide (NIPAAm), N-isopropylmethacrylamide (NIPMAm), N,N'-diethylacrylamide (DEAAm), N-(L)-(1-hydroxymethyl)propylmethacrylamide (HMPMAm), N,N'-dimethylethylmethacrylate (DMEMA), 2-(2-methoxyethyl)-2-methylpropanol (DMPA), 2-(2-methylpropanol ... The optimal density of a ligand having adjuvant properties, such as PRRa, attached to a polymer based on a comonomer selected from dihydroxyethyl methacrylate (DEGMA) or a substituted poly(phosphoester) is 1-25%, for example, the density of the adjuvant attached to the polymer can be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%.

[0279] In some embodiments, the hydrophobic molecule (H) is an amphiphilic AB diblock copolymer in which one block is hydrophobic and the other block is hydrophilic. In some embodiments, when the hydrophobic molecule (H) is an amphiphilic AB diblock copolymer and the ligand is hydrophobic, e.g., an imidazoquinoline TLR-7 / 8 agonist, the ligand is preferably linked to the hydrophobic block at a density of about 1 to 50 mol%, typically about 1 to 20 mol%. In some embodiments, when the hydrophobic molecule (H) is an amphiphilic AB diblock copolymer and the ligand is hydrophilic, the ligand is preferably linked to the hydrophilic block at a density of about 1 to 20 mol% or to the hydrophilic end of the hydrophilic block, and thus the AB diblock copolymer is semi-telechelic with respect to the ligand. In a non-limiting example, the hydrophobic molecule (H) comprises a temperature-responsive AB-type diblock copolymer comprising an HPMA block and a DEGMA block, with an imidazoquinoline TLR-7 / 8 agonist linked to the DEGMA block at a density of between about 1-5 mol%. In a further non-limiting example, the hydrophobic molecule (H) comprises an AB-type diblock polymer comprising an HPMA copolymer hydrophobic block and an HPMA homopolymer hydrophilic block, with an imidazoquinoline TLR-7 / 8 agonist linked to the HPMA copolymer hydrophobic block at a density of about 20 mol%. In some embodiments, the hydrophobic molecule is a triblock copolymer, e.g., ABA, or other multiblock copolymer composition.

[0280] The unexpected discovery reported herein is that peptide antigens (A) linked to AB-type diblock copolymers containing an HPMA hydrophilic block linked to a ligand with adjuvant properties assemble into nanoparticle micelles of uniform size regardless of peptide antigen (A) composition, and this increased robustness of nanoparticle micelle formation was associated with increased magnitude of T cell immunity. In a non-limiting example, peptide antigens (A) were linked to a diblock copolymer composed of an HPMA copolymer hydrophobic block (i.e., p[(HPMA)-co-(MA-b-Ala-2B)]) and an HPMA homopolymer hydrophilic block (i.e., p(HPMA)) via a triazole linker (Lys(N3)-DBCO) to form the peptide antigen conjugate p{[(HPMA)-co-(MA-b-Ala-2B)]-bp(HPMA)}-DBCO-(Lys(N3))-A (where 2B is TLR-7 / 8a, also referred to as Compound 1). In a further embodiment, the peptide antigen (A) is linked to a diblock copolymer composed of a DEGMA copolymer hydrophobic block (i.e., p[(DEGMA)-co-(MA-b-Ala-2B)]) and an HPMA homopolymer hydrophilic block (i.e., p(HPMA)) by a triazole linker (Lys(N3)-DBCO) to form the peptide antigen conjugate p{[(DEGMA)-co-(MA-b-Ala-2B)]-bp(HPMA)}-DBCO-(Lys(N3))-A (where 2B is TLR-7 / 8a, also referred to as Compound 1).In another embodiment, the peptide antigen (A) is linked by a triazole linker (Lys(N3)-DBCO) to a diblock copolymer composed of a DEGMA homopolymer (i.e., 2BXy-p[(DEGMA)) and an HPMA homopolymer hydrophilic block (i.e., p(HPMA)) linked to a ligand with adjuvant properties to form the peptide antigen conjugate 2BXy-p{[(DEGMA)-co-(MA-b-Ala-2B)]-bp(HPMA)}-DBCO-(Lys(N3))-A, where the peptide antigen (A) and 2BXy (TLR-7 / 8a, also referred to as Compound 2) are linked to a single site at both ends of the polymer, making the polymer heterotelechelic.

[0281] In some embodiments, the hydrophobic molecule (H) is a poly(amino acid)-based polymer composed of glutamic acid or aspartic acid and comonomers of aromatic and / or hydrophobic amino acids, such as phenylalanine, aminophenylalanineamine (or "phenylalanineamine"), tryptophan, tyrosine, benzylglutamate, histidine, leucine, isoleucine, norleucine, and valine, to which one or more ligands having adjuvant properties, such as PRRa, are attached by the gamma carboxylic acid of glutamic acid or the beta carboxylic acid of aspartic acid. In a preferred embodiment, the hydrophobic molecule (H) is a poly(amino acid)-based polymer composed of comonomers of glutamic acid and tryptophan, to which one or more ligands having adjuvant properties, such as PRRa, are linked by the gamma carboxylic acid to the glutamic acid residue. In a further embodiment, the hydrophobic molecule (H) is a poly(amino acid)-based polymer composed of comonomers of lysine and aromatic and / or hydrophobic amino acids, such as phenylalanine, aminophenylalanine, histidine, tryptophan, tyrosine, benzylglutamate, leucine, isoleucine, norleucine, and valine, wherein one or more ligands having adjuvant properties, such as PRRa, are linked to the polymer via the epsilon amine of the lysine. In a preferred embodiment, the hydrophobic molecule (H) is a poly(amino acid)-based polymer composed of comonomers of lysine and tryptophan, wherein one or more ligands having adjuvant properties, such as PRRa, are linked to the lysine via the epsilon amine. In a preferred embodiment, the hydrophobic molecule (H) is a poly(amino acid) copolymer linked to one or more ligands with adjuvant properties, such as PRRa, the polymer is between 5 and 30 amino acids in length and the adjuvant is attached at a density of 20 to 100 mol%, for example, 30%, 50%, 60%, 80% and 100 mol%.In a further embodiment, the ligand is attached to only one end of the poly(amino acid) polymer, i.e., semi-telechelic polymer. Herein, the inventors report the unexpected discovery that a hydrophobic molecule (H) composed of a poly(amino acid)-based copolymer and further comprising an aromatic group linked to the polymer, such as an aromatic amino acid (e.g., phenylalanine, aminophenylalanine, histidine, tryptophan, tyrosine, benzyl glutamate) or an aromatic ligand (e.g., imidazoquinoline), provides unexpected improvements in manufacturability due to improved organic solvent solubility and improved particle stability and biological activity of peptide-antigen conjugates compared to poly(amino acids) composed primarily of aliphatic amino acids or aliphatic ligands. Thus, in a preferred embodiment, a hydrophobic molecule (H) composed of a poly(amino acid) or other class of polymer comprises one or more aromatic amino acids and / or a ligand containing an aromatic group.

[0282] In a further embodiment, the hydrophobic molecule (H) is a poly(amino acid)-based polymer composed entirely of glutamic acid, aspartic acid, or unnatural amino acid residues having a carboxylic acid, wherein a ligand having adjuvant properties is linked to all of the glutamic acid, aspartic acid, or unnatural amino acid residues, i.e., the ligand having adjuvant properties is bound at a density of 100 mol%. In a further embodiment, the hydrophobic molecule (H) is a poly(amino acid)-based polymer composed entirely of lysine or unnatural amino acids having free amines, wherein a ligand having adjuvant properties is linked to all of the lysine or unnatural amino acid residues, i.e., the adjuvant is bound at a density of 100 mol%. In a further embodiment, a PEGylated comonomer, such as γ-(2-methoxyethoxy)esteryl-L-glutamate, is incorporated to impart temperature-responsive properties to the copolymer. In other embodiments, a temperature-responsive polymer can be grafted to the pendant side chains of a poly(amino acid) to form a graft copolymer. In further embodiments, a temperature-responsive polymer can be linked to the termini of a poly(amino acid) polymer to form a temperature-responsive diblock polymer. In still further embodiments, a hydrophobic second polymer can be linked by pendant side groups to a poly(amino acid) polymer that is linked to a ligand with adjuvant properties to form a graft copolymer, or can be linked to the termini of such a poly(amino acid) to form a diblock copolymer.

[0283] In some embodiments, the hydrophobic molecule (H) is a poly(amino acid) based polymer linked to a ligand, such as a hydrophobic ligand with adjuvant properties, and has the formula: [ka] It has.

[0284] In formula I, R 2is typically selected from one of hydrogen, hydroxyl, or amine. In some embodiments, R 2 is linked to a ligand or another polymer by any suitable linker molecule. The number of methylene units, y3, is typically 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. The N-terminal amine of the poly(amino acid) of Formula I is typically linked to a linker precursor, X2, or may be linked to a peptide antigen (A), either directly or via an extension (B1 or B2). The repeating number of the monomer is indicated by k, typically between 3 and 300. Any suitable linker, X, is used to link a ligand, such as a hydrophobic ligand with adjuvant properties, to the poly(amino acid) backbone. In some embodiments, the linker, X, can be linked to a second polymer that is linked to a ligand. In some embodiments, the monomer k can be linked to two or more different ligands.

[0285] In some embodiments, the poly(amino acid) based polymer of Formula I is [ka] (wherein y4 is any integer, for example, 0 to 6, such as 0, 1, 2, 3, 4, 5, or 6). The ligand can be linked to the functional group (FG) by any suitable means, where FG is any suitable functional group, including an amine, carboxylic acid, thiol, hydroxyl, azide, alkyne, hydrazine, aldehyde, or ketone, for attachment, i.e., linkage, of the ligand, either directly or via a linker.

[0286] When y3 is equal to 1, the poly(amino acid) based polymer of Formula I is [ka] is.

[0287] The N-terminus of the poly(amino acid) of Formula I can be linked to the peptidic antigen (A) via a linker (L) by any suitable means. In some embodiments, the N-terminus of the poly(amino acid) of Formula I is linked directly to the C-terminus of the peptidic antigen (A) or to the C-terminus of the B2 extension via an amide bond. In other embodiments, the N-terminus of the poly(amino acid) of Formula I is linked to a linker precursor (X2) that reacts with a linker precursor (X1) that is linked to the peptidic antigen (A) directly or via an extension (B1 or B2). In some embodiments, a cyclooctyne (e.g., DBCO)-containing linker precursor (X2) is attached to the N-terminus of the poly(amino acid) of Formula I and reacts with an azide-containing linker precursor (X1) to form a triazole bond.

[0288] In some embodiments, the poly(amino acid)-based hydrophobic molecule (H) may comprise a hydrophobic amino acid (e.g., an aromatic amino acid), or a hydrophobic amino acid (e.g., an aromatic amino acid) linked to a ligand and an amino acid, preferably an additional amino acid, e.g., a charged or hydrophilic amino acid, that is useful for complementing or adjusting the physical and chemical properties of the hydrophobic molecule (H) to produce a material that is soluble during manufacture in organic solvents but capable of forming particles in aqueous conditions. Thus, in some embodiments, the hydrophobic molecule (H) may have the formula: [ka] It is a poly(amino acid)-based polymer having the formula:

[0289] The poly(amino acid)-based polymer of Formula II comprises the normal monomer l and the optional monomers m, n, and o. R 3 is typically selected from one of hydrogen, hydroxyl, or amine. In some embodiments, R 3is a ligand, e.g., a ligand with adjuvant properties, or another polymer, linked to a hydrophobic molecule (H) by any suitable linker molecule. The number of methylene units is indicated by y5, y6, y7, and y8, and is typically 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. The N-terminal amine of the poly(amino acid) of Formula I is typically linked to a linker precursor X2, or may be linked to a peptidic antigen (A), either directly or via an extension (B1 or B2). In an exemplary embodiment, the poly(amino acid)-based polymer of Formula II comprises 1, a monomer selected from any natural or unnatural amino acid, where R 4 is selected from lower alkyl or aromatic groups and imparts hydrophobic properties to the polymer backbone. In some embodiments, R in Formula II 4 teeth, [ka] [ka] [ka] You can choose from: 4 X is any suitable linker.

[0290] In some embodiments, the poly(amino acid) based polymer of Formula II includes an optional comonomer, m, which can be any natural or unnatural amino acid, e.g., a PEG amino acid spacer (e.g., m in Formula II can be -NH-(CH-CH-O) y9 -(CH2) y10 -(CO)-, where y9 is an integer usually between 1 and 24, and y10 is an integer usually between 1 and 3), or an amino acid with a small substituent, e.g., R 5 is selected from hydrogen, lower alkyl, or lower alkyl containing hydroxyl, and is provided to increase spacing or flexibility of the polymer backbone. 5 teeth, [ka] You can choose from:

[0291] In some embodiments, the poly(amino acid)-based polymer of Formula II includes an optional comonomer, n, selected from any natural or unnatural amino acid, where R 6 is selected from any group containing a functional group that carries a charge, either permanently or at a specific pH. In some embodiments, R in Formula II 6 teeth, [ka] You can choose from:

[0292] In some embodiments, the poly(amino acid)-based polymer of Formula II comprises an optional comonomer o selected from any natural or unnatural amino acid, wherein a ligand is linked to this monomer o by any suitable linker X. The ligand may be a ligand with adjuvant properties. The ligand linked to the poly(amino acid) of Formula II may be hydrophobic, hydrophilic, amphiphilic, charged, or neutral in nature. The poly(amino acid)-based polymer of Formula II comprising monomer o may further comprise monomer units 1, m, and n that complement the properties of the ligand attached to monomer o.

[0293] In the poly(amino acid)-based polymer of Formula II, the repeating number of monomers is represented by l, m, n, and o, and the sum of l, m, n, and o is typically any integer between 3 and 300. Each of the different types of monomers, l, m, n, or o, can be the same or different. The monomer represented by "l" confers hydrophobicity to the poly(amino acid)-based polymer of Formula II, i.e., makes the polymer a water-insoluble hydrophobic molecule (H). The hydrophobic monomers, l, can be the same or different and typically contain aromatic rings. In a preferred embodiment, the hydrophobic monomer l contains heterocyclic and / or amine-substituted aromatic rings. Optional comonomers, represented by "m," can be used to increase the flexibility or spacing of different monomers comprising the polymer backbone. Optional comonomers, represented by "n," contain charged functional groups. Optional comonomers, represented by "o," are used to bind ligands, such as PRR agonists. In some embodiments, the ligand linked to the monomer o by any suitable linker is a PRR agonist. In some embodiments, the monomer o is linked to a ligand that is positively or negatively charged and is adjacent to the oppositely charged monomer n. In some embodiments, the charged comonomer n is located adjacent to the comonomer o that contains a functional group of opposite charge to that of the functional group containing the comonomer n, and these opposite charges result in a net charge of zero, and thus the monomer n functions to neutralize the charge of the ligand bound to the monomer o.

[0294] The percentages of monomers l, m, n, and o that make up the poly(amino acid)-based polymer of Formula II depend on the specific application. In some embodiments, the poly(amino acid)-based polymer of Formula II is composed entirely of monomer l. In other embodiments, the poly(amino acid)-based polymer of Formula II is composed of comonomers l and o, e.g., between 5 and 95 mol% of monomer l and about 95 to 5 mol% of monomer o. In some embodiments, the poly(amino acid)-based polymer of Formula II includes comonomers l and m, where m provides spacing, i.e., distance, between the hydrophobic monomer l and can reduce the rigidity of the polymer. In other embodiments, the poly(amino acid)-based polymer of Formula II includes monomers l, m, and o, where monomer m provides spacing between the bulky substituents that make up monomers l and o. In other embodiments, the poly(amino acid)-based polymer of Formula II includes monomers l and o, and optionally monomers m and n, where monomer n is used to adjust the charge of the polymer backbone. In certain embodiments, the poly(amino acid)-based polymer of Formula II is composed entirely of monomers m and o. In other embodiments, the poly(amino acid)-based polymer of Formula II is composed entirely of monomers m, n, and o, or only n and o. In yet other embodiments, the poly(amino acid)-based polymer of Formula II comprises monomers l, m, n, and o.

[0295] When the poly(amino acid)-based polymer of Formula II includes an adjuvant, the percentage of monomers comprising the polymer, represented by o, which is the monomer linked to the adjuvant via any suitable linker, is typically 10-60%, e.g., between 2 and 12 amino acids of a polymer 20 amino acids in length are monomer o. In some embodiments of the poly(amino acid) polymer of Formula II, 1 is the predominant monomer unit. In further embodiments, the poly(amino acid)-based polymer of Formula II is composed entirely of 1 monomers, i.e., all monomers are 1 monomers, in which case the adjuvant is optionally attached to the termini of the poly(amino acid), either directly or indirectly via a second polymer or any suitable linker molecule.

[0296] When the polymer of Formula II is a copolymer containing o monomers, the ligands may be linked to pendant functional groups (FG) distributed along the polymer backbone as shown here: [ka] (wherein y11 represents the number of methylene units and is any integer, for example, 0 to 6, such as 0, 1, 2, 3, 4, 5 or 6.) The functional group (FG) is any suitable functional group, including amine, carboxylic acid, thiol, hydroxyl, azide, alkyne, hydrazine, aldehyde or ketone, that allows for the attachment, i.e., linkage, of a ligand, either directly or via a linker.

[0297] In some embodiments, y5, y6, y7, and y8 are equal to 1, and the polymer of Formula II is [ka] is.

[0298] A ligand having adjuvant properties may be linked to any of the hydrophobic molecules (H) of the present disclosure. In certain embodiments, the ligand having adjuvant properties is linked to a polymer of Formula II. The ligand having adjuvant properties may be linked to a poly(amino acid)-based polymer of Formula II through a pendant functional group (FG) on the o monomer, at the end of the polymer, or indirectly through another molecule or polymer grafted to the pendant functional group (FG) or at the end of the polymer. In preferred embodiments, the functional group (FG) of monomer o covalently links the ligand having adjuvant properties, or another ligand molecule, to the poly(amino acid) backbone. In some embodiments, the FG comprising monomer o of Formula II can be linked to a second polymer. The FG included in Formula II can be selected from a carboxylic acid, an aldehyde, a ketone, an amine, a hydrazine, a thiol, an azide, or an alkyne, or any suitable functional group that can be used to link a ligand or another polymer to the polymer backbone.

[0299] In preferred embodiments, the N-terminus of the poly(amino acid) of Formula II is linked to the peptidic antigen (A) by a linker (L), either directly or through an extension (B1 or B2), through reaction of linker precursors X1 and X2. In some embodiments, the N-terminus of the poly(amino acid) of Formula II is linked directly to the C-terminus of the peptidic antigen (A) or to the C-terminus of the B2 extension by an amide bond (i.e., the linker (L) is not present). In other embodiments, the N-terminus of the poly(amino acid) of Formula II is linked to a cyclooctyne (DBCO)-containing linker precursor (X2) that reacts with an azide-containing linker precursor (X1) that is linked to the peptidic antigen (A) directly or through an extension (B1 or B2).

[0300] The poly(amino acid) of Formula I or Formula II is a hydrophobic molecule (H) that can be linked to a peptidic antigen (A) either directly through an N-terminal amine, a C-terminal carboxylic acid, or through an optional side chain, e.g., a functional group of a comonomer, or indirectly through a linker (L) or extension (B1 or B2). In some embodiments, the poly(amino acid) of Formula I or Formula II is a hydrophobic molecule (H) that is linked to a peptidic antigen (A), resulting in a peptidic antigen conjugate of the formula [C]-[B1]-A-[B2]-[L]-H, or [B1]-A-[B2]-[L]-H(C), where [ ] indicates that the group is optional.

[0301] In a preferred embodiment, the poly(amino acid) of Formula I or Formula II is a hydrophobic molecule (H) linked at its N-terminus to a linker (L), which is linked to a C-terminal extension (B2), which is linked to the C-terminus of a peptide antigen (A), which is linked at its N-terminus to an N-terminal extension (B1), which is linked to a charged molecule (C).

[0302] For example, C-B1-A-B2-TH

[0303] In further embodiments, the charged molecule (C) can be linked directly to the hydrophobic molecule (H) comprised of a poly(amino acid) of Formula I or II, or via a linker (L) that is linked to the peptidic antigen (A) via an extension, where the bracket notation for A-B2-L(C)-H is intended to indicate that L is linked to both C and H.

[0304] For example, A-B2-L(C)-H or A-B2-LH(C)

[0305] In a further embodiment, the poly(amino acid) of Formula I or Formula II is a hydrophobic molecule (H) linked at its N-terminus to a linker (L) which is linked to an optional charged (C) molecule and to a C-terminal extension (B2) linked to the C-terminus of the peptidic antigen (A).

[0306] For example, A-B2-(CL)-H

[0307] In a preferred embodiment, the hydrophobic block comprises a poly(amino acid) with adjuvant ligands attached to side chains distributed along the backbone of the poly(amino acid). The adjuvant ligands may be hydrophobic or hydrophilic in nature, charged or uncharged. In a preferred embodiment, the adjuvant ligands are PRR agonists.

[0308] In some embodiments, the ligand with adjuvant properties can be a pattern recognition receptor (PRR) agonist. Non-limiting examples of pattern recognition receptor (PRR) agonists include TLR-1 / 2 / 6 agonists (e.g., lipopeptides and glycolipids, e.g., Pam2cys or Pam3cys lipopeptides), TLR-3 agonists (e.g., dsRNA, e.g., poly I:C, and nucleotide base analogs), TLR-4 agonists (e.g., lipopolysaccharide (LPS) derivatives, e.g., monophosphoryl lipid A (MPL), and small molecules such as derivatives of pyrimidoindole or analogs), TLR5 agonists (e.g., flagellin), TLR-7 and -8 agonists (e.g., ssRNA and nucleotide base analogs, including derivatives of imidazoquinoline, hydroxy-adenine, benzonaphthyridine, and loxoribine), and TLR-9 agonists (e.g., unmethylated CpG), stimulator of interferon genes (STING) agonists (e.g., cyclic dinucleotides, e.g., cyclic diadenylate monophosphate (cyclic di-DMP), cyclic nucleotide analogs, e.g., ... diadenylate monophosphate), C-type lectin receptor (CLR) agonists (e.g., various mono-, di-, tri-, and polymeric sugars, which may be linear or branched, such as mannose, Lewis-X trisaccharide, etc.), RIG-I-like receptor (RLR) agonists, and NOD-like receptor (NLR) agonists (e.g., peptidoglycan and structural motifs from bacteria, such as meso-diaminopimelic acid and muramyl dipeptide), and combinations thereof. In some embodiments, the pattern recognition receptor agonist can be a TLR agonist, such as an imidazoquinoline-based TLR-7 / 8 agonist. For example, the ligand with adjuvant properties can be imiquimod (R837) or resiquimod (R848), which are FDA-approved for use in humans.

[0309] In some embodiments, the ligand with adjuvant properties can be a TLR-7 agonist, a TLR-8 agonist, and / or a TLR-7 / 8 agonist. Numerous such agonists are known, including many different imidazoquinoline compounds.

[0310] Imidazoquinolines are used in the methods disclosed herein. Imidazoquinolines are synthetic immunomodulators that act by binding to Toll-like receptors 7 and 8 (TLR-7 / TLR-8) on antigen-presenting cells (e.g., dendritic cells) and structurally mimic the viral single-stranded RNA that is the natural ligand for these receptors. Imidazoquinolines are heterocyclic compounds containing a fused quinoline-imidazole backbone. Derivatives, salts (including hydrates, solvates, and N-oxides), and prodrugs thereof are also contemplated by the present disclosure. Certain imidazoquinoline compounds are known in the art; see, for example, U.S. Pat. No. 6,518,265 and U.S. Pat. No. 4,689,338. In some non-limiting embodiments, the imidazoquinoline compound is not imiquimod and / or not resiquimod.

[0311] In some embodiments, the ligand with adjuvant properties can be a small molecule having a 2-aminopyridine fused to a five-membered nitrogen-containing heterocyclic ring, such small molecules including, but not limited to, imidazoquinoline amines and substituted imidazoquinoline amines, such as amide-substituted imidazoquinoline amines, sulfonamide-substituted imidazoquinoline amines, urea-substituted imidazoquinoline amines, aryl ether-substituted imidazoquinoline amines, heterocyclic ether-substituted imidazoquinoline amines, amide ether-substituted imidazoquinoline amines, sulfonamide-substituted imidazoquinoline amines, sulfonamide-substituted imidazoquinoline amines, Ether-substituted imidazoquinoline amines, urea-substituted imidazoquinoline ethers, thioether-substituted imidazoquinoline amines, hydroxylamine-substituted imidazoquinoline amines, oxime-substituted imidazoquinoline amines, 6-, 7-, 8-, or 9-aryl, heteroaryl, aryloxy, or arylalkyleneoxy-substituted imidazoquinoline amines, and imidazoquinoline diamines; amide-substituted tetrahydroimidazoquinoline amines, sulfonamide-substituted tetrahydroimidazoquinoline amines, urea-substituted tetrahydroimidazoquinoline amines, aryl amines, thioether-substituted tetrahydroimidazoquinoline amines, hydroxylamine-substituted tetrahydroimidazoquinoline amines, oxime-substituted tetrahydroimidazoquinoline amines, and tetrahydroimidazoquinoline diamines, including, but not limited to, aryl ether-substituted tetrahydroimidazoquinoline amines, heterocyclic ether-substituted tetrahydroimidazoquinoline amines, amido ether-substituted tetrahydroimidazoquinoline amines, sulfonamide ether-substituted tetrahydroimidazoquinoline amines, urea-substituted tetrahydroimidazoquinoline ethers, thioether-substituted tetrahydroimidazoquinoline amines, hydroxylamine-substituted tetrahydroimidazoquinoline amines, oxime-substituted tetrahydroimidazoquinoline amines, and tetrahydroimidazoquinoline diamines. imidazopyridine amines, including, but not limited to, amido-substituted imidazopyridine amines, sulfonamido-substituted imidazopyridine amines, urea-substituted imidazopyridine amines, aryl ether-substituted imidazopyridine amines, heterocyclic ether-substituted imidazopyridine amines, amido ether-substituted imidazopyridine amines, sulfonamido ether-substituted imidazopyridine amines, urea-substituted imidazopyridine ethers, and thioether-substituted imidazopyridine amines; 1,2-bridged imidazoquinoline amines;6,7-fused cycloalkylimidazopyridine amines; imidazonaphthyridine amines; tetrahydroimidazonaphthyridine amines; oxazoloquinoline amines; thiazoloquinoline amines; oxazolopyridine amines; thiazolopyridine amines; oxazolonaphthyridine amines; thiazolonaphthyridine amines; pyrazolopyridine amines; pyrazoloquinoline amines; tetrahydropyrazoloquinoline amines; pyrazolonaphthyridine amines; tetrahydropyrazolonaphthyridine amines; as well as 1H-imidazo dimers fused with pyridine amines, quinoline amines, tetrahydroquinoline amines, naphthyridine amines, or tetrahydronaphthyridine amines.

[0312] In some embodiments, the ligand having adjuvant properties has the formula: [ka] It is an imidazoquinoline having the formula:

[0313] In formula III, R 7 is selected from one of hydrogen, optionally substituted lower alkyl, or optionally substituted lower ether; R 8 is selected from one of an optionally substituted arylamine or an optionally substituted lower alkylamine. 8 may be optionally substituted to become a linker that connects to the polymer. 8 In some compounds where R is selected from lower alkyl amines, the compound may also contain R selected from aryl amines. 8 The results showed that the adjuvant of Formula III produced a better quality response than the adjuvant of Formula III, although it was less potent. Thus, the moderate potency adjuvant of Formula III produced a better quality response. Note that the adjuvant of Formula III is a type of ligand and may also be referred to as an adjuvant of Formula III or a ligand with adjuvant properties.

[0314] In some embodiments, R in Formula III 7, hydrogen, [ka] You can choose from:

[0315] In some embodiments, R 8 of, [ka] (In the formula, e represents the number of methylene units and is an integer of 1 to 4.) You can choose from:

[0316] In some embodiments, R 8 teeth, [ka] It is possible.

[0317] In some embodiments, R 8 teeth, [ka] It is possible.

[0318] In some embodiments, R 7 teeth, [ka] It is possible, R 8 teeth, [ka] It is possible.

[0319] Non-limiting examples of hydrophobic molecules (H) comprised of poly(amino acids) of Formula I linked to adjuvants of Formula III include: [ka] (where k is between 3 and 300). For example, when k=5, the peptide is composed of 5 amino acids linked to an adjuvant of Formula III. In some embodiments, a hydrophobic molecule (H) composed of a poly(amino acid) of Formula I linked to an adjuvant of Formula III can be linked, either directly or indirectly via a linker (L) and / or extension (B1 or B2), to a peptide antigen (A), which is optionally linked by any suitable means to a charged molecule (C), to form a peptide antigen conjugate. In some embodiments, the N-terminus of the poly(amino acid) of Formula I linked to an adjuvant of Formula III is linked directly to the C-terminus of the peptide antigen (A) or to the C-terminus of the B2 extension via an amide bond. In other embodiments, the N-terminus of the poly(amino acid) of Formula I linked to the adjuvant of Formula III is linked to a clickable linker precursor X2, such as an alkyne or DBCO, or a thiol-reactive linker precursor X2, such as a maleimide, which reacts with a linker precursor (X2) linked to the peptidic antigen (A) either directly or through an extension (B1 or B2). In a preferred embodiment, a DBCO linker precursor X1 is attached to the N-terminus of the poly(amino acid) of Formula I linked to the adjuvant of Formula III and used to react with an azide-containing linker precursor X2 linked to the peptidic antigen (A) either directly or through an extension (B1 or B2).

[0320] Non-limiting examples of hydrophobic molecules (H) comprised of poly(amino acids) of Formula II linked to adjuvants of Formula III include: [ka] Examples include:

[0321] for example, [ka] (wherein the comonomer l is typically an integer between 3 and 300, the optional comonomer o is typically an integer between 3 and 300 amino acid residues, and the sum of l and o is typically between about 3 and 300. Alternatively, o is 0 and the polymer is composed entirely of l, i.e., the polymer is a poly(tryptophan) polymer that is not linked to an adjuvant. In some embodiments, the N-terminus of the poly(amino acid) of Formula II that is linked to an adjuvant of Formula III is linked to the peptidic antigen (A) by any suitable means, either directly or indirectly through a linker (L) and / or extension (B1 or B2). In some embodiments, the N-terminus of the poly(amino acid) of Formula II that is linked to an adjuvant of Formula III is linked directly to the C-terminus of the peptidic antigen (A) or to the C-terminus of the B2 extension by an amide bond. In other embodiments, the N-terminus of the poly(amino acid) of Formula II linked to the adjuvant of Formula III is linked to a clickable linker precursor X2, such as DBCO, or a thiol-reactive linker precursor X2, such as maleimide, which reacts with a linker precursor X1 linked to the peptide antigen (A) directly or through an extension (B1 or B2). In a preferred embodiment, a DBCO linker precursor X2 is attached to the N-terminus of the poly(amino acid) of Formula II linked to the adjuvant of Formula III and used to react with a linker precursor X1 bearing an azide functional group.

[0322] An unexpected discovery disclosed herein is that the length of the poly(amino acid) of either Formula I or Formula II linked to the adjuvant of Formula III that constitutes the hydrophobic molecule (H) of the peptide-antigen conjugate, i.e., the number of monomer units of such poly(amino acid), is a major determinant affecting the magnitude of the T-cell response generated against the peptide antigen (A). Accordingly, it has been found that peptide-antigen conjugates composed of hydrophobic molecules (H) composed of poly(amino acids) of Formula I or Formula II having five or more monomer units promote higher quality and magnitude of T-cell responses compared to poly(amino acids) of the same formula that are less than five amino acids in length. A further discovery was that the number of adjuvants of Formula III linked to a poly(amino acid) of Formula I or Formula II also affected the magnitude of the immune response generated, with peptide-antigen conjugates comprising a hydrophobic molecule (H) of Formula I or II containing three or more adjuvants of Formula III resulting in larger T cell responses compared to peptide-antigen conjugates comprising a hydrophobic molecule (H) of Formula I or II containing fewer than three adjuvants of Formula III. A non-limiting explanation for these findings is that increasing the length of the hydrophobic molecule (H) of Formula I or Formula II linked to an adjuvant of Formula III ensures the formation of micelles or other supramolecular structures, even when linked to hydrophilic peptide antigens, and that the formation of such particles leads to improved immune responses, possibly due to improved pharmacokinetics and cellular uptake resulting from particles rather than soluble material. A further non-limiting explanation is that increasing the length of the hydrophobic molecule (H) comprised of a poly(amino acid) of Formula I or Formula II linked to the adjuvant of Formula III will improve the stability, e.g., kinetic stability, of the particles formed by the peptide antigen conjugate in aqueous buffer; improved particle stability ensures that the particles remain intact, thereby delaying clearance (either renal or hepatic) of the peptide antigen conjugate comprising the particles and promoting uptake by antigen-presenting cells.

[0323] A further unexpected discovery disclosed herein is that the potency of an adjuvant of formula III linked to a poly(amino acid) of formula I or formula II that constitutes the hydrophobic molecule (H) of a peptide-antigen conjugate has been found to be inversely related to the magnitude and breadth of the T cell response generated against the peptide antigen after multiple immunizations. Accordingly, as disclosed herein, the inventors have developed an adjuvant of formula III, also referred to as Compound 1, wherein: R 7 = [ka] and R 8 = [ka] ) is an adjuvant of formula III, also referred to as Compound 2, wherein R 7 = [ka] and R 8 = [ka] ) results in a greater magnitude and breadth of T cell response compared to poly(amino acids) of Formula I linked to .

[0324] A non-limiting example is an adjuvant of formula III, where R 7 = [ka] ) is found to be less potent than the adjuvant of formula III (wherein R 7 = [ka] ) results in less inflammation, and the moderate inflammation induced by lower potency agonists leads to less exhaustion of the T cell response, thereby resulting in a greater magnitude and breadth of T cell response overall.

[0325] Based on these findings, a preferred embodiment of a hydrophobic molecule (H) comprised of a poly(amino acid) of formula I linked to an adjuvant of formula III is: [ka] is.

[0326] In the present disclosure, we have identified an adjuvant of formula III, compound 1, where R 7 = [ka] and R 8 = [ka] We report the unexpected discovery that hydrophobic molecules (H) based on poly(amino acid)s of Formula II linked to comonomers o of Formula III result in significant increases in the magnitude and quality of T cell responses when the poly(amino acid) polymer is composed of between 5 and 10 amino acids and between 60 and 100% of the copolymer is composed of comonomers o linked to adjuvants of Formula III. In preferred embodiments, comonomers o constitute between 20 and 60%, e.g., 60%, of the monomer units of the poly(amino acid) of Formula II.

[0327] for example, [ka]

[0328] The unexpected data disclosed herein demonstrate that the length of the polymer containing the hydrophobic molecule (H) and the number and potency of the attached ligands with adjuvant properties (i.e., the TLR-7 / 8 agonist of Formula III) are key determinants of the magnitude and quality of the immune response generated against a peptide antigen (A) delivered as a peptide antigen conjugate. These data suggest that a hydrophobic molecule (H) composed of a poly(amino acid) composed of hydrophobic amino acids and / or amino acids linked to a ligand should be of sufficient length to enable particle formation when linked to any peptide antigen (A), including highly hydrophilic peptide antigens (A) that negate the tendency of poly(amino acid)-based hydrophobic molecules (H) to drive particle assembly. Poly(amino acids) of insufficient length, e.g., less than three amino acids in length, may not provide sufficient hydrophobic surface area to promote particle formation in aqueous conditions when linked to certain peptide antigens, particularly hydrophilic peptide antigens with high charge density. Thus, as disclosed herein, poly(amino acids) of Formula I or Formula II should be greater than 3 amino acids in length, preferably between 5 and 30 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 20, or 30 amino acids in length. Longer poly(amino acids), e.g., greater than 30 amino acids in length, e.g., about 30, 40, or 50 amino acids in length, can be produced, for example, by solid-phase peptide synthesis. As an alternative to solid-phase peptide synthesis, solution polymerization reactions may be used to produce long chain poly(amino acids) composed of hydrophobic monomers.

[0329] Herein, we disclose the further unexpected discovery that Compound 1, which has a potency for in vitro determined TLR-7 activity (i.e., EC50) of 10 nanomolar, results in enhanced T cell responses after repeated immunizations compared to Compound 2, a more potent agonist with a potency for in vitro determined TLR-7 activity (i.e., EC50) of 18.7 nanomolar. These data suggest that the potency of adjuvants included in immunogenic compositions can be adjusted to optimize immune responses, and that adjuvant-mediated innate immune activation can be modified to provide the appropriate level of stimulation required to optimize T cell immunity. Peptide-antigen conjugates can be used to modulate the level of innate immune stimulation by delivering multiple, e.g., three or more, doses of a moderately potent agonist (i.e., an agonist with an EC50 > 100 nanomolar) or fewer than three doses of a more potent agonist (i.e., an agonist with an EC50 < 100 nanomolar).

[0330] The potencies of various TLR-7 agonists, TLR-8 agonists, and combined TLR-7·TLR-8 agonists can be readily ascertained from the literature. Imidazoquinoline-based and adenine-based TLR-7 and TLR-7 / 8 agonists have been described (see, for example, Shukla et al., J. Med. Chem., 53:4450-4465, 2010; Gerster et al., J. Med. Chem., 2005; U.S. Pat. No. 6,069,149; and Hirota et al., J. Med. Chem., 45:5419-5422, 2002, which are incorporated herein by reference), and the R of the adjuvant of Formula III 2 Aromatic linkers, such as benzyl and xylyl linkers, selected from R 2 This demonstrates that the compound has increased potency against TLR-7 compared to compounds selected from lower alkyls.

[0331] Based on the unexpected discoveries described herein, preferred embodiments of hydrophobic molecules (H) comprised of poly(amino acids) of Formula I or Formula II linked to adjuvants of Formula III are typically between 5 and 30 amino acids in length, when the density of comonomers linked to the adjuvants of Formula III is between 20 and 100 mol %. Optionally, the adjuvants of Formula III are selected from lower alkyl groups, such as R 8 For example, in the case of Compound 1, the density of amino acids linked to Compound 1 should be between 40 and 100%, for example 60%. Optionally, the adjuvant of Formula III may comprise an R selected from aromatic 8 For example, in the case of Compound 2, the density of amino acids linked to Compound 2 should be less than 20%, or between 1-2 molecules of Compound 2 delivered on each polymer.

[0332] In a preferred embodiment, a polymer having a molecular weight of less than about 10,000 g / mol is selected from the group consisting of N-2-hydroxypropyl(methacrylamide) (HPMA), hydroxyethyl(methacrylate) (HEMA), styrene, vinylpyrrolidone (PVP), N-isopropylacrylamide (NIPAAm), N-isopropylmethacrylamide (NIPMAm), N,N'-diethylacrylamide (DEAAm), N-(L)-(1-hydroxymethyl)propylmethacrylamide (HMPMAm), N,N'-dimethylethylmethacrylate (DMEMA), 2-(2-methylethyl)methacrylamide ( ... The density of the adjuvant of Formula III linked to a polymer based on a comonomer selected from (ethoxyethoxy)ethyl methacrylate (DEGMA) or a substituted poly(phosphoester) is about 5-100 mol %, for example, the density of the adjuvant bound to the polymer can be about 5-6%, about 6-7%, about 8-9%, about 9-10%, about 10-11%, about 11-12%, about 12-13%, about 13-14%, about 15-16%, about 17-18%, about 18-20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0333] In a preferred embodiment, polymers having a molecular weight greater than 10,000 g / mol are selected from the group consisting of N-2-hydroxypropyl(methacrylamide) (HPMA), hydroxyethyl(methacrylate) (HEMA), styrene, vinylpyrrolidone (PVP), N-isopropylacrylamide (NIPAAm), N-isopropylmethacrylamide (NIPMAm), N,N'-diethylacrylamide (DEAAm), N-(L)-(1-hydroxymethyl)propylmethacrylamide (HMPMAm), N,N'-dimethylethylmethacrylate (DMEMA), 2-(2-methoxyethyl)-2-methylpropanol (DMPA), 2-(2-hydroxy ... The density of the adjuvant of Formula III linked to a polymer based on a comonomer selected from dihydroxyethyl methacrylate (DEGMA) or a substituted poly(phosphoester) is about 1-25 mol %, for example, the density of the adjuvant bound to the polymer can be about 1-2%, about 2-3%, about 3-4%, about 5-6%, about 6-7%, about 7-8%, about 8-9%, about 9-10%, about 10-11%, about 11-12%, about 13-14%, about 14-15%, about 16-17%, about 17-18%, about 19-20%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%.

[0334] In a further embodiment, a second polymer composed of hydrophobic and / or temperature-responsive monomers is linked to either the end or side chain of a poly(amino acid) of Formula I or Formula II that is linked to an adjuvant of Formula III.

[0335] A non-limiting example of a hydrophobic molecule (H) comprised of a temperature-responsive DEGMA-based polymer grafted to a poly(amino acid) of Formula I linked to an adjuvant of Formula III is provided here for clarity: [ka] (wherein comonomer k is typically an integer between 3 and 300, comonomer k' is typically an integer between 3 and 300 amino acid residues, and the sum of k and k' is typically between about 3 and 300). In a preferred embodiment, k is between about 3 and 10, and k is between about 1 and 10. The linker, X, can be any suitable linker molecule, and m is typically between about 10 and 300 monomer units. The N-terminus of the poly(amino acid) is linked to the peptide antigen (A) by any suitable means, either directly or indirectly via a linker (L) and / or extension (B1 or B2), to form a peptide antigen conjugate, which may include an additional charged molecule (C).

[0336] A non-limiting example of a hydrophobic molecule (H) comprised of a temperature-responsive DEGMA-based polymer linked to the terminus of a poly(amino acid) of Formula I, which is linked to an adjuvant of Formula III, is provided here for clarity: [ka] (wherein the comonomer k is typically an integer between 3 and 100 monomer units). The linker, X, can be any suitable linker molecule. In a preferred embodiment, k' is between about 3 and 10 monomer units. The N-terminus of the poly(amino acid) is linked to the peptide antigen (A) by any suitable means, either directly or indirectly via a linker (L) and / or extension (B1 or B2), to form a peptide antigen conjugate, which may include an additional charged molecule (C).

[0337] A non-limiting example of a hydrophobic molecule (H) comprised of a polyphosphoester-based polymer linked to the terminus of a poly(amino acid) of Formula I, which is linked to an adjuvant of Formula III, is provided here for clarity: [ka] (wherein the comonomer k is typically an integer between 3 and 100 monomer units). The linker, X, can be any suitable linker molecule. In preferred embodiments, k is between about 5 and 10 monomer units and m is between about 10 and 300 monomer units. The N-terminus of the poly(amino acid) is linked to the peptide antigen (A) by any suitable means, either directly or indirectly via a linker (L) and / or extension (B1 or B2), to form a peptide antigen conjugate, which may include an additional charged molecule (C).

[0338] A non-limiting example of a hydrophobic molecule (H) comprised of a poly(benzyl glutamate)-based polymer linked to the terminus of a poly(amino acid) of Formula I, which is linked to an adjuvant of Formula III, is provided here for clarity: [ka] (wherein the comonomer k is typically an integer between 3 and 100 monomer units, and m is typically an integer between 50 and 300 amino acid residues). The linker can be any suitable linker molecule. In a preferred embodiment, x is between about 5 and 10 monomer units, and m is between about 10 and 300 monomer units. The N-terminus of the poly(amino acid) is linked to the peptide antigen (A) by any suitable means, either directly or indirectly via a linker (L) and / or extension (B1 or B2), to form a peptide antigen conjugate, which may include an additional charged molecule (C).

[0339] In a non-limiting example, a peptide antigen (A) is linked to a particle (P) or a hydrophobic molecule (H), which may further include an optional extension (B1 and / or B2) and an optional linker (L) to obtain a peptide antigen conjugate of formula IV, where [ ] indicates that the group is optional. [B1]-A-[B2]-[L]-P, [B1]-A-[B2]-[L]-H, P-[L]-[B1]-A-[B2] or H-[L]-[B1]-A-[B2] Formula IV

[0340] The peptide antigen (A) of Formula IV is composed of an integer number of amino acids, n, where n is typically between 7 and 35, e.g., 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, or 35 amino acids, and the hydrophobic molecule (H) is typically a poly(amino acid) of Formula I or II linked to an adjuvant of Formula III.

[0341] A non-limiting example of a peptide-antigen conjugate of Formula IV, consisting of a peptide antigen (A) optionally linked at its N-terminus to a cathepsin-cleavable tetrapeptide extension (B1=Lys-Pro-Leu-Arg SEQ ID NO: 8) and at its C-terminus to a combined immunoproteasome-cathepsin-cleavable hexapeptide extension (B2=Gly-Gly-Ser-Leu-Val-Arg SEQ ID NO: 13) linked to a triazole linker (L) which is linked to a hydrophobic molecule (H) composed of a poly(amino acid) of Formula I linked to an adjuvant of Formula III, is shown here by way of example: [ka]

[0342] Optionally, charged molecules (C) The peptide antigen conjugates disclosed herein are composed of a peptide antigen (A) linked to a particle (P) or a hydrophobic molecule (H), and may further include optional extensions (B1 and / or B2), an optional linker (L), and an optional charged molecule (C), where C= represents a charged molecule having a functional group that confers an electrostatic charge. The immunogenic compositions disclosed herein, which include a peptide antigen linked to a particle (P) or to a hydrophobic molecule (H) that assembles into particles in aqueous conditions, may aggregate if there is not sufficient surface charge to stabilize the particles. Therefore, the charged molecule (C) may be optionally linked to the peptide antigen conjugate as a means of stabilizing the particles and preventing aggregation, or alternatively, the charged molecule (C) may be incorporated into particles containing the peptide antigen conjugate as a means of stabilizing the particles. Thus, the purpose of the charged molecule (C) is to control the net charge of particles formed by the peptide antigen conjugate as a means of promoting the stability of these particles.

[0343] A charged molecule (C) refers to any molecule having one or more functional groups that are positively or negatively charged in an aqueous buffer solution at a pH of about 7.4. The functional groups that make up a charged molecule (C) can have partial or full charge integer values. A charged molecule (C) can be a molecule with a single charged functional group or multiple charged functional groups. The net charge of a charged molecule (C) can be positive, negative, or neutral. The charges of the functional groups that make up a charged molecule (C) can be dependent or independent of the pH of the solution in which the charged molecule (C) is dispersed, such as in the case of tertiary amines and quaternary ammonium compounds, which are pH-dependent and pH-independent, respectively. The charge of a molecule can be easily estimated based on the Lewis structure of the molecule and accepted methods known to those skilled in the art. Charge can also arise as a result of inductive effects; for example, atoms bonded to each other with different electron affinities can result in polar covalent bonds that result in some atoms being negatively charged and some atoms being positively charged. For example, nitrogen bonding to a hydrogen results in a negative partial charge on the nitrogen and a positive partial charge on the hydrogen atom. Alternatively, an atom in a molecule can be considered to have a full charge integer value if the number of electrons assigned to that atom is less than or equal to the atomic number of that atom. The charge of a molecule is determined by adding up the charges of each atom that makes up the molecule. Those skilled in the art are familiar with how to estimate the charge of a molecule by adding up the formal charges of each atom in the molecule.

[0344] The charged molecule (C) can bear either a net negative, net positive, or neutral charge, depending on the net charge of the peptide-antigen conjugate required for the particular application of the present invention disclosed herein. For example, it is known that most cell surfaces bear a net negative charge. Therefore, particles with a positive net charge may interact with all cell surfaces without a high degree of specificity. In contrast, particles with a negative net charge, while experiencing electrostatic repulsion from most cell surfaces, have been shown to promote selective uptake by certain antigen-presenting cell populations. For example, positively charged particles delivered intravenously into the circulation have been found to accumulate in the liver and lungs as well as in antigen-presenting cells in the spleen, while negatively charged particles have been found to preferentially accumulate in antigen-presenting cells in the spleen after intravenous administration. Therefore, the net charge of the charged molecule (C) can be tailored to meet the specific requirements of the application.

[0345] In some embodiments, the charged molecule (C) has a net negative charge and is composed of a functional group that is negatively charged at physiological pH, about pH 7.4. Suitable charged molecules (C) that have a net negative charge include molecules that have a functional group that exists as the conjugate base of an acid at physiological pH, about pH 7.4 (e.g., a functional group with a pKa of less than about 6.5). These include, but are not limited to, molecules with carboxylate, sulfate, phosphate, phosphoramidate, and phosphonate. Carboxylate-containing charged molecules (C) can be, but are not limited to, glutamic acid, aspartic acid, pyruvic acid, lactic acid, glycolic acid, glucuronic acid, citrate, isocitrate, alpha-keto-glutarate, succinate, fumarate, malate, and oxaloacetate, and derivatives thereof. In preferred embodiments, the negatively charged molecule (C) is comprised of a molecule having between 1 and 20 negatively charged functional groups, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 negatively charged functional groups, but typically 16 or fewer negatively charged functional groups. In some embodiments, the charged molecule (C) is a poly(glutamic acid) peptide between 2 and 6 amino acids in length. Poly(glutamic acid) sequences comprised of 1, 2, 3, 4, 5, or 6 amino acids are expected to carry negative charges of -1, -2, -3, -4, -5, and -6 at pH 7.4, respectively. In further embodiments, the charged molecule (C) is phosphoserine or sulfoserine.

[0346] In certain embodiments, the charged molecule (C) has a net negative charge and is composed of one or more negatively charged amino acids. In a preferred embodiment, the charged molecule (C) having a net negative charge is composed of between 1 and 20 negatively charged amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 negatively charged amino acids. In a non-limiting example, the charged molecule (C) is composed of 16 aspartic acid monomers, for example, Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 25), and is used to prepare the charged molecule (C) having a net negative charge of -16; A charged molecule (C) composed of p-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 26) is used to prepare a charged molecule (C) having a net negative charge of -15; a charged molecule (C) composed of 14 aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 27), has a net negative charge of -14. A charged molecule (C) composed of 13 aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 28), is used to prepare a charged molecule (C) having a net negative charge of -13; a charged molecule (C) composed of 12 aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp A charged molecule (C) composed of Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 29) is used to prepare a charged molecule (C) having a net negative charge of -12; a charged molecule (C) composed of 11 aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 30), is used to prepare a charged molecule (C) having a net negative charge of -11;A charged molecule (C) composed of 10 aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 31), is used to prepare a charged molecule (C) with a net negative charge of −10; a charged molecule (C) composed of 9 aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 32), prepares a charged molecule (C) with a net negative charge of −9. a charged molecule (C) composed of eight aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 33), is used to prepare a charged molecule (C) having a net negative charge of −8; a charged molecule (C) composed of seven aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 34), is used to prepare a charged molecule (C) having a net negative charge of −7; A charged molecule (C) composed of two aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 35), is used to prepare a charged molecule (C) having a net negative charge of −6; a charged molecule (C) composed of five aspartic acid monomers, e.g., Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 36), is used to prepare a charged molecule (C) having a net negative charge of −5; a charged molecule (C) composed of four aspartic acid monomers, e.g., Asp-Asp -Asp-Asp (SEQ ID NO: 37), is used to prepare a charged molecule (C) with a net negative charge of -4; a charged molecule (C) with three aspartic acid monomers, e.g., Asp-Asp-Asp, is used to prepare a charged molecule (C) with a net negative charge of -3; a charged molecule (C) with two aspartic acid monomers, e.g., Asp-Asp, is used to prepare a charged molecule (C) with a net negative charge of -2;A charged molecule (C) composed of one aspartic acid monomer, e.g., Asp, is used to prepare a charged molecule (C) having a net negative charge of −1. The aspartic acid (Asp) in the above example can be replaced with any suitable negatively charged amino acid, including but not limited to glutamic acid, sulfo-serine, or phosphor-serine, where the negatively charged amino acid can be the same or different;

[0347] In some embodiments, the charged molecule (C) has a net positive charge and is composed of a positively charged functional group. Suitable positively charged molecules (C) include those having a functional group that is positively charged at physiological p...

Claims

[Claim 1] The invention described in the specification.

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